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Module 1
Introduction
The aim of this website is to describe cell signalling within cells. One cell releases a chemical stimulus (e.g. a neur-
its biological context. There has been an explosion in the otransmitter, hormone or growth factor), which then al-
characterization of signalling components and pathways. ters the activity of target cells. The latter have receptors
The next major challenge is to understand how cells exploit capable of detecting the incoming signal and transferring
this large signalling toolkit to assemble the specific sig- the information to the appropriate internal cell signalling
nalling pathways they require to communicate with each pathway to bring about a change in cellular activity.
other. The primary focus is the biology of cell signalling.
The emerging information on cell signalling pathways is Communication through electrical
integrated and presented within the context of specific cell signals
types and processes. The beauty of cell signalling is the Communication through electrical signals is found mainly
way different pathways are combined and adapted to con- in excitable systems, particularly in the heart and brain. It
trol a diverse array of cellular processes in widely different is usually fast and requires the cells to be coupled together
spatial and temporal domains. through low-resistance pathways such as the gap junc-
The first half of the website characterizes the compon- tions (Module 1: Figure cell communication). In addition
ents and properties of the major cell signalling pathways, to passing electrical charge, the pores in these gap junctions
with special emphasis on how they are switched on and are large enough for low-molecular-mass molecules such
off. Attention is also focused on the spatial and temporal as metabolites and second messengers to diffuse from one
aspects that determine how information is encoded and cell to another.
distributed to precise cellular locations. The second half of
the website deals with the way these different signalling Communication through chemical
pathways are employed to control the life history of cells signals
from their birth during the process of cell proliferation, Cells are enclosed within a lipophilic plasma membrane,
their differentiation into specific cell types to carry out which represents a formidable barrier that has to be
different cell functions, and finally their death through crossed by all incoming signals. Hydrophobic hormones,
processes such as apoptosis. Cell signalling orchestrates such as the steroid hormones, can simply diffuse across
all these cellular processes. Many of the same signalling this cell-surface barrier to gain access to protein receptors
systems that control development come into play again to located in either the cytoplasm or the nucleus. More
regulate a wide range of specific processes in adult cells, elaborate mechanisms are required for the water-soluble
such as contraction, secretion, metabolism, proliferation, stimuli (e.g. hormones, neurotransmitters and growth
information processing in neurons and sensory perception. factors) that cannot cross the plasma membrane (Module
These examples illustrate how cell signalling pathways are 1: Figure cell communication). The basic concept of a cell
adapted and co-ordinated to regulate many different cellu- signalling pathway, therefore, concerns the mechanisms
lar processes. This intimate relationship between cell sig- responsible for receiving this external information and
nalling and biology is providing valuable insights into the relaying it through internal cell signalling pathways to
underlying genetic and phenotypic defects responsible for activate the sensor and effector mechanisms that bring
many of the major human diseases. about a change in cellular responses (Module 1: Figure
cell signalling mechanism). Cell signalling is a dynamic
Overview of cell signalling mechanisms process in that there are ON mechanisms during which
Cells in organisms such as us constantly communicate with information flows down the signalling pathway in re-
each other. This cellular discourse occurs through both sponse to external stimuli (the green arrows in Module
electrical and chemical signals (Module 1: Figure cell com- 1: Figure cell signalling mechanism), opposed by the
munication). Communication through electrical signals OFF mechanisms that are responsible for switching off
is very fast and depends upon the presence of gap junctions the signalling system once external stimuli are withdrawn
to allow information to pass directly from one cell to its (the red arrows in Module 1: Figure cell signalling mech-
neighbour. Communication through chemical signals is anism). Some of these basic principles of cell signalling
by far the major form of information transfer between are explored in this introductory module, which will also
briefly outline the contents of the other modules.
Green text indicates links to content within this module; blue text ON mechanisms
indicates links to content in other modules Most signalling pathways begin with the arrival of external
Please cite as Berridge, M.J. (2014) Cell Signalling Biology; cell stimuli usually in the form of a chemical signal, which
doi:10.1042/csb0001001 is received by receptors at the cell periphery that function
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2014 Portland Press Limited www.cellsignallingbiology.org
Licensed copy. Copying is not permitted, except with prior permission and as allowed by law.
Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r2
CHEMICAL
ELECTRICAL
COMMUNICATION
COMMUNICATION
Receptor
Cell signalling
pathway
E nucl
Ecyto Ecyto
STIMULI
Receptors
Transducers
Amplifiers
Signalling
Messengers pathway
CELLULAR RESPONSES
as molecular antennae embedded in the plasma membrane ON mechanisms responsible for transmitting information
(Module 1: Figure cell signalling mechanism). These re- into the cell are counteracted by the OFF mechanisms that
ceptors then function to transfer information to a variety of switch off this flow of information once stimuli are with-
transducers and amplifiers to produce intracellular mes- drawn. A related OFF mechanism is receptor desensitiz-
sengers. These messengers stimulate the sensors and ef- ation, whereby receptors lose their sensitivity to external
fectors responsible for activating cellular responses. These stimuli.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r3
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r4
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r5
that can function as such or is a precursor that is cleaved mune responses, particularly during inflammation. There
by proteases such as the ADAM proteases to release the are over 40 members of the family that seem to function
soluble stimulus through a process known as ectodomain through two main receptor types (Module 1: Figure cy-
shedding. Many stimuli, such as the eicosanoids, nitric ox- tokines).
ide (NO), ATP and sphingosine 1-phosphate (S1P), are There are a number of peptides such as atrial natriur-
formed within the cytoplasm and leave the cell by diffus- etic peptide (ANP), brain-type natriuretic peptide (BNP),
ing across the plasma membrane. In some cases, this exit C-type natriuretic peptide (CNP) and guanylin that act
from the cell is facilitated by special transporters such as through the particulate guanylyl cyclases (pGCs).
the ATP-binding cassette (ABC) transporters (Module 3: There are a number of stimuli capable of opening ion
Figure ABC transporters). Many stimuli are synthesized as channels (Module 1: Figure stimuli for ion channels). In
large precursors, such as pro-opiomelanocortin (POMP), this case, the ion channel carries out all the signalling func-
which then undergo extensive, tissue-specific processing tions.
as they are cleaved into a number of individual hormones Most of the stimuli described above arrive at the cell sur-
or neuropeptides. face through a process of diffusion. In some cases, however,
Many stimuli such as hormones as neurotransmitters stimuli can be presented to receptors by special molecules.
are packaged in to vesicles where they are stored before A classic example is the role of MHCII on the antigen-
being released by exocytosis. These stimuli then have dif- presenting cell that binds fragments of antigen that are
ferent modes of action (juxtacrine, autocrine, paracrine and then presented to the T cell receptor (Module 9: Figure
endocrine), which have been defined on the basis of how TCR signalling). Another example is the role of CD14 in
far they travel to reach their cellular receptor targets. presenting lipopolysaccharide (LPS) to the Toll-like re-
Once these cell stimuli reach their targets they use a ceptor (TLR) (Module 2: Figure Toll receptor signalling).
diverse number of cell signalling pathways to control cel-
lular activity (Module 2: Figure cell signalling pathways). Juxtacrine
One way of describing this diversity is to consider the Direct cell-to-cell signalling achieved by a membrane-
nature of the stimuli that feed into different cell signalling anchored stimulus in one cell acting on receptors located
pathways. Some signalling mechanisms are used by many in a neighbouring cell (Module 1: Figure formation and
different signalling pathways, whereas other pathways re- action of cell stimuli). The following are examples where
spond to a specific set of stimuli. An example of the former information is transmitted between cells through such a
is the cyclic AMP signalling pathway, which was the first direct mechanism:
signalling pathway to be clearly defined (Module 1: Fig-
ure stimuli for cyclic AMP signalling). The major stim- Notch signalling pathway (Module 2: Figure Notch sig-
uli for this signalling pathway fall into two main classes: nalling).
neurotransmitters and hormones. They all act by enga- Eph receptor signalling (Module 1: Figure Eph receptor
ging G protein-coupled receptors (GPCRs), which use signalling).
heterotrimeric GTP-binding proteins (G proteins) to ac- Classical cadherin signalling (Module 6: Figure classical
tivate the amplifier adenylyl cyclase that converts ATP into cadherin signalling).
the second messenger cyclic AMP (for further details see Activation of the Fas receptor (FasR) by the Fas lig-
Module 2: Figure cyclic AMP signalling). Some of the stim- and (FasL) as occurs during activation of the extrinsic
uli that activate this signalling pathway belong to a group pathway of apoptosis (Module 11: Figure apoptosis).
of lipid-derived stimuli known as the eicosanoids that in- The inflammatory mediator TNF can function in its
clude the prostaglandins, thromboxanes and leukotrienes transmembrane pro-form.
(Module 1: Figure eicosanoids). The endocannabinoids are
another group of lipid stimuli such as anandamide and 2- Autocrine
arachidonylglycerol (2-AG). This is a local signalling mechanism whereby the cell that
The inositol 1,4,5-trisphosphate (InsP3 )/diacylglycerol releases a stimulus has receptors capable of responding to
(DAG) signalling pathway is also used by a very large that stimulus (Module 1: Figure formation and action of
number of stimuli that are mainly neurotransmitters and cell stimuli). An example of such an autocrine response is
hormones (Module 1: Figure stimuli for InsP3 /DAG sig- found in blood platelets that release eicosanoids that feed
nalling). These external stimuli bind to GPCRs, which are back to influence the progress of the platelet activation
coupled to G proteins to activate the amplifier phospholi- sequence (Module 11: Figure platelet activation).
pase C (PLC). PLC hydrolyses an inositol lipid to generate Adenosine is another example of an autocrine factor that
the two second messengers, InsP3 and DAG (for further may function as an endogenous sleep-regulatory molecule.
details see Module 2: Figure InsP3 and DAG formation).
This signalling pathway is also used by other groups of Paracrine
stimuli such as the growth and survival factors (Module Paracrine refers to a local signalling process whereby one
1: Figure stimuli for enzyme-linked receptors) and some cell releases a stimulus that diffuses away to act locally on
of the Wnt stimuli that control development (Module 1: cells in the immediate neighbourhood (Module 1: Figure
Figure stimuli for developmental signalling). formation and action of cell stimuli). The following are
The cytokines are a diverse group of stimuli that func- but a few of the many examples of paracrine signalling
tion mainly in the control of haematopoiesis and im- processes:
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r6
Membrane-anchored
stimulus Ectodomain
shedding
ADAM Growth factors
Cytokines
Blood vessel
Eicosanoids;
NO; ATP; S1P
Stimuli
Hormones
Neuro-
transmitters
Autocrine
Juxtacrine Paracrine Endocrine
Neurotransmitters transfer information from the syn- sensing by the carotid body (Module 10: Figure carotid
aptic ending to the postsynaptic membrane during body chemoreception).
neuronal information transfer (see Step 3 in Module 10:
Figure kinetics of neurotransmission).
Acetylcholine (ACh) released from motor neurons trig- Orexin
gers excitation-contraction coupling in skeletal muscle There are two orexins (orexin-A and orexin-B), which are
(Module 7: Figure skeletal muscle E-C coupling). peptide neurotransmitter that are synthesized and released
Release of chemokines controls neutrophil chemotaxis from approximately 7,000 orexin neurons located in the
during inflammatory responses (Module 11: Figure in- lateral hypothalamus (Module 10: Figure brain anatomy).
flammation) and the migration of haematopoietic stem They are synthesized as a prepro-orexin precursor peptide
cells (HSCs) in the bone barrow (Module 8: Figure bone that is then processed to form orexin-A (a 33-amino acid
marrow). peptide) and orexin-B (a 28-amino acid peptide). Orexin-A
The H2 O2 released from wounds during wound healing acts through the OX1 R that is coupled to Gq/11 to activate
may function as a paracrine signal to attract leucocytes. phospholipase C (PLC) to give InsP3 and DAG (Module 2:
Release of nitric oxide (NO) to control smooth muscle Figure InsP3 /DAG recycling), whereas the orexin-B acts
cells through the nitric oxide (NO)/cyclic GMP sig- through OX2 R that is also coupled to Gq/11 , but can also act
nalling pathway (Module 2: Figure NO and cyclic GMP through Gi/o to inhibit the cyclic AMP signalling pathway.
signalling). Orexin has a central role in regulating the sleep and wake
Release of sphingosine 1-phosphate (S1P), which regulatory mechanisms (Module 10: Figure sleep/wake
is released from endothelial cells as part of the cycle regulation).
sphingomyelin signalling pathway (Module 2: Figure
sphingomyelin signalling) controls the activity of peri-
cytes (Module 9: Figure angiogenesis signalling). Endocrine
Release of the growth factor platelet-derived growth During endocrine signalling, the stimulus is usually a hor-
factor B (PDGF-B) from tip cells during angiogenesis mone that is released from one cell to enter the blood
acts locally to control the proliferation of pericytes stream to be carried around the body to act on cells ex-
(Module 9: Figure angiogenesis signalling). pressing the appropriate receptors. There are numerous
ATP and acetylcholine (ACh) released from globus cells examples of how cellular activity is regulated through such
act locally to excite sensory nerve endings during O2 endocrine control mechanisms:
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r7
EGF
EGF
AR AR
HB-EGF HB-EGF
HB-EGF
TGF BTC
TGF EPR
BTC BTC
EPR EPR NRG1 NRG3
NRG2 NRG4
Heparin-binding
domain
TK TK TK TK
ADAM
X
Kinase
Pro-AR Pro-TGF domain
Pro-EGF Pro-HB-EGF Pro-BTC
Pro-EPR
ErbB-1 ErbB-2 ErbB-3 ErbB-4
(HER1 (HER2 (HER3) (HER4)
EGFR) Neu)
During the operation of the metabolic energy network, (TNF) receptor. Mutations in the cleavage site of the
endocrine cells such as the insulin secreting -cells and TNF receptor that prevents its cleavage are the cause
glucagon-secreting -cells release insulin and glucagon of TNF-receptor-associated periodic febrile syndrome
respectively to alter the metabolic activity of liver and (TRAPs).
muscle cells (Module 7: Figure metabolic energy net-
work). Eicosanoids
During the control of food intake and body weight, The eicosanoids are a group of stimuli that are derived
various endocrine cells in the gut release gut hormones from the metabolism of arachidonic acid (AA). AA is a
that alter the activity of the neural circuits that control fatty acid that is located on the sn 2 position of many
the feeding and satiety centres in the brain (Module 7: phospholipids. The enzyme phospholipase A2 (PLA2 ),
Figure control of food intake). which is activated by Ca2 + and by the mitogen-activated
protein kinase (MAPK) signalling pathway, releases AA,
Ectodomain shedding which is then metabolized via two pathways to produce
Many cell stimuli such as growth factors and cytokines the prostanoids (prostaglandins and thromboxanes) and
are released from cells through a process of ectodomain the leukotrienes (Module 1: Figure eicosanoids). The first
shedding (Module 1: Figure formation and action of cell step in the formation of the prostanoids is the enzyme cyc-
stimuli). The precursor of the growth factor is inserted looxygenase (COX) that converts AA into the unstable
into the surface membrane and is then cleaved by pro- cyclic endoperoxides prostaglandins PGG2 and PGH2 .
teases to release the extracellular region as shown for the The latter is a precursor that is converted by various iso-
epidermal growth factor (EGF) stimuli (Module 1: Fig- merases into the prostaglandins PGI2 , PGD2 , PGE2 and
ure EGF stimuli and receptors). The different members of PGF2 , and thromboxane A2 (TXA2 ). The formation of
the EGF family are transported to the plasma membrane the leukotrienes begins with the enzyme 5-lipoxygenase
where they are anchored through a transmembrane re- (5-LO) that converts AA into the hydroxyperoxide 5-
gion. ADAM proteases such as ADAM-12 hydrolyse the hydroperoxyeicosatetraenoic acid (5-HPETE). The activ-
protein chain near the membrane to shed the extracellular ity of 5-LO depends upon a 5-lipoxygenase-activating
region that is then free to diffuse away to act on the EGF protein (FLAP) that may function by presenting AA to
receptors on neighbouring cells. 5-LO. The 5-HPETE is converted into leukotriene A4
Ectodomain shedding can also be used to inactiv- (LTA4 ), which is the precursor for two enzymes. An LTC4
ate cell surface receptors by cleaving off the extracellu- hydrolase converts LTA4 into LTB4 , whereas an LTC4
lar domains as occurs for the tumour necrosis factor synthase converts LTA4 into LTC4 . This synthetic step
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r8
NEUROTRANSMITTERS HORMONES
Acetylcholine (Muscarinic) Glutamate
Amylin 5-Hydroxytryptamine (5-HT) Adenine nucleotides Lysophosphatidic
Adrenomedullin (ADM) Melanocyte stimulating (ATP, ADP) acid (LPA)
Calcitonin gene-related hormone (-MSH; -MSH) Adenosine Sphingosine 1-
peptide (CGRP) Neuropeptide Y Adrenaline phosphate (S-1-P)
Corticotropin-releasing Opioids (Met-enk; Leu-enk; Adrenocorticotropic Melatonin
factor (CRF) dynorphins; -endorphin) hormone (ACTH) Prostaglandins
Dopamine Somatostatin Angiotensin Uridine triphosphate
Noradrenaline Pituitary adenylyl cyclase Chemokines (UTP)
Tetrahydrocannabinol activating peptide (PCAP) Glucagon Vasopressin
-Aminobutyric acid (GABA) Vasoactive intestinal Glucagon-like
Galanin peptide (VIP) peptide 1 (GLP-1
Histamine
GPCR
Transducer
Amplifier AC
Cell stimuli that act through the cyclic AMP signalling pathway.
A large number of neurotransmitters and hormones act through G protein-coupled receptors (GPCRs) to engage the cyclic AMP signalling pathway.
The GPCR acts through a heterotrimeric G protein, which has and subunits, to stimulate the amplifier adenylyl cyclase (AC) to generate the
second messenger cyclic AMP.
NEUROTRANSMITTERS HORMONES
Adenine nucleotides Melatonin
Acetylcholine (Muscarinic) Neurokinin A (ATP, ADP) Neurotensin
Dopamine Neurokinin B Angiotensin Oxytocin
Noradrenaline Neuromedin B (NMB) Bradykinin Platelet-activating
Galanin Neuromedin C (NMC) Chemokines factor (PAF)
Glutamate Orexins (A and B) Cholecystokinin (CCK) Prostaglandins
5-Hydroxytryptamine (5-HT) Pituitary adenylyl Endothelins (ET-1;ET-2; Sphingosine 1-
Gastrin-releasing cyclase activating ET-3) phosphate (S-1-P)
peptide (GRP) peptide (PCAP) Histamine Thromboxanes
Substance P Leukotrienes Uridine triphosphate
Lysophosphatidic acid (LPA) (UTP)
Vasopressin
GPCR
Transducer
Amplifier PLC
Cell stimuli that act through the inositol 1,4,5-trisphosphate (InsP3 )/diacylglycerol (DAG) signalling pathway.
A large number of neurotransmitters and hormones act through G protein-coupled receptors (GPCRs) to engage the InsP3 /DAG signalling pathway.
The GPCRs that sense these different stimuli all feed into a family of heterotrimeric G proteins, which have and subunits that stimulate the
amplifier phospholipase C (PLC) to generate the two second messengers, InsP3 and DAG.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r9
Endoplasmic reticulum
Phospholipid PLA 2 lyso-Phospholipid Platelet-activating
factor (PAF)
PGG 2 AA 5-HPETE
PGH 2 LTA 4
GS Gq Gq
depends upon the conjugation of LTA4 with glutathione Both of these products have a role in forming stimuli for
to form LTC4 , which is thus referred to as a cysteinyl- cell signalling. If the lysophospholipid is derived from
containing leukotriene, as are its derivatives LTD4 and phosphatidylcholine with an alkyl linkage in the sn 1 po-
LTE4 . sition, it functions as a precursor for an acetyltransferase
These eicosanoids are lipophilic and can thus diffuse that converts it into platelet-activating factor (PAF). The
out from their cell of origin to act on neighbouring free fatty acid that is released from the sn 2 position is
cells. Also shown in Module 1: Figure eicosanoids are often arachidonic acid (AA), which is a precursor for the
the family of G protein-coupled receptors (GPCRs) that synthesis of the eicosanoids, such as the prostaglandins,
detect these eicosanoids. These receptors are connected thromboxanes and leukotrienes.
to either the cyclic AMP signalling pathway (Module 1: There is a large family of PLA2 enzymes that function
Figure stimuli for cyclic AMP signalling) or the inos- to provide these two signalling precursors. Humans have
itol 1,4,5-trisphosphate (InsP3 )/diacylglycerol (DAG) sig- about 15 enzymes that differ with regard to their cellu-
nalling pathway (Module 1: Figure stimuli for InsP3 /DAG lar distribution and how they are activated. Some of the
signalling). enzymes are secreted (sPLA2 ). The cytosolic forms fall
Inflammatory cells such as macrophages, mast cells and into two main groups: the Ca2 + -sensitive (cPLA2 ) and
blood platelets produce large amounts of these eicosanoids Ca2 + -insensitive (iPLA2 ) groups. In the case of cPLA2 ,
during an inflammatory response. In macrophages, there is enzyme activity is activated by Ca2 + that acts through
an increase in the expression of COX that results in an in- Ca2+ /calmodulin-dependent protein kinase II (CaMKII),
crease in the release of mediators such as platelet-activating which phosphorylates Ser-515 causing the enzyme to
factor (PAF) and PGE2 (Module 11: Figure macrophage translocate to internal membranes such as the endoplasmic
signalling). In blood platelets, PGI2 acting on its receptor reticulum. Full activation of the enzyme is also dependent
activates cyclic AMP formation, which then inhibits plate- upon the mitogen-activated protein kinase (MAPK) sig-
let activation (Module 11: Figure platelet activation) nalling pathway. Mast cells provide an example of how
PLA2 functions to generate cell signalling stimuli (Module
Phospholipase A2 (PLA2 ) 11: Figure mast cell signalling). PLA2 may also func-
Phospholipase A2 (PLA2 ) functions to cleave the sn 2 es- tion in osmosensing by responding to cell swelling to
ter bond of phospholipids to release the fatty acid to leave produce lipid messengers that activate channels such as
behind a lysophospholipid (Module 1: Figure eicosanoids). TRPV4.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r10
AC Target cell
Lipocalcin Lipocalcin
ATP
-
Et
Et
cAMP CB1 h h
h
h
Et Eth Et
Gi/o
G i/o
Ca 2+
N-type and
- P/Q-type
+ Eth Anandamide
GIRK
K+
NAPE PA
TRPV1
Ca 2+ PC
NAPE
NAT P P Eth
PLD
PE
P Eth
CB2
+
An ansp
Ch Anandamide
tr
an or
P
+
da ter
h
Et
mi
de
Ca 2+ Arachidonic acid
Eth Ethanolamine
FAAH
Endocannabinoids Anandamide
The active ingredient in the Cannabis plant is 9 - Anandamide [N-arachidonylethanolamine (AEA)] is one
tetrahydrocannabinol (THC), which has marked psy- of the major endocannabinoids that functions in the
choactive properties. The brain contains the cannabin- control of a number of important processes, includ-
oid receptor CB1, which not only responds to THC ing white fat cell insulin resistance (Module 12: Fig-
but also responds to the endogenous cannabinoids (en- ure insulin resistance), and contributes to the process of
docannabinoids). The two main endocannabinoids appear Ca2+ and synaptic plasticity during learning and memory
to be anandamide and 2-arachidonoylglycerol (2-AG), in neurons (Module 10: Figure Ca2+ -induced synaptic
but other related molecules such as noladin ether, N- plasticity). Like many other endocannabinoids, anand-
arachidonoyldopamine and virodhamine have been iden- amide contains an arachidonyl moiety that is derived
tified. A feature of all of these endocannabinoids is that from phosphatidylcholine (PC) in the plasma membrane
they contain an arachidonyl moiety. (Module 1: Figure anandamide). Anandamide forma-
The signalling function of the endocannabinoids are car- tion begins with an N-acetyltransferase (NAT) removing
ried out by two cannabinoid receptors (CB1 and CB2), this arachidonyl moiety from phosphatidylcholine (PC)
which are typical G-protein-coupled receptor (GPCR) and transferring it to the ethanolamine head group of
(Module 1: Table G protein-coupled receptors). Most of phosphatidylethanolamine (PE) to form N-arachidonyl-
the CB1 receptors are located in the brain, but can occur phosphatidylethanolamine (NAPE). This anandamide
elsewhere. On the other hand, the CB2 receptors are found precursor is then cleaved by a specific NAPE phospholi-
mainly on immune cells. In the brain the endocannabinoid pase D (PLD) to leave phosphatidic acid (PA) in the mem-
retrograde signalling mechanism is particularly import- brane and releasing anandamide. Since it is lipophilic, the
ant (Module 10: Figure endocannabinoid retrograde sig- anandamide can easily cross the plasma membrane where
nalling). it appears to associate with lipocalcin that acts as a vehicle
Dysregulation of the endocannabinoid signalling system to carry anandamide to neighbouring cells where it can
occurs during obesity and may contribute to metabolic carry out its paracrine functions.
abnormalities such as insulin resistance and the onset of The formation of anandamide is sensitive to Ca2 + ,
Type 2 diabetes. which acts by stimulating NAT.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r11
At its target cells, anandamide can act through a num- Heparin-binding EGF-like growth factor (HB-EGF)
ber of receptors, both classical G-protein-linked receptors Neuregulin
(GPCRs) and receptor-operated channels such as TRPV1 Transforming growth factor- (TGF)
and TRPV4. There are two cannabinoid receptors CB1
Insulin-like growth factors (IGFs)
and CB2. The CB1 receptor, which is coupled to the het-
erotrimeric G-proteins Gi or Go , acts by dissociating the IGF-I
G-protein complex to form Gi or Go and the dimer IGF-II
that have different functions. The Gi or Go act to in-
hibit adenylyl cyclase (AC) to reduce the activity of the Fibroblast growth factors (FGFs)
cyclic AMP signalling pathway, whereas either inhibits Hepatocyte growth factor (HGF)
Ca2 + channels, such as the N-, P/Q- and L-type chan- Platelet-derived growth factors (PDGFs)
nels, or it activates the GIRK K + channels. Anandam- PDGF-A
ide can also act directly on TRPV1 and TRPV4 channels PDGF-B
that introduce Ca2 + into the cell. These various actions PDGF-C
of anandamide in neurons seem to play a role in trigger- PDGF-D
ing long-term depression (LTD). It is of interest that low
levels of dietary omega-3 fatty acids, which provide the Vascular endothelial growth factors (VEGFs)
arachidonic acid precursor for endocannabinoid forma- VEGF-A
tion, causes a decrease in LTD and might be responsible VEGF-B
for various neuropsychiatric diseases such as depression. VEGF-C
The action of anandamide is terminated by a two-step VEGF-D
process. First, it is brought back into cells by an anand- Placental growth factor (PLGF)
amide transporter, which accelerates its transport across
the plasma membrane (Module 1: Figure anandamide). Angiopoietin growth factors
The next step is for the anandamide to be hydrolysed There are four angiopoietins (Ang14) that function in
by fatty acid amide hydrolase (FAAH) located on inner controlling a variety of processes:
membranes.
The primary function of the angiopoietins is to con-
2-Arachidonoylglycerol (2-AG) trol angiogenesis. The maturation and stability of en-
2-Arachidonylglycerol (2-AG) is one of the dothelial cells is controlled by pericytes that release
endocannabinoids that appears to be formed through two angiopoietin 1 (Ang1), which acts through TIE2 re-
mechanisms. First, it is produced during phosphoinositide ceptors (see step 7 in Module 9: Figure angiogenesis
metabolism when PtdIns4,5-P2 is hydrolysed by signalling). Angiopoietin 2 (Ang2) released by prolif-
phospholipase C (PLC) to give InsP3 and DAG (Module erating endothelial cells facilitates the loosening of cell
2: Figure InsP3 /DAG recycling). These two second contacts by inhibiting the activation of the TIE2 recept-
messengers are then recycled back to the precursor lipid ors by Ang1 (see step 9 in Module 9: Figure angiogenesis
PtdIns4,5P2 through a series of steps. The DAG can be signalling).
converted into phosphatidic acid (PA) by DAG kinase or Cell adhesion of haematopoietic stem cells (HSCs) to
it can be hydrolysed by a Ca2 + -sensitive DAG lipase to the stem cell niche is provided by an interaction between
form 2-AG. Ang1 and its TIE2 receptor (Module 8: Figure HSC
The action of 2-AG is terminated by a monoacylglycerol regulation).
lipase (MAGL) (Module 2: Figure InsP3 /DAG recycling). Epidermal growth factors (EGFs)
There are a number of EGF stimuli that have a shared
Growth factors function of activating EGF receptors (Module 1: Figure
There are a large number of growth factors that act by EGF stimuli and receptors). The founder member of this
stimulating the cell cycle signalling mechanisms respons- family is EGF, but there are a number of related pro-
ible for inducing cell proliferation (Module 9: Figure cell teins such as amphiregulin (AR), betacellulin (BTC), epi-
cycle signalling mechanisms). As indicated below, many of regulin (EPR), heparin-binding EGF-like growth factor
these growth factors are grouped together into families: (HB-EGF), neuregulins (NRGs) and transforming growth
Angiopoietin growth factors factor- (TGF). These EGFs are also characterized by
having an extracellular EGF motif that consists of six
Ang1 spatially conserved cysteine residues that form three in-
Ang2 tramolecular disulphide bonds. In addition, amphiregulin
Ang3 (AR) and heparin-binding EGF-like growth factor (HB-
Ang4 EGF) have an N-terminal heparin-binding domain. All
these EGF stimuli are made as precursors that are anchored
Epidermal growth factors (EGFs)
to the plasma membrane through a transmembrane do-
Amphiregulin (AR) main. A process of ectodomain shedding is then respons-
Betacellulin (BTC) ible for releasing the extracellular region through the action
Epiregulin (EPR) of proteases such as the ADAM proteases.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r12
These EGF stimuli act through the epidermal growth can be released under appropriate conditions as occurs for
factor receptors (EGFRs) by promoting both homo- IGFBP-4 (see below). In many cases, the IGFBPs are dif-
and heterodimerization of the ErbB receptor subunits, ferentially regulated during development suggesting that
which are typical protein tyrosine kinase-linked recept- they may have specific functions in controlling different
ors (PTKRs). events during the developmental sequence.
There are six IGFBPs (IGFBP16):
Neuregulins (NRGs)
The neuregulins (NRGs) are coded for by four genes that IGFBP-1. One of its functions is to control bone form-
give rise to the neuregulin family that act through the EGF ation.
receptor family (Module 1: Figure EGF stimuli and recept- IGFBP-2. It is secreted by osteoblasts to regulate the
ors). The neuregulin family are derived from four genes function of IGF in controlling bone development.
(NRG1, NRG2, NRG3 and NRG4). Most information is IGFBP-2 functions in bone remodelling where it may
available for NRG1, which exist in numerous splice forms play a unique role in facilitating the interaction between
(Types I to VI). IGF-II and its receptor. When IGFBP-2 binds to IGF-II
Type I NRG1: this isoform is also known as hereg- it undergoes a conformational change that enhances its
ulin, Neu differentiation factor (NDF) or acetylcholine affinity for the glycosaminoglycans in the bone extra-
receptor inducing activity (ARIA). The last name relates cellular matrix (ECM) and thus positions IGF-II to act
to a role for NRG1 in activating ErbB receptors to drive on its receptor. IGFBP-2 also modulates the action of
the expression of nicotinic acetylcholine receptors during IGF-I in regulating brain development. IGF-I has both
synapse formation at neuromuscular junctions in skeletal neuroprotective and myelinogenetic actions so it is of
muscle. interest that the levels of IGFBP-2 are increased in vari-
Type II NRG1: this isoform is also known as glial ous pathological conditions such as multiple scelerosis.
growth factor-2 (GGF2). In the brain, neuregulin-1 may IGFBP-4 inhibits IGF action in a variety of cell types
act through ErbB to control the expression of NMDA including bone cells. In the latter case, the IGFBP-4
receptors and PSD95 (Module 12: Figure schizophrenia). IGF complex functions as a reservoir of IGF that is re-
There are numerous reports linking the NRG1 gene to leased following proteolysis of IGFBP-4 by pregnancy-
schizophrenia. associated plasma protein (PAPP-A).
IGFBP-5. This isoform is the most highly conserved
Insulin-like growth factors (IGFs)
of the IGFBP family and is not normally found in the
The insulin-like growth factors (IGFs), which are pro-
circulation, but is located mainly in the bone matrix
duced in a variety of cells such as the liver, brain and in
where it can influence IGF activity. There are indica-
osteoblasts. In the case of the liver, the formation and re-
tions that the binding of IGFBP-5 to the extracellular
lease of IGFs is stimulated by growth hormone (GH).
matrix (ECM) reduces its IGF affinity thus releasing
There are two IGFs: IGF-I and IGF-II. The IGFs are
this growth factor. IGFBP-5 is also susceptible to hy-
particularly important for controlling early development.
drolysis by various proteases such as ADAM-9, which
IGF-II is mainly responsible for placental growth of mul-
would also help to release IGF.
tiple foetal organs whereas IGF-I regulates growth of the
brain in both the foetus and adult. A remarkably feature
of this IGF control system is the way it can adjust growth Fibroblast growth factors (FGFs)
to the supply of nutrients. IGF-I has both neuroprotect- There is a large family of fibroblast growth factors (FGFs).
ive and myelinogenetic actions. IGF-II is one of the most These FGFs are characterized by their ability to bind
abundant growth factors and is stored in bone where it heparin and heparan sulphate proteoglycans (HSPG) that
regulates bone formation by the osteoblasts (Module 7: function as cofactors for the effective activation of FGF
Figure osteoblast function). IGF-I also has an important receptors. The diffusion of FGFs is limited through their
role in controlling protein synthesis during both physiolo- affinity for HSPG and are thus likely to act in a paracrine
gical cardiac hypertrophy and during the pathological hy- manner on cells close to their site of release. In humans
pertrophy responsible for heart disease (Module 12: Figure there are 22 FGF genes FGF 123 with FGF 15 missing
physiological and pathological hypertrophy). IGF-I also because the gene in mouse was found to be an ortho-
function in muscle repair and regeneration by stimulat- logue of human FGF19. The numbering system was in-
ing protein synthesis in satellite cells (Module 8: Figure troduced to clear up difficulties that arose from individual
satellite cell activation). FGFs having multiple names often reflecting different cel-
The action of IGFs is tightly regulated by the lular origins. For example FGF1 has been called acidic
IGF-binding proteins (IGFBPs), which are found in both FGF (aFGF), endothelial cell growth factor (ECGF),
the serum and in the bone matrix. retina-derived growth factor (RDGF), eye-derived growth
factor-II (EDGF-II) and brain-derived growth factor-II
IGF-binding proteins (IGFBPs) (BDGF-II). Similarly, FGF2 has been called basic FGF
IGF-binding proteins act by preventing IGF from bind- (bFGF), eye-derived growth factor-I (EDGF-I) and brain-
ing to its IGF receptors. For the most part, therefore, derived growth factor-I (BDGF-I).
they are thought of as negative regulators of the IGFs. FGF23 is produced by osteoblasts and released to act on
However, there are indications that they may also help in the kidney where it reduces the reabsorption of phosphate.
cell activation by functioning as a local store of IGFs that The expression of FGF23 is controlled by the vitamin
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r13
D hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] trate the cellular processes required for tissue remodelling
(Module 7: Figure vitamin D receptor activation). is potentially dangerous because it can have pathological
FGFs act through FGFR receptors (FGFR14), which consequences when control over this HGF/MET pathway
are members of the protein tyrosine kinase-linked recept- is taken over by tumour cells to drive metastasis.
ors (PTKRs) (Module 1: Figure tyrosine kinase-linked
receptors). The FGFs are particularly important in the Platelet-derived growth factors (PDGFs)
control of development. Not only do they promote cell There are four platelet-derived growth factor genes that
growth but they also regulate cell survival, migration and code for the four isoforms (PDGF-A, PDGF-B, PDGF-
differentiation. During limb development, for example, the C and PDGF-D). The biologically active form of PDGF
mesenchyme releases FGF10 to induce the formation of depends on these four different proteins combining to
the ectoderm ridge, which then releases FGF8 to feed in- form either homodimers (AA, BB, CC, DD) or het-
formation back to the mesoderm. A large number of these erodimers (AB) that are connected together by disulph-
FGFs are expressed in the eye where they control lens ide bonds. These dimeric forms then function to activ-
development and function. They also function in wound ate the platelet-derived growth factor receptor (PDGFR)
healing and tissue repair and are important regulators of by bringing together the two subunits (Module 1: Figure
haematopoietic stem cell (HSC) self renewal (Module 8: PDGFR activation).
Figure HSC regulation). The PDGFs function in the development of connective
tissue by stimulating the proliferation of smooth muscle
Heparan sulphate proteoglycans (HSPG) cells and the pericytes of blood vessels. As such, it plays an
The heparan sulphate (HS) proteoglycans (HSPGs) play important role in angiogenesis where PDGF-B is released
an important role in cell signalling by binding growth by the tip cell to stimulate the proliferation and migration
factors such as fibroblast growth factor (FGF) and of pericytes (Module 9: Figure angiogenesis signalling).
platelet-derived growth factor (PDGF), which thus serves PDGF also stimulates the proliferation of mesangial cells
to restrict their action to areas close to their sites of re- (Module 7: Figure mesangial cell).
lease. An example of this is seen during angiogenesis where Progranulin (PGRN)
PDGF released from the tip cells functions locally to con- Progranulin (PGRN) is a secreted glycoprotein that has
trol the proliferation of the pericytes (Module 9: Figure an- been implicated in a number of cellular processes such
giogenesis signalling). HSPG is a sulphated linear polymer as inflammation, cell proliferation, neurite growth and cell
containg repeating disaccharide subunits of D-glucosamine survival. The protein consists of repeating granulin (GRN)
and hexuronic acid. These polymers are found both in the peptides each of which has six cysteines and these can in-
extracellular matrix and on the surface of cells. teract to form six intramolecular disulphide bonds. The
The heparan sulphate proteoglycans syndecan-3 and correct folding of the protein is very dependent on an
syndecan-4 are expressed in satellite cells. ER chaperone network consisting of protein disulphide
isomerases (PDIs) such as ERp5 and ERp57, which inter-
Hepatocyte growth factor (HGF) act with Ca2 + -binding proteins such as calreticulin. The
Hepatocyte growth factor (HGF), which is also known individual granulin peptides of PGRN are released by pro-
as scatter factor, belongs to the plasminogen family and teases such as elastase, which can be regulated by secretory
is a mesenchymal-derived heparin-binding growth factor leukocyte protease inhibitor (SLPI). PGRN can also be
that acts through the hepatocyte growth factor receptor cleared by binding to sortilin 1 (SORT1), which is a traf-
(HGFR), which is also known as the MET receptor. ficking protein that transports PGRN to the lysosomes.
The closely related ligand macrophage-stimulating pro- Just how PGRN acts is unclear. No obvious PRGN
tein (MSP) acts on the RON receptor, which resembles signalling receptors have been identified, but there are in-
the MET receptor. HGF is released as a 97 kDa precursor dications that it can bind to tumour necrosis factor (TNF)
that is then cleaved by proteases such as urokinase-type receptors. Some of the actions of PGRN appear to depend
plasminogen activator (uPA) to give the active disulphide- on activation of the MAPK signalling and PtdIns 3-kinase
linked heterodimer. Each monomer has a hairpin loop signalling pathways. A decrease in PGRN levels has also
followed by four kringle domains, which are double- been associated with an upregulation of the Fz2 receptor
looped structures stabilized by disulphide bridges.HGF that is coupled to the non-canonical Wnt/Ca2+ signalling
is released mainly from mesenchymal cells and acts in system.
a paracrine manner on neighbouring epithelial cells to PGRN has both neurotrophic and anti-inflammatory
induce invasive growth. Such invasive growth is a nor- responses. Such an action is particularly important in the
mal part of processes that occur during embryonic de- brain where it acts to inhibit microglial inflammatory re-
velopment and tissue repair. For example, HGF controls sponses.
the proliferation of satellite cells during repair of skeletal Frontotemporal dementia (FTD) has been linked to
muscle (Step 2 in Module 8: Figure satellite cell function). mutations in the GRN gene that encodes PGRN.
HGF also plays an important role in the control of male
and female gonodal function. In carrying out its normal Vascular endothelial growth factor (VEGF)
actions it can stimulate proliferation, disrupt intercellu- The vascular endothelial growth factor (VEGF) fam-
lar junctions, induce migration and protect cells against ily, which has five members, functions in angiogenesis
apoptosis. This multitasking capacity of HGF to orches- (Module 9: Figure angiogenesis).
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r14
Ghrelin
1
NH 2 G S S F L S P E H Q R
V
O Q
S E K R Q
C O
K 28
HCH
K P P A K L Q P R COOH
HCH
HCH
HCH
HCH
HCH
HCH
H
n-octanoyl group
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r15
its levels decrease in obesity but increases during weight (GPCR), that is coupled through Gq to activate the inositol
loss. The precise function of adiponectin is unclear, but it 1,4,5-trisphosphate (InsP3 ) signalling cassette.
appears to have a role in protecting cells against glucose in- Ghrelin is a multifunction hormone capable of stimu-
tolerance and insulin resistance (Module 12: Figure insulin lating a variety of processes:
resistance).
There are two adiponectin receptors (AdipoR1 and Ghrelin that is released in anticipation of eating func-
AdipoR2). The AdipoR1 is found in skeletal muscle tions in the control of food intake and body weight
whereas the type 2 receptor is expressed in liver. These by activating the NPY/AgRP neurons that control the
AdipoRs appear to act by promoting the entry of ex- feeding centre (Module 7: Figure control of food intake).
ternal Ca2 + , which then acts through CaMKK to phos- Ghrelin acts on pituitary somatotrophs to release
phorylate AMPK as part of the AMP signalling pathway growth hormone (GH).
(Module 2: Figure AMPK control of metabolism). Ghrelin may act on the stomach to control acid secretion
and gastric movement.
Resistin
Resistin, which appears to be released from macrophages High levels of ghrelin have been found in both anorexia
and stromal cells located in adipose tissue, has been implic- nervosa and in Prader-Willi syndrome (PWS).
ated in insulin resistance and the development of diabetes Glucagon-like peptide 1 (GLP-1)
during obesity. The precursor pre-proglucagon is synthesized and released
by intestinal L cells following feeding (Module 7: Fig-
Gut hormones ure L cell). This precursor is then processed by prohor-
In addition to its function in digestion and nutrient absorp- mone convertase 1 and 2 to different hormones depending
tion, the gastrointestinal tract is also an endocrine organ in on the tissue. In the pancreas, it is converted into gluca-
that it contains a large number of cells that synthesize and gon, whereas in the CNS and intestine it is processed to
secrete gut hormones. The stomach has various endocrine form glucagon-like peptide 1 (GLP-1) and oxyntomodulin
cells that contribute to the neural and endocrine functions (OXM). GLP-1 acts on the GLP-1 receptor (GLP-1R) to
of the stomach: activate the cyclic AMP signalling pathway. The ability of
Enterochromaffin-like cells (ECLs) release histamine. GLP-1 to reduce food intake might be mediated through
D cells secrete somatostatin. GLP-1Rs located in the hypothalamus.
G cells secrete gastrin. One of the main targets of GLP-1 is the insulin-secreting
-cell where it facilitates the glucose-induced release of
The intestine also has endocrine cells: insulin (Module 7: Figure -cell signalling).
Enterochromaffin cells release 5-hydroxytryptamine Circulating GLP-1 is inactivated by dipeptidyl pepti-
(5-HT). dase 4 (DPP4).
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r16
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r17
Pro-opiomelanocortin (POMC)
-MSH
ACTH
-MSH
-Lipotropin
-Lipotropin
-Endorphin
-MSH
Pro-opiomelanocortin (POMC).
Pro-opiomelanocortin (POMC) is a large polypeptide hormone precursor that is cleaved into a number of biologically active peptides that can act as
hormones or neurotransmitters.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r18
Toll receptor signalling pathway (Module 2: Figure Toll factors that function in the JAK/STAT signalling pathway
receptor signalling). (Module 2: Figure JAK/STAT function).
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r19
The IL-10 receptor (IL-10R) consists of two IL-10R1 paracrine manner to stimulate their IFN receptors to activ-
subunits and two IL-10R2 subunits. Upon binding IL-10, ate a battery of genes that help combat the infection. One
these receptor subunits recruit the transducers Jak1 and of the gene products is double-stranded RNA-dependent
tyrosine kinase 2 (Tyk2) that function to activate STAT3 protein kinase (PKR), which phosphorylates and inhib-
(Module 2: Figure JAK/STAT heterogeneity) which is a its the initiation factor eIF2 and the resulting decrease in
transcription factor that functions in the JAK/STAT sig- protein synthesis helps to reduce viral replication (Module
nalling pathway (Module 2: Figure JAK/STAT function). 9: Figure regulation of eIF-2 cycling). IFN- and IFN-
also activate the 25A synthetase, which produces the
Interleukin-17 (IL-17) oligoadenylate that switches on a latent ribonuclease that
Interleukin-17 (IL-17) released from CD4 + T helper 17 degrades double-stranded RNA (ssRNA).
(Th17) cells coordinates the recruitment of neutrophils.
Interferon- (IFN-)
Interleukin-21 (IL-21) Interferon- (IFN-) consists of two peptide chains (143
Interleukin-21 (IL-21) is a potent regulator of various im- amino acids) that have two N-linked glycosylations. It is
mune cells such as germinal centre B-cells, natural killer produced by helper T cells and NK cells that have been ac-
(NK) cells and cytotoxic T-cells. The IL-21 receptor (IL- tivated by interleukin-2 (IL-2) and interleukin-12 (IL-12).
21R), which resembles that of other type I cytokines such It acts through the type II interferon- receptor (IFNR),
as IL-2R (Module 1: Figure type I cytokine receptors), which is coupled to the JAK/STAT signalling pathway ,
responds to IL-21 by activating the JAK/STAT signalling to influence many different responses such as an increased
pathway ( Module 2: Figure JAK/STAT function). It uses expression of the class I MHC complex and an increase in
Jak1 and Jak3 to induce the STAT3 homodimer to activate the activity of macrophages, neutrophils and NK cells.
its target genes. The main function of IL-21 is to stim- An important function of IFN- is to regulate the ex-
ulate proliferation as occurs during B-cell differentiation pression of the Vitamin D receptor (VDR). Expression of
in the lymph node (Module 8: Figure B cell maturation the VDR is reduced in a large proportion of the popu-
signalling). lation in the Mediterranean island of Sardinia that suffer
from Multiple sclerosis (MS). These MS patients have re-
Thymic stromal lymphopoietin (TSLP)
duced expression of the Ifng gene that encodes IFN-.
Thymic stromal lymphopoietin (TSLP) is a cytokine that
has been implicated in Atopic dermatitis (AD) where it
plays a major role in activating the itch sensation. TSLP Cardiotrophin (CT-1)
is generated in the keratinocytes in the skin and in the Cardiotrophin (CT-1) was originally discovered in the
bronchial epithelial cells in asthma. The keratinocytes have heart, but is now known to function in many other cell
proteinase-activated receptor 2 (PAR2 ) that are coupled types. In the heart, it can activate proliferation and sur-
through Gq/11 to activate phospholipase C3 (PLC3). vival. It can also have pathological consequences by con-
The PLC3 then generates inositol 1,4,5-trisphosphate tributing to myocyte hypertrophy and collagen synthesis
(InsP3 ) that releases Ca2 + from the endoplasmic retic- that are part of the cardiac remodelling events that result
ulum (ER) that then results in activation of Orai1, which in heart disease. The cardiotrophin receptor (CT-1R) is a
is a store-operated channel (SOC) that promotes the entry member of the IL-6 subfamily of receptors that are char-
of external Ca2 + (Module 3: Figure SOC signalling com- acterized by sharing the transducing subunit glycoprotein
ponents). The increase in cytosolic Ca2 + then activates 130 (gp130) (Module 1: Figure type I cytokine receptors).
NFAT that enters the nucleus where it acts to increase the The CT-1R has two CT-1R subunits and it uses LIFR
expression of TSLP. The latter then diffuses out to interact and gp130 as transducing subunits. Upon binding CT-1,
with receptors on the sensory neurons in the skin to induce the CT-1R recruits Jak1 to activate STAT3 (Module 2:
the itch sensation by activating TRPA1 channels (Module Figure JAK/STAT heterogeneity), which is a transcription
10: Figure Itch signal transduction mechanism). factor that functions in the JAK/STAT signalling pathway
(Module 2: Figure JAK/STAT function).
Interferons (IFNs)
The interferons are cytokines that modulate immune re- Ciliary neurotrophic factor (CNTF)
sponses and have a special role in dealing with viral in- Ciliary neurotrophic factor (CNTF) is expressed primarily
fections. There are three main IFNs. The type I IFNs, in the nervous system where it functions as a neurotrophic
interferon- (IFN-) and interferon- (IFN-), which factor. It is also a potent survival factor for both neur-
have a relatively high antiviral potency, are released from ons and astrocytes. CNTF has been tested as a therapeutic
endothelial cells and macrophages in responses to infection agent to control certain neurodegenerative disorders such
by viruses and bacteria. Interferon- (IFN-) is a type II as motor neuron disease, but patients were found to suf-
IFN. fer severe weight loss suggesting that CNTF may play a
role in the control of food intake and body weight. CNTF
Interferon- (IFN-) has been implicated in neurogenesis where it may mediate
Type I interferon- (IFN-) and interferon- (IFN-) the ability of dopamine to stimulate proliferation in both
are produced by endothelial cells and macrophages in re- the subventricular zone and the dentate gyrus. The CNTF
sponses to viral infections (Module 2: Figure viral recog- receptor (CNTFR) is a member of the IL-6 subfamily of
nition). Once released they act in both an autocrine and receptors that are characterized by sharing the transducing
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r20
subunit glycoprotein 130 (gp130) (Module 1: Figure type Granulocyte-macrophage colony-stimulating factor
I cytokine receptors). The CNTFR has two CNTFR (GM-CSF)
subunits and it uses LIFR and gp130 as transducing sub- Granulocyte-macrophage colony-stimulating factor
units. Upon binding CNTF, the CNTFR recruits Jak1 (GM-CSF) is one of the colony-stimulating factors
to activate STAT3 (Module 2: Figure JAK/STAT hetero- (CSFs) and is also known as CSF-2. GM-CSF was first
geneity), which is a transcription factor that functions identified as one of the haematopoietic cytokines that
in the JAK/STAT signalling pathway (Module 2: Figure functions to control haematopoiesis (Module 8: Figure
JAK/STAT function). haematopoietic cytokines). It not only operates as a
growth factor to control the proliferation of the early
progenitor cells but it also guides the subsequent differ-
Erythropoietin (EPO) entiation and maturation of the neutrophilic granulocytes
Erythropoietin (EPO) is an example of a haematopoietic and macrophages.
cytokine that functions to control haematopoiesis (Module The GM-CSF receptor (GM-CSFR) is a heterotetramer
8: Figure haematopoietic cytokines). EPO is synthesized composed of two GM-CSF and two c subunits (Module
predominantly by the proximal kidney tubule cells and is 1: Figure type I cytokine receptors). This c is a promiscu-
released into the circulation to control the differentiation ous transducing subunit that is also used by IL-3 and IL-5.
and maturation of erythrocytes. Upon binding GM-CSF, this c subunit recruits the trans-
The EPO receptor (EPOR) is composed of two identical ducer Jak2 to activate STAT5a and STAT5b (Module 2:
subunits (Module 1: Figure type I cytokine receptors). Figure JAK/STAT heterogeneity). These STATs are tran-
The typical cytokine receptor modules that make up the scription factors that function in the JAK/STAT signalling
extracellular domains co-operate with each other to bind a pathway (Module 2: Figure JAK/STAT function).
single EPO molecule to induce the conformational changes
in the intracellular box motifs that recruit and activate Jak2. Growth hormone (GH)
The latter then activates STAT5a and STAT5b (Module 2: Growth hormone (GH) consists of a single protein chain
Figure JAK/STAT heterogeneity), which are transcription (approximately 190 amino acids), which is synthesized and
factors that function in the JAK/STAT signalling pathway released by somatotrophs (Module 10: Figure somato-
(Module 2: Figure JAK/STAT function). troph regulation). It acts to promote growth, but it can
also modulate various metabolic processes (protein, lipid
Ftl ligand (FL) and carbohydrate). The action of GH can be either direct
FL is one of the haematopoietic cytokines that functions (e.g. in fat cells where it induces triacylglycerol breakdown
in the control of haematopoiesis (Module 8: Figure haema- and reduces their ability to take up lipids) or indirect, as
topoietic cytokines). It is an example of a membrane- occurs through its ability to stimulate liver cells to produce
anchored stimulus that functions through a juxtacrine and release insulin-like growth factor I (IGF-I)
mechanism to control stem cells and progenitors cells.
In addition, it controls the subsequent differentiation and Leukaemia inhibitory factor (LIF)
maturation of dendritic cells. Leukaemia inhibitory factor (LIF) is a multifunctional cy-
The Ftl ligand (FL) acts on the FL receptor (FLR), which tokine that was found first through its ability to suppress
is a typical protein tyrosine-linked receptors (PTKRs) the proliferation of a myeloid leukaemic cell line. However,
(Module 1: Figure tyrosine kinase-linked receptors). it was found subsequently to have many other functions: it
maintains embryonic stem cells in an undifferentiated state,
it facilitates the implantation of blastocysts, it functions in
Granulocyte colony-stimulating factor (G-CSF) the autonomic nervous system, adipocytes, liver, osteo-
Granulocyte colony-stimulating factor (G-CSF), which is clasts and it assists in the release of adrenocorticotropic
also known as colony-stimulating factor-3 (CSF-3), is one hormone (ACTH) by the corticotrophs.
of the haematopoietic cytokines that functions to con- The LIF receptor (LIFR) is a member of the IL-6 sub-
trol haematopoiesis (Module 8: Figure haematopoietic cy- family of receptors that are characterized by sharing the
tokines). One of its primary functions is to control the transducing subunit glycoprotein 130 (gp130) (Module
differentiation and maturation of the neutrophils. 1: Figure type I cytokine receptors). The LIFR has two
The G-CSF receptor (G-CSFR) is composed of two LIFR subunits and the transducing subunits LIFR
identical subunits (Module 1: Figure type I cytokine re- and gp130. Upon binding LIF, the LIFR recruits Jak1
ceptors). The extracellular region has a number of do- to activate STAT3 (Module 2: Figure JAK/STAT hetero-
mains: a terminal immunoglobulin-like domain, a cytokine geneity), which is a transcription factor that functions
receptor module and three fibronectin type-III-like do- in the JAK/STAT signalling pathway (Module 2: Figure
mains. Some of these domains are used to bind G-CSF to JAK/STAT function).
induce the conformational change in the intracellular box During osteoclastogenesis, LIF acts like PTH to increase
motifs that recruit and activate Jak2. The latter then activ- the expression of RANKL by the supporting cells.
ates STAT5a and STAT5b (Module 2: Figure JAK/STAT
heterogeneity), which are transcription factors that func- Oncostatin (OSM)
tion in the JAK/STAT signalling pathway (Module 2: Fig- Oncostatin (OSM) is a cytostatic cytokine that inhibits cell
ure JAK/STAT function). proliferation and may thus act as a tumour suppressor. It
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r21
was discovered through its ability to inhibit the growth ceptor modules, functions together to bind a single EPO
of melanoma cells in culture. It is a 28 kDa glycoprotein molecule to induce a conformational change in the in-
that is released from T cells and monocytes. There are tracellular box motifs that recruit and activate Jak2. The
two OSM receptors that are members of the IL-6 sub- latter then activates STAT5a and STAT5b (Module 2: Fig-
family of receptors that are characterized by sharing the ure JAK/STAT heterogeneity), which are transcription
transducing subunit glycoprotein 130 (gp130) (Module 1: factors that function in the JAK/STAT signalling pathway
Figure type I cytokine receptors). The OSMR has two (Module 2: Figure JAK/STAT function).
OSMR subunits that then combine with two transdu-
cing subunits, one of these is gp130 and the other is
either LIFR (Type I OSMR) or OSMR (Type II OSMR). Chemokines
Upon binding OSM, these OSMRs recruit the Jak trans- The chemokines are low-molecular-mass proteins that
ducers to activate STAT3 (Module 2: Figure JAK/STAT have a variety of functions. There are approximately 50
heterogeneity), which is a transcription factor that func- chemokines that have been divided into four families:
tions in the JAK/STAT signalling pathway (Module 2: Fig- CXC (), CC (), C () and CX3C () (Module 1: Figure
ure JAK/STAT function). chemokines). These families are subdivided on the basis of
the position of conserved cysteine residues located in the
Prolactin (PRL) N-terminal region (see the models for each family). For
Prolactin (PRL) is a small protein (19 amino acids), example, the CXC family has two cysteine residues (C)
which has certain structural similarities to growth hor- separated by a single non-cysteine amino acid residue (X).
mone (GH). It is released into the circulation from the This nomenclature is used to refer to both the ligands (L)
lactotrophs (Module 10: Figure lactotroph regulation). and their receptors (R). Consequently, CXCL1 refers to
PRL functions to stimulate lactation and can also influ- the first ligand of the CXC family and CXCR1 is the first
ence maternal behaviour. However, it is made in other receptor of the same family. The same terminology applies
locations and has many other functions. The prolactin re- to the other families. The CX3C () family is unusual in
ceptor, which is located primarily in mammary gland, liver that it has a single member CX3CL1 that is tethered to
and ovary, is a member of the JAK/STAT signalling path- membranes through its C-terminal region. It can act on its
way (Module 2: Figure JAK/STAT heterogeneity). CX3CR1 either in this tethered form or as a freely diffus-
ible molecule after it has been released from the membrane
Stem cell factor (SCF) by proteases such as A Disintigrin or the ADAM protease
Stem cell factor (SCF), which is also known as steel factor, family.
is produced by many cell types (bone marrow stromal Chemokines first came to prominence as chemoattract-
cells, endothelial cells, placental cells). SCF functions dur- ants during inflammation, but subsequently were found
ing the early stages of haematopoiesis where it contrib- to have signalling functions in many other cellular pro-
utes to maintain haematopoietic cell self renewal (Module cesses:
8: Figure HSC regulation) and continues to control the
formation of erythroid cells and mast cells (Module 8: Fig- Chemokines establish gradients to attract various in-
ure haematopoietic cytokines). SCF is also released from flammatory cells such as neutrophils (Module 11: Figure
keratinocytes during the control of melanogenesis by the inflammation).
melanocytes (Module 7: Figure melanogenesis). Chemokines play a role in haematopoiesis where they
SCF is the ligand for the c-KIT receptor (also known regulate the migration of cells in lymphoid organs,
as CD117), which is one of the protein tyrosine-linked re- bone marrow and thymus. For example, haematopoietic
ceptors (PTKRs) (Module 1: Figure tyrosine kinase-linked stem cell (HSC) migration is controlled by CXCL12
receptors). [stromal-derived factor-1 (SDF-1)] that acts through the
Mutations in SCF or its receptor c-KIT cause CXCR4 chemokine receptor (Step 1 in Module 8: Figure
piebaldism. Mutations in c-KIT are also found in many bone marrow). SDF-1 plays a role in renal cell carcinoma
gastrointestinal stromal tumours. (RCC).
The chemokine CXCL12 controls preosteoclast chemo-
Thrombopoietin (TPO) taxis (Module 8: Figure preosteoclast chemotaxis).
Thrombopoietin (TPO) is produced predominantly by T cell chemotaxis is driven by a chemokine gradient
hepatocytes but can also be released from kidney prox- (Module 9: Figure T cell chemotaxis).
imal tubule cells, fibroblasts and endothelial cells. TPO is Microglia use chemokines to develop neural inflam-
an example an haematopoietic cytokine that functions to matory responses (Module 7: Figure microglia interac-
control haematopoiesis (Module 8: Figure haematopoietic tions). A chemokine signalling network contributes to
cytokines). It operates very early to regulate the prolifera- the neuronal-microglial interactions (Module 7: Figure
tion of the stem and progenitor cells. Later on it guides the neuronal chemokine function) that may contribute to
subsequent differentiation and maturation of the megaka- neuropathic pain.
ryocytes and blood platelets. A relationship between chemokines and cancer has re-
The TPO receptor (TPOR) is composed of two identical vealed that chemokines such as CXCL12 and its re-
subunits (Module 1: Figure type I cytokine receptors). ceptor CXCR4 may play a critical role in the onset
Each extracellular domain, which has typical cytokine re- of metastasis. In addition, the chemokine CCL19 may
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r22
CCL3 CC () C COOH
CCL5 CCL5
CCL7 CCL7 NH2 C C
CCL8 CCL8 CCL2
C
CCL13 CCL11 CCL8
CCL14 CCL2 CCL13 CCL3 CCL CCL13
CCL15 CCL7 CCL15 CCL4 CCL19
CCL16 CCL8 CCL24 CCL17 CCL5 CCL19 CCL27 CCL21
CCL23 CCL13 CCL26 CCL22 CCL8 CCL20 CCL21 CCL1 CCL25 CCL28 CCL25
CCR1 CCR2 CCR3 CCR4 CCR5 CCR6 CCR7 CCR8 CCR9 CCR10 CCR11
C () CX3CL1 C
CX3C ()
C COOH
XCL1 NH2 C X X X C
NH2 C XCL2 CX3CL1
C
XCL ADAM
XCR1 COOH CX3CR1
STEROID HORMONES
Aldosterone
Cortisol
Progesterone
Oestrogen
Testosterone
Transducers
SHR
Receptor,
transducer
and AC PI 3-K PLC
Amplifiers
messenger
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r23
Cholesterol O O O
O Aldosterone
3--HSD
HO O Progesterone C H2 OH
C O
Pregnenolone HO OH
CYP17
Cortisol
CH3 C H2 OH O
C O C O
OH OH
function to attract leukaemic T-cells into the brain in tion. The non-genomic action depends on steroids binding
patients with T cell acute lymphoblastic leukaemia. to cell-surface receptors that are then coupled to conven-
Retinal pigmented epithelial cells release the chemokines tional intracellular signalling pathways.
eotaxin-1 (CCL11), eotaxin-2 (CCL24) and eotaxin- Aldosterone and cortisol biosynthesis illustrates how
3 (CCL26) to activate the CCR3 receptors on the steroids are synthesized from cholesterol through a series
choroidal endothelial cells to stimulate angiogenesis. of reactions carried out by enzymes associated with either
Abnormal activation of this signalling pathway may the mitochondrion or the smooth endoplasmic reticulum
be responsible for the choroidal neovascularization re- (Module 1: Figure aldosterone and cortisol biosynthesis).
sponsible for age-related macular degeneration (AMD).
The chemokine monocyte chemoattractant protein-1 Aldosterone and cortisol biosynthesis
(MCP-1) is released by white fat cells to attract mac- Aldosterone and the corticosteroids (cortisol and corticos-
rophages to adipose tissue where they release TNF terone) are synthesized in the zona glomerulosa and the
that sets up an inflammatory response that contributes zona fasciculata/reticularis cells of the adrenal gland re-
to insulin resistance (Module 12: Figure insulin resist- spectively (Module 7: Figure adrenal gland). Like other
ance). steroids, they are synthesized in a series of steps carried
In the germinal centre the chemokines CXCL12 and out by a number of different enzymes (Module 1: Figure
CXCL13 direct the migration of B-cells between the aldosterone and cortisol biosynthesis):
dark and light zones during B-cell differentiation in the
lymph node (Module 8: Figure germinal centre).
CYP11A1 is a side chain cleavage enzyme that initiates
the process by cleaving off the side chain of cholesterol
Steroids to produce pregnenolone.
Steroid hormones are hydrophobic stimuli responsible for 3-HSD is a short-chain hydroxysteroid dehydro-
regulating a number of physiological processes such as genase that converts pregnenolone into progesterone.
reproduction (oestrogen and testosterone), glucose meta- The two synthetic pathways now diverge.
bolism and stress responses (cortisol) and salt balance CYP21A is a steroid 21-hydroxylase that converts pro-
(aldosterone). Steroids have two main modes of action, gesterone into 11-deoxycorticosterone.
genomic and non-genomic (Module 1: Figure steroid stim- CYP11B2 (11-hydroxylase), which is highly expressed
uli). The genomic action depends on the fact that the ster- in the zona glomerulosa cells, is an aldosterone syn-
oids are hydrophobic and thus can pass through the plasma thase that carries out the last three steps of converting
membrane to bind to intracellular receptors, which are 11-deoxycorticosterone into corticosterone, 18-OH-
transcription factors capable of activating gene transcrip- corticosterone and finally aldosterone.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r24
CYP17 is a steroid 17-hydroxylase and 17,20-lyase oestrone (E1), oestradiol (E2) and oestriol (E3). Most at-
that converts progesterone into 17-progesterone that tention has focused on E2 that acts through oestrogen
begins the synthetic sequence that results in the forma- receptor- (ER) and oestrogen receptor- (ER) en-
tion of cortisol. coded by the Esr1 and Esr2 genes respectively, which are
CYP21A converts 17-progesterone into 11- typical of the nuclear receptors (Module 4: Table nuclear
deoxycortisol. receptor toolkit).
CYP11B1, which is strongly expressed in the zona fas- In addition to these genomic actions mediated by nuc-
ciculata/reticularis carries out the final step of cortisol lear receptors, E2 also has a non-genomic action in that it
synthesis by converting 11-deoxycortisol into cortisol. can stimulate the G protein coupled receptor 20 (GPR20).
Such an action seems to be particularly important in regu-
Aldosterone lating both the expression and the channel gating of plasma
Aldosterone is mainly synthesized and released by the membrane epithelial Na+ channels (ENaC).
zona glomerulosa cells in the cortical region of the ad-
renal gland (Module 7: Figure adrenal gland). However, it Receptors
can also be produced by other cells located in the nervous In order to respond to the myriad cell stimuli outlined
system, heart, kidney and blood vessels. This extra-adrenal in the previous section, cells have evolved an equally im-
production of aldosterone may be particularly important pressive battery of cell-surface receptors. These receptors
for tissue repair. The enzymes responsible for aldosterone have two main functions: they have to detect incoming
and cortisol biosynthesis are located on the mitochondrion stimuli and then transmit this information to the internal
and smooth endoplasmic reticulum (Module 1: Figure al- transducers that initiate the signalling pathway (Module 1:
dosterone and cortisol biosynthesis). One of the primary Figure cell signalling mechanism). Receptors vary enorm-
genomic actions of aldosterone is to regulate Na + reab- ously in the way they carry out this transfer of information
sorption by the distal convoluted tubule (DCT) (Module across the plasma membrane. The diverse receptor types
7: Figure kidney tubule function) and the colon (Module can be separated into two main groups depending on the
7: Figure colon function). number of membrane-spanning regions they have to em-
Cortisol bed them into the membrane:
Cortisol is one of the glucocorticoids produced by the Single membrane-spanning receptors
cortisol biosynthetic mechanisms located in zona fascicu-
lata/reticularis cells of the adrenal gland (Module 1: Fig- Protein tyrosine kinase-linked receptors (PTKRs)
ure aldosterone and cortisol biosynthesis). It has multiple Serine/threonine kinase-linked receptors (S/TKRs)
functions in the cardiovascular, neural and immunolo- Particulate guanylyl cyclases (pGCs)
gical systems. It has both immunosuppressive and anti- Non-enzyme-containing receptors
inflammatory responses. Multi-membrane-spanning receptors
The glucocorticoids have anti-inflammatory effects me-
diated in part by an increase in the transcription and syn- G protein-coupled receptors (GPCRs)
thesis of inhibitor of nuclear factor B (NF-B) (IB) that Ion channel receptors
then functions to inhibit NF-B by promoting its reten- Sigma receptors
tion in the cytosol. There are a large number of single membrane-spanning
Glucocorticoids also increase the production of proteins, which fall into two groups:
other anti-inflammatory molecules such as interleukin-10
(IL-10), TNF-, COX2 and phospholipase A2 (PLA2 ). Type I transmembrane proteins, which are orientated
Through their ability to modulate the immune system, with an extracellular N-terminus and intracellular C-
glucocorticoids such as prednisone, dexamethasone and terminus. Most of the single-spanning receptors belong
hydrocortisone are used to treat many inflammatory con- to this group.
ditions such as asthma, allergies, dermatitis, rheumatoid Type II transmembrane proteins, which are orientated
arthritis, leukaemias and lymphomas. with an extracellular C-terminus and intracellular N-
terminus.
Corticosterone
Corticosterone is produced by the cortisol biosynthetic Protein tyrosine kinase-linked receptors (PTKRs)
mechanisms located in zona fasciculata/reticularis cells The protein tyrosine kinase-linked receptors (PTKRs) are
of the adrenal gland (Module 1: Figure aldosterone and the classical example of single-membrane-spanning re-
cortisol biosynthesis). It functions to increase glycogen ceptors that contain an enzymatic activity (Module 1:
formation by enhancing the conversion of amino acids Figure stimuli for enzyme-linked receptors). There are
into carbohydrates. about 20 subfamilies of these PTKRs, which have the
same basic structural features (Module 1: Figure tyr-
Oestrogens osine kinase-linked receptors). The extracellular domain
Oestrogens are the female sex hormones that act primarily is responsible for binding the growth or survival factors,
in sexual development and reproduction. However, they whereas the cytosolic region contains the tyrosine kinase
have many other functions, particularly in the cardiovas- domain that is the transducer responsible for initiating the
cular system. There are a number of oestrogens such as process of signal transduction. One of the characteristics
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r25
Tyrosine Serine/threonine
kinase kinase
PTKRs S/TKRs
Grb2 Transducer
Transducers Ras TK TK and amplifier
SOS
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r26
of these receptors is that they can transmit information The way in which these receptors function resembles
down a number of cell signalling pathways by assembling that for many of the other PTKRs as exemplified for
a number of transducers and amplifiers. There are a num- the platelet-derived growth factor receptor (PDGFR)
ber of variations on this basic structural organization as (Module 1: Figure PDGFR activation). The various EGFs
illustrated by the following examples of some of the main induce two chains to form both homo- and heterodimers.
PTKRs: This dimerization then induces the receptor phosphoryla-
tion events that initiate the onset of cell signalling. The Cbl
Anaplastic lymphoma kinase (ALK) receptor down-regulation of cell signalling components mechanism
Epidermal growth factor receptor (EGFR) (Module 1: Figure receptor down-regulation) is respons-
Colony-stimulating factor-1 receptor (CSFR-1) ible for the down-regulation of these ErbB receptors.
Fibroblast growth factor receptor (FGFR) The EGF receptor family play an important role in
Hepatocyte growth factor receptor (HGFR) mammary gland development where ErbB1 controls out-
Insulin receptor growth of the ducts, whereas ErbB2 and ErbB3 regulate
Insulin-like growth factor receptor (IGFR) alveolar morphogenesis and lactation.
Platelet-derived growth factor receptor (PDGFR) Members of the ErbB family of receptors are often amp-
TIE receptors lified or activated through mutations in various cancers.
Trk receptors
Vascular endothelial growth factor receptor (VEGFR) Fibroblast growth factor receptor (FGFR)
The fibroblast growth factor receptors (FGFRs) are typical
Anaplastic lymphoma kinase (ALK) receptor protein tyrosine kinase-linked receptors (PTKR) (Module
The anaplastic lymphoma kinase (ALK) receptor belongs 1: Figure tyrosine kinase-linked receptors). There are four
to the protein tyrosine kinase-linked receptors (PTKRs) FGFRs (FGFR14). The cytosolic region of the molecule
family (Module 1: Figure tyrosine kinase-linked recept- has a split tyrosine kinase domain. The extracellular do-
ors). ALK is expressed mainly in the central and peri- main has three immunoglobulin-like (Ig-like) domains.
pheral nervous system. Little is known about the stimulus The affinity of the different FGFRs for the large FGF fam-
responsible for activating this receptor. However, it has ily is varied by splicing events in the third Ig-like domain.
come to prominence as a major predisposition gene for The first two Ig-like domains are separated by a stretch
childhood neuroblastomas. of acidic amino acids and a heparin-binding domain that
interacts with heparan sulphate proteoglycans (HSPGs)
Epidermal growth factor receptor (EGFR) that also bind to the family of fibroblast growth factors
The epidermal growth factor receptor (EGFR), which (FGFs). The HSPG facilitates the interaction between the
is one of the protein tyrosine kinase-linked receptors FGFs and their FGFRs to induce the dimeric receptor
(PTKR) (Module 1: Figure tyrosine kinase-linked recept- complexes necessary to recruit and engage different sig-
ors), has four members (Module 1: Figure EGF stimuli nalling pathways.
and receptors). There is a problem with regard to termin-
ology in that they have been given different names and Hepatocyte growth factor receptor (HGFR)
some of these are shown on the Figure. In much of the There are two closely related hepatocyte growth factor re-
literature, the ErbB terminology is now preferred and will ceptors (HGFRs), MET and RON. MET belongs to the
be used in this case. All of these receptors have a sim- family of protein tyrosine kinase-linked receptors (PTKR)
ilar domain structure: they are single membrane-spanning (Module 1: Figure tyrosine kinase-linked receptors). MET
proteins with an N-terminal extracellular ligand-binding is the receptor that responds to hepatocyte growth
domain and a C-terminal region that has a kinase domain factor (HGF) whereas RON responds to macrophage-
and numerous tyrosine docking sites that participate in the stimulating protein (MSP). Most attention has been fo-
process of signal transduction. The external ligand-binding cused on MET that is coded for by the p190 c-met proto-
domain has two cysteine-rich regions. Despite these sim- oncogene. MET is a disulphide-linked heterodimer that
ilarities, these receptors have different properties. For ex- originates from a single protein. A small part is cleaved off
ample there are marked differences in the way they respond to form the -subunit that is attached to the transmem-
to the large family of epidermal growth factors (EGFs) brane chain. (Module 1: Figure tyrosine kinase-linked
(Module 1: Figure EGF stimuli and receptors). ErbB1 is receptors). The extracellular region of this chain has
the most catholic in its tastes and responds to most of the a Sema domain, a cysteine-rich domain called the Met-
ligands. There is no known ligand for ErbB2, which thus related sequence (MRS) and four immunoglobulin-like
is an example of an orphan receptor. However, there is (Ig-like) structures (IPT domain). The intracellular part
much interest in c-ErbB2 because it is one of the common has the catalytic tyrosine kinase domain and regulatory
oncogenes found in breast cancer. ErbB3, which interacts domain, which has tyrosine residues at 1349 and 1356 that
with the neuregulins NRG1 and NRG2, is unusual in that provide docking sites to recruit the components of sig-
its kinase domain is non-functional. This does not mean nalling pathways.
that ErbB3 is prevented from signalling because it does so The juxtamembrane region of the chain has two
by forming functional dimers by interacting with one of phosphorylation sites that function in receptor down-
the other ErbB receptors as described later. ErbB4 binds regulation. Phosphorylation of Ser-985 inhibits the activ-
to a number of EGFs. ity of the receptor tyrosine kinase whereas Tyr-1003
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r27
PDGF
PDGFR PDGF
monomers
PDGFR
P 589 P Src
PtdIns3,4,5P3
MAP kinase
P 581 P
signalling pathway
Ras P
Grb2 P 716
ERK1/2 P 740 P p85 PI 3-K PtdIns 3-kinase
Raf SOS
P 751 P signalling pathway
P 763 P
P 771 P
P P 775 P
ERK1/2 PtdIns4,5P2
P P 778 P
Tyr kinase
Tyr kinase
Tyr kinase
Tyr kinase
DAG
InsP3
P 1009 P
P 1021 P PLC
InsP3 /DAG
signalling pathway
Ephrin-A Reverse
signalling
Ephrin-B
Cell
contact
EphA
receptor
Forward
signalling
EphB
receptor
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r28
phosphorylation is a binding site for the ubiquitin ligase nalling cassette that contributes to cell proliferation
Cbl that functions in the Cbl down-regulation of cell sig- (Module 2: Figure PLC structure and function).
nalling components (Module 1: Figure receptor down-reg-
ulation). Ephrin (Eph) receptor signalling
Both the MET and RON receptors play an important The ephrin (Eph) receptors constitute one of the
role in the invasive growth of carcinomas. largest family of protein tyrosine kinase-linked recept-
ors (PTKRs) (Module 1: Figure tyrosine kinase-linked re-
Insulin-like growth factor receptor (IGFR) ceptors). These receptors function to transfer information
There are two insulin-like growth factor receptors (IG- between cells that come into contact with each other and
FRs) that are capable of responding to insulin-like growth are particularly important for spatial patterning during a
factors (IGFs). The IGF type I receptor (IGF-IR) re- variety of developmental processes such as axonal guid-
sembles the insulin receptor both in its structure and mode ance, cell morphogenesis and bone cell differentiation. The
of activation (Module 2: Figure insulin receptor). Like Eph receptor family is divided into ten A types (EphA1
the insulin receptor, IGF-IR has a heterotetrameric 2 2 EphA10) and six B types (EphB1EphB6). The stimuli for
structure. The two IGFs (IGF-I and IGF-II) bind with these Eph receptors are the ephrins, which are expressed
equal affinities to the two extracellular -subunits to in- on the surface of cells (Module 1: Figure Eph receptors).
duce the conformational change responsible for receptor These ephrins are divided into A and B types determ-
activation. The -subunits, which are mostly intracellular, ined by their ability to bind to either the EphA or EphB
have the tyrosine kinase domain and motifs that interact receptors. There are six ephrin-A ligands (ephrin-A1
with components of the different signalling pathways ac- ephrin-A6), which are attached to the membrane through
tivated by these receptors. a glycosylphosphatidylinositol (GPI) anchor, and three
The IGF type II receptor (IGF-IIR) is a single protein transmembrane ephrin-B ligands (ephrin-B1ephrin-B3).
that is embedded in the membrane through a transmem- The ephrin-B ligands, which have a cytoplasmic domain,
brane region. The extracellular domain has fifteen cysteine- are unusual in that they have a dual function. Not only do
based repeats. The primary function of the IGF-IIR, which they function as a ligand to activate the EphB receptors, but
is strongly expressed during embryonic development, is to also they function as a receptor in that the cytoplasmic do-
remove IGF-II and is thus a negative regulator of this IGF main can convey information in the reverse direction. The
isoform. The IGF-IIR gene is maternally imprinted. Eph receptor/ephrin complex is a bidirectional signalling
Platelet-derived growth factor receptor (PDGFR) system and, through the forward and reverse signal-
The platelet-derived growth factor receptor (PDGFR) is ling modes, information can be conveyed to both inter-
a classical protein tyrosine kinase-linked receptor (PTKR) acting cells.
(Module 1: Figure tyrosine kinase-linked receptors). There Craniofrontonasal syndrome (CFNS) is caused by a
are two isoforms, and , which have different affin- loss-of-function mutation of the gene that encodes ephrin-
ities for platelet-derived growth factor (PDGF). The ex- B1.
tracellular region has five immunoglobulin-like (Ig-like) The Eph receptors have an N-terminal ephrin-binding
domains that function in ligand binding (domains IIII) domain followed by a cysteine-rich region and then two
and receptor dimerization (domain IV) (Module 1: Figure fibronectin type III domains (Module 1: Figure Eph
PDGFR activation). The intracellular region has a split tyr- receptor signalling). These ectodomains are connected
osine kinase domain and numerous tyrosine residues that through a typical transmembrane domain to the cytoplas-
are phosphorylated during receptor activation, which is mic domains. There is a relatively long juxtamembrane
initiated when a dimeric PDGF molecule brings together segment that connects to the kinase domain. At the C-
two PDGFRs. Once dimerization occurs, a transphos- terminal region there is a SAM domain that may participate
phorylation process begins whereby the kinase domain in receptor dimerization. Finally there is a PDZ domain-
on one chain phosphorylates numerous tyrosine residues binding motif.
on the neighbouring chain. These phosphorylated tyrosine When cells approach each other, the ephrin-A ligands
residues then provide docking sites for various signalling bind to the EphA receptors and the ephrin-B ligands bind
components to generate multiple output signals (Module to the EphB receptors and the resulting interactions in-
1: Figure PDGFR activation): duce dimerization as is typical of other PTKRs (Module 1:
Figure stimuli for enzyme-linked receptors). In addition to
Growth factor receptor-bound protein 2 (Grb2) recruits dimers, the Eph receptor/ephrin complex can form higher-
components of the extracellular-signal-regulated kinase order aggregates, and this clustering may be facilitated by
(ERK) pathway (Module 2: Figure ERK signalling) that interactions between the fibronectin type III domains and
promotes proliferation. the SAM domain. As the receptors are brought together,
The p85 subunit of Class I PtdIns 3-kinase recruits the transphosphorylation by the kinase domains results in the
PtdIns 3-kinase signalling cassette (Module 2: Figure phosphorylation of multiple sites, which then provides the
PtdIns 3-kinase signalling) that promotes survival and binding motifs to recruit a range of signalling transducers.
protein synthesis. One of the main functions of Eph receptor signalling is
Phospholipase C (PLC) induces both the inositol to modulate the dynamics of cell movement by altering
1,4,5-trisphosphate (InsP3 )/Ca2+ signalling cassette and both actin remodelling and cell adhesion. A number of the
the diacylglycerol (DAG)/protein kinase C (PKC) sig- downstream signalling pathways are thus directed towards
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r29
Reverse signalling
PTP-BL
Grb4 P P
Src
P P
PtdIns
Globular ephrin-
binding domain EphA EphB
receptor receptor
Fibronectin type III
repeats
P P Src P P
P P P P
P Cbl
Kinase
Kinase
Kinase
Cdc42 Intersectin
Kinase
Ephexin
Wasp Abl
P
Rho Kalirin
Arp2/3
SAM ROCK Rac
PAK
GRIP Syn
Actin Growth
Forward Growth cone Dendritic Receptor
assembly spine cone
signalling collapse
morphology AMPAR collapse degradation
the control of actin assembly. There are subtle differences nalling elements, acts through Rac and the p21-activated
in the action of EphA and EphB receptors that appear to kinase (PAK) to control spine morphogenesis (Step 2 in
be adapted to control different cellular processes. Module 10: Figure postsynaptic density).
One of the functions of EphA receptors in retinal gan-
TIE receptors
glion neurons is to induce growth cone collapse by activat-
The two angiopoietin receptors TIE1 and TIE2 respond
ing the Rho signalling mechanism (Module 2: Figure Rho
to the angiopoietin growth factors (Ang14) that control
signalling). The activated EphA receptor binds to the Rho
angiogenesis. The TIE receptors are typical protein tyr-
guanine nucleotide exchange factor (GEF) ephexin, which
osine kinase-linked receptors (PTKRs) (Module 1: Figure
is responsible for stimulating Rho (Module 1: Figure Eph
tyrosine kinase-linked receptors) that function to generate
receptor signalling). The RhoGTP then activates the Rho
a number of cell signalling pathways (Module 1: Figure
kinase (ROCK) that stimulates the actinmyosin contrac-
stimuli for enzyme-linked receptors).
tions responsible for collapsing the growth cone. Aggreg-
ating platelets communicate with each other through the Trk receptors
bidirectional Eph signalling system (Step 11 in Module 11: The Trk receptors, which mediate the action of the differ-
Figure platelet activation). ent neurotrophins (BDNF, NGF, NT-3 and NT-4/5), are
Most information is available for the forward signalling typical protein tyrosine kinase-linked receptors (PTKRs)
pathways initiated by the EphB receptors. While the EphA (Module 1: Figure tyrosine kinase-linked receptors). There
receptors are mainly linked to Rho activation, the EphB re- are three Trk receptors:
ceptors are coupled to the Rho GEFs kalirin and intersectin
that activate Rac (Module 2: Figure Rac signalling) and TrkA responds to nerve growth factor (NGF)
Cdc42 (Module 2: Figure Cdc42 signalling) respectively. TrkB responds to brain-derived neurotrophic factor
Cdc42 acts through Wiskott-Aldrich syndrome protein (BDNF) and neurotrophin-4/5 (NT-4/5)
(WASP) and the actin-related protein complex (Arp2/3 TrkC responds to neurotrophin-3 (NT-3)
complex) to regulate actin assembly (Module 1: Figure Eph Like other PTKRs, the Trk receptors act through a num-
receptor signalling). Kalirin, which is found in neuronal ber of cell signalling pathways (Module 1: Figure stimuli
dendrites as part of the postsynaptic density (PSD) sig- for enzyme-linked receptors).
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r30
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r31
Caspase-3 JNK
Caspase-6 Ceramide DAG InsP3
Messengers STATs NF-B P38
Caspase-8
NF-B Ca 2+
have an external ligand-binding domain and an internal respond to pathogens by releasing inflammatory mediat-
cytoplasmic domain that lack enzyme activity. The IL- ors (Module 7: Figure microglia interactions).
1R and TLRs are included within this TLR/IL-1R family
by virtue of the fact that they display considerable ho- Tumour necrosis factor (TNF) receptor (TNF-R)
mology with regard to their cytoplasmic domains. They The superfamily of tumour necrosis factor (TNF) recept-
belong to the non-enzyme-containing receptors that func- ors (TNF-Rs) has many members (e.g. TNF-R itself, Fas,
tion by recruiting various signal transducing components DR3-6, p75 neurotrophin receptor (p75NTR ) and RANK.
(Module 1: Figure cytokines). This cytoplasmic domain As a group, they are responsible for controlling many cel-
has the Toll/IL-1R (TIR) domain that has three conserved lular processes such as apoptosis and various inflammatory
boxes that are highly conserved in all of the receptors. The responses. They are fairly versatile receptors in that they
main difference between them lies in the external domain. can relay information out through a number of signalling
In the case of the IL-1R, the external domain contains three pathways (Module 1: Figure cytokines). The TNF-R has
immunoglobulin-like domains. By contrast, this region of two subunits, TNF-R1 and TNF-R2, which are usually co-
the TLRs contains a number of leucine-rich repeat (LRR) expressed in cells. Some members of this family are often
motifs. It is this region of the molecule that is responsible referred to as death receptors because one of their actions is
for binding the pathogen-associated molecular patterns to stimulate caspase-8 to induce apoptosis (Module 11: Fig-
(PAMPs), which are the breakdown products of patho- ure TNF apoptotic signalling). However, these receptors
gens. The TLRs are also sensitive to damage-associated can also contribute to inflammatory responses through
molecular patterns (DAMPS). When the TLRs detect these their ability to activate the nuclear factor B (NF-B) sig-
DAMPS and PAMPs, they alert cells to the existence of nalling pathway (Module 2: Figure NF-B activation).
damaging pathogens (Module 11: Figure formation and ac- The DR6 receptor responds to the N-terminal amyloid
tion of PAMPs). Information from the TLRs on the plasma precursor protein (APP) fragment (N-APP) that functions
membrane is relayed through the Toll receptor signalling in both axonal pruning and neuronal apoptosis (see step 11
pathway (Module 2: Figure Toll receptor signalling). The in Module 12: Figure amyloid cascade hypothesis). Altera-
TLRs located on the endosomal membranes function in tions in the activity of the DR6 activity has been implicated
virus recognition and antiviral responses (Module 2: Fig- in Alzheimers disease as part of the amyloid cascade hy-
ure virus recognition). The TLRs on microglia in the brain pothesis.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r33
Immunoglobulin- TPOR
like domain EPOR IL-2R IL-4R
Cytokine receptor
module
LIFR
GM-CSFR IL-6R CNTFR
G-CSFR IL-11R
IL-3R
IL-5R
GM-CSFR
IL-3R IL-6R LIFR
IL-5R IL-11R CNTFR
main and a short cytoplasmic region, which is associ- factor NFATc1 by the well-established mechanism that de-
ated with DNAX-activating protein 12 (DAP12), which pends upon the Ca2 + -dependent activation of calcineurin
is also known as TYRO protein tyrosine kinase binding (Module 4: Figure NFAT activation).
protein (TYROBP). The latter has a typical immunore- Mutations in the DAP12 gene (TYROBP) and in the
ceptor tyrosine-based activation motif (ITAM) that asso- TREM-2 gene have been linked to polycystic lipomem-
ciates with various amplifiers such as phospholipase C branous osteodysplasia with sclerosing leukoencephalo-
(PLC) (Module 1: Figure cytokines). The nature of the pathy syndrome (PLOSL), which is also known as Nasu-
stimuli that act through TREM-2 are still somewhat un- Hakola disease (NHD). Since PLOSL causes disorders of
certain. It appears to bind to surface ligands on pathogens both bone and the central nervous system, it is evident that
and it may also interact with Hsp60 on the surface of as- the TREM2/DAP12 signalling pathway may also have an
trocytes. important function in the development and maintenance
The TREM-2 expressed on developing osteoclasts func- of brain function.
tions in osteoclastogenesis by switching on the Ca2 + sig- A variant of TREM-2 may contribute to the
nal that activates the transcription factor nuclear factor inflammation in Alzheimers disease (Module 12: Figure
of activated T cells (NFAT) (Module 8: Figure osteo- Inflammation and Alzheimers disease).
clastogenesis). The related receptor signal-regulatory pro-
tein (SIRP1) has a similar role, and their actions will Integrin signalling
thus be considered together. When activated, TREM-2 The integrins are cell-surface receptors that function in
and SIRP1 interact with DNAX-activating protein 12 cell adhesion either to the extracellular matrix (ECM) or
(DAP12), which is an adaptor that has a typical im- to specific cell-surface ligands during cellcell interactions
munoreceptor tyrosine-based activation motif (ITAM) (Module 1: Figure integrin receptor). Integrins have two
(Module 8: Figure osteoclastogenesis). This ITAM region main functions. Firstly, they provide a link between the
has tyrosine residues that are phosphorylated by the non- internal cytoskeleton and the ECM as occurs at focal adhe-
receptor tyrosine kinase Src. These specific phosphotyr- sion complexes and podosomes. Secondly, they have a very
osine residues recruit Syk that then activates phospholipase important signalling function that is unusual because they
C1 (PLC1) to switch on the inositol 1,4,5-trisphosphate can signal in both directions. In the conventional outside-in
(InsP3 )/Ca2+ signalling cassette. The resulting Ca2 + signal mode, external stimuli activate the integrin receptor, which
appears as a typical series of repetitive Ca2 + transients then engages various transducing elements to transmit sig-
(Module 8: osteoclast Ca2+ oscillations). These Ca2 + os- nals into the cell. In the inside-out mode, intracellular
cillations are responsible for activating the transcription signals coming from other receptors, often growth factor
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r34
High
affinity
Low
affinity
Integrin Outside-in Inside-out
receptor
Grb2 P P
FAK TK FAK FAK TK
Transducers Ras SOS P TK P
GEFs
Src
receptors, induce a conformational change in the integrin 7 /1 is expressed in cardiac cells, where it functions
receptors that greatly enhance their affinity for their ex- in the link between the sarcolemma and the sarcomeres
ternal ligands. An example of inside-out signalling is found (Module 12: Figure cardiac contractile elements)
in osteoclasts, where the colony-stimulating factor-1 re- 5 /1 responds to the cytoxin-associated gene A (CagA)
ceptor (CSF-1R) functions to sensitize the integrin recept- ligand (CagL) on the end of the nano-syringe that is used
ors (Module 8: Figure osteoclastogenesis). by Helicobacter pyloris to inject proteins into its host
Integrins are composed of transmembrane - and - cell (Module 12: Figure H pylori nano-syringe). CagA
subunits that come together to form a functional heterodi- is thought to be a bacterial oncoprotein responsible for
mer (Module 1: Figure integrin receptor). There are 18 inducing stomach cancer.
-subunits and eight -subunits that can combine to form
24 different heterodimers that have their own binding spe- The integrins have large extracellular regions that inter-
cificities and characteristic expression patterns (Module 1: act with specific sequences on the extracellular matrix pro-
Figure integrin heterodimeric complexes). The following teins (cellmatrix interactions) or specific cell-surface lig-
examples illustrate the expression of different combina- ands (cellcell interactions). The intracellular cytoplasmic
tions in specific cell types: domain is relatively short (4070 amino acids) and lacks
enzyme activity. There is considerable information on in-
2 /1 and IIb/3 are expressed in blood platelets tegrin structure and the dramatic conformational changes
(Module 11: Figure platelet activation). The calcium and that occur during the formation of adhesion complexes
integrin-binding protein 1 (CIB1) appears to regulate (Module 1: Figure integrin receptor structure). The struc-
IIb/3 by binding to its cytoplasmic tail. ture of the L subunit illustrates the large cytoplasmic head
v /3 is expressed in osteoclasts and functions in bone that consists of a series of domains beginning with an I do-
resorption (Module 7: Figure osteoclast function) main followed by the -propeller region and then three
L /2 , which is also known as lymphocyte function- domains that end in the transmembrane domain. One of
associated antigen-1 (LFA1), is expressed on lympho- the linkers between these three domains is known as genu
cytes and contributes to the immunological synapse (knee), because it is the region where the molecule bends
4 /7 , which is also known as very late antigen 4 over when it assumes the low-affinity state. Finally, there
(VLA4), is expressed on lymphocytes is a short cytoplasmic domain, which has a GFFKR hinge
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r35
E E-Cad Fg
7
Fn
Lm Coll
IIb
Lm ICAM
Fn Coll Vn
VCAM
4 3 Lm
3
2
Fn Vn M L
5 1 5
4 6 1 V 2
11 6
7 D X
Vn
Lm 8 9 10 8
Coll Coll
Fn Fn
Tn Lm
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r36
motif and the KK motif that binds to RAPL. The 2 be- external ligands, which usually bind to a pocket formed
gins with an I-like domain followed by a hybrid domain, by the external regions of some of the transmembrane
which is linked through a genu to four integrin epidermal domains, induce a conformational change in the receptor
growth factor (I-EGF) repeats. The latter are connected to that is then transmitted through the membrane to activate
a -tail, which connects to the transmembrane region and the GTP-binding proteins (G proteins). These G proteins
the cytoplasmic tail region. The latter has a hinge region fall into two main groups: the heterotrimeric G proteins
and a NPXF motif that binds to talin. and the monomeric G proteins. It is the heterotrimeric
External stimuli such as intercellular adhesion molecule G proteins that are the main transducers responsible for
(ICAM) in the case of the L /2 integrin in lymphocytes, transferring information from the GPCRs to a number
bind to the I domain of the L subunit through a reac- of signalling pathways. This transduction process is dis-
tion that depends upon Mg2 + occupying the metal-ion- cussed in further detail in Module 2 (see Module 2: Figure
dependent adhesion site (MIDAS). The molecule then un- heterotrimeric G protein signalling). The receptor activ-
dergoes the large conformational changes that bring about ates the G proteins by functioning as a guanine nucleotide
the changes in affinity. exchange factor (GEF) to induce the exchange of GDP
In order to transmit information into the cell, the rel- for GTP (Module 2: Figure G protein binary switching).
atively short cytoplasmic domains of the integrin recept- When the G protein is bound to GTP it activates a variety
ors function as a signalling platform to assemble various of downstream effectors including adenylyl cyclase and
transducing elements (Module 1: Figure integrin receptor). phospholipase C.
An important component of the transducing mechanism Many oncogenic growth factors are known to act
are kinases such as the integrin-linked kinase (ILK) and through GPCRs and many human cancers have mutations
focal adhesion kinase (FAK), which can relay informa- in these GPCRs.
tion down conventional signalling pathways such as the
mitogen-activated protein kinase (MAPK) signalling path- Ca2 + -sensing receptor (CaR)
way, the PtdIns 3-kinase signalling pathway, the inositol The Ca2 + -sensing receptor (CaR) belongs to the family of
1,4,5-trisphosphate (InsP3 )/Ca2+ signalling cassette and G protein-coupled receptors (GPCRs) (Module 1: Table
the signalling pathways activated by the monomeric G G protein-coupled receptors). The primary function of the
proteins (Rho, Rac and Cdc42) that are critical for remod- CaR is to regulate parathyroid hormone (PTH) synthesis
elling the actin cytoskeleton. and release, but it is also expressed in many other cell
More detailed information on the signalling and skeletal types, such as bone cells, neurons, intestine, kidney, skin,
functions of integrins is presented in the following sec- pancreas and heart. It is a typical GPCR with the usual
tions: seven transmembrane domains, a large extracellular N-
terminal domain and a C-terminal domain of 216 amino
Blood platelet aggregation (Steps 810 in Module 11:
acids, some of which have potential phosphorylation sites
Figure platelet activation)
for protein kinase C (PKC) and protein kinase A (PKA).
Cardiac cell contractile and cytoskeletal elements
The CaR operates as a dimer, with the two subunits linked
(Module 12: Figure cardiac contractile elements)
together by two disulfide bonds (Module 7: Figure PTH
Osteoclast activation in bone resorption (Step 1 in
secretion). In the parathyroid gland, the CaR dimers are
Module 7: Figure osteoclast function)
located on caveolae where they appear to be linked to both
Focal adhesion complex formation (Module 6: Figure
caveolin and the scaffolding protein filamin. The CaR can
integrin signalling)
also form heterodimers with the mGluR1 and mGluR5
Glanzmanns thrombasthenia is a bleeding disorder that receptors in the brain. Unlike many other GPCRs, the
has been linked to mutations in the 3 integrin subunit. CaR does not desensitize and responds continuously to
the prevalling Ca2 + concentration.
G protein-coupled receptors (GPCRs) In addition to responding to Ca2 + , CaR is also sensit-
The G protein-coupled receptors (GPCRs) represent a ive to a number of other agonists that fall into three main
very large superfamily of receptors that are capable of groups. The first group consists of related inorganic ions
responding to an enormous number and variety of extra- (Mg2 + and Gd3 + ) or organic polycations (neomycin and
cellular stimuli (light, odorants, neurotransmitters, hor- spermine) that act in much the same way as Ca2 + to dir-
mones and proteases) (Module 1: Table G protein-coupled ectly activate the receptor. The other two groups function
receptors). As their name implies, they are coupled to indirectly as allosteric regulators that alter the affinity of
the heterotrimeric G proteins that function as the trans- the receptor, either positively (calcimimetics) or negatively
ducers to relay information to different signalling path- (calcilytics).
ways such as the cyclic AMP signalling pathway (Module In the parathyroid gland, CaR functions as a calcio-
1: Figure stimuli for cyclic AMP signalling) and the inos- stat in that it is very sensitive to small fluctuations in
itol 1,4,5-trisphosphate (InsP3 )/diacylglycerol (DAG) sig- the plasma level of Ca2 + . It has the potential of re-
nalling pathway (Module 1: Figure stimuli for InsP3 /DAG laying information to the parathyroid cell through dif-
signalling). ferent signalling pathways. The main mechanism ap-
The GPCRs are characterized by having seven- pears to be through the inositol 1,4,5-trisphosphate
membrane-spanning regions with the N-terminus facing (InsP3 )/Ca2+ signalling cassette. It may also act to inhibit
the outside and the C-terminus lying in the cytoplasm. The the Ca2 + -inhibitable isoform of adenylyl cyclase (AC)
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r38
Neurotensin receptors
NT1 Gq/11 Stimulate phospholipase C
NT2 Gq/11 Stimulate phospholipase C
Sphingosine 1-phosphate receptors (see Module 2: Figure sphingomyelin signalling
EDG-1 Gi Inhibit adenylyl cyclase
EDG-3 Gq/11 Stimulate phospholipase C
EDG-5 Gq/11 Stimulate phospholipase C
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r39
Melatonin receptors
MT1 Gq/11 Stimulate phospholipase C
MT2 Gi Inhibit adenylyl cyclase
MT3 Gq/11 Stimulate phospholipase C
Olfactory receptors (ORs)
Approximately 1000 OR genes have been Gs Stimulate adenylyl cyclase
identified
PP-fold peptides
Y1 Gi Inhibit adenylyl cyclase
Y2 Gi Inhibit adenylyl cyclase
Y3 Gi Inhibit adenylyl cyclase
Y4 Gi Inhibit adenylyl cyclase
Y5 Gi Inhibit adenylyl cyclase
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r41
Hedgehog
Wnts
Sonic hedgehog (Shh)
Indian hedgehog (Ihh) Wnt5A Wnt8
Desert hedgehog (Dhh) Wnt11
LRP5/6
Wnt Wnt
PTC SMO
Fz Fz
Dsh Dsh
Transducers
SU
GSK3 DAAM1
KIF7 SUFU RHO RAC
Stimuli that regulate development act through a number of cell signalling pathways.
The Hedgehog proteins and Wnts are stimuli that control development. They both use a seven-membrane-spanning receptor that resembles the G
protein-coupled receptor used by other signalling systems, but in this case the receptor has evolved to activate other signalling pathways. In the case
of Hedgehog, the smoothened (SMO) receptor engages a transducing complex that activates the transcription factor GLI1. The signalling pathways
for Wnts are carried out by Frizzled (Fz) receptors that are coupled to different signalling pathways. Some of the Wnts activate the transcription factor
-catenin, whereas other Wnts bind to Fz receptors that relay information to other signalling pathways.
Finally, the ion channel receptors are an example where Sigma receptors
a single protein combines the function of receptor, trans- There are two sigma ( ) receptors: sigma-1 recept-
ducer and amplifier (Module 1: Figure stimuli for ion chan- ors (Sig-1R) and sigma-2 receptors (Sig-2R). These re-
nels). ceptors, which are widely distributed in the brain and
These different transducers and amplifiers produce in many peripheral organs, are located mainly in the
many different intracellular messengers that carry in- mitochondrial-associated ER membranes (MAMs) that
formation to the internal sensors and effectors. The cell are specialized functional zones where regions of the en-
signalling pathways responsible for producing these mes- doplasmic reticulum (ER) come into close contact with
sengers are described in more detail in Module 2: Cell the mitochondria (Module 5: Figure mitochondrial-asso-
Signalling Pathways. ciated ER membranes). Most information is available for
Sig-1R, which has two transmembrane domains with the
N- and C-termini located in the lumen of the ER. There
Ion channel receptors are two steroid-binding domains that come together to
Ion channels play a crucial role in many aspects of cell form a pocket, which is the binding site for a number
signalling. One of their most obvious roles is to func- of agents such as neurosteroids, neuroleptics, dextroben-
tion as receptors for a number of external stimuli (Module zomorphans, methamphetamine and cocaine. Within the
1: Figure stimuli for ion channels). These receptors are MAM, the Sig-1R interacts with both the inositol 1,4,5-tri-
multifunctional in that they detect the incoming stimu- sphosphate receptors (InsP3 Rs) on the ER membrane and
lus, they transduce the information into channel open- the Ca2 + -sensitive chaperone protein BiP, which is located
ing and, by virtue of conducting large amounts of charge, within the ER lumen. When the luminal level of Ca2 + de-
they markedly amplify the signal. Such amplification is clines, the Sig-1R dissociates from BiP and then acts as
the reason such channel receptors are such effective trans- a chaperone to stabilize the activity of the InsP3 R and
ducers of sensory information. thus regulates the transfer of Ca2 + from the ER to the
Module 3: Ion Channels describes the properties of these mitochondrion. A similar response occurs following stim-
ion channel receptors and it also describes how some ion ulation by Sig-1R agonists such as progesterone and this
channels are important effectors for different intracellular also results in a relocation of the receptor to the plasma
messengers. Their sensitivity to cyclic nucleotides such as membrane where it regulates the activity of a number of
cyclic AMP and cyclic GMP is a critical component of channels (Module 5: Figure mitochondrial-associated ER
sensory transduction. membranes):
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-
_
Na + Cl
+
V
Messengers Ca 2+ Ca 2+
V
NMDA receptor (NMDAR) and effectors (Module 1: Figure cell signalling mechanism).
Different members of the large family of These messengers can take many forms. The concept of an
voltage-dependent K+ (Kv ) channels (Module 3: internal messenger first emerged in the cyclic AMP sig-
Table voltage-dependent K+ channels). There are a nalling pathway (Module 1: Figure stimuli for cyclic AMP
number of examples where the Sig-1R supresses the signalling) where the external stimulus was considered to
K + current and would thus lead to excitation. In the be the first messenger, whereas the cyclic AMP formed dur-
case of small cell lung carcinoma cells, Sig-1Rs inhibited ing information transduction was referred to as the second
the KV 1.3 delayed rectifier K + channel resulting in cell messenger. However, the term second messenger can be
cycle arrest. This observation is consistent with the confusing because there are examples where there are addi-
observation that the Kv 1.3 channel palys a critical role tional messengers within a signalling pathway. Therefore,
in maintaining the membrane potential that promotes to avoid confusion, the term intracellular messenger will
the T cell receptor (TCR) Ca2+ signalling that drives cell be used to refer to the agents that carry information within
proliferation (Module 9: Figure T cell Ca2+ signalling). the cell.
Intracellular messengers can take many different
The fact that these receptors are sensitive to psychos- forms:
timulants, such as methamphetamine and cocaine, has at-
tracted considerable interest as to how they may function 1. Ca2 + is one of the major intracellular messengers that
in drug addiction. Stimulation of sigma receptors increases transmits information for the Ca2+ signalling pathways
dopamine (DA) transmission in the shell of the nucleus ac- (Module 2: Figure Ca2+ modules).
cumbens (NAc), which is a part of the brain responsible 2. Cyclic AMP that functions in the cyclic AMP sig-
for the reinforcing effects of drugs such as cocaine that are nalling pathway (Module 2: Figure cyclic AMP
abused by humans. signalling).
In addition to drug addiction, the Sig-1Rs have also 3. Ca2 + -mobilizing second messengers:
been linked to a number of disease states such as
Inositol 1,4,5-trisphosphate (InsP3 ) that functions as
Alzheimers disease (AD), amnesia, amyotrophic lateral
a messenger linking receptors to the Ca2 + signalling
sclerosis (ALS), retinal degeneration, and cancer
pathway (Module 2: Figure InsP3 and DAG forma-
tion).
Intracellular messengers Cyclic ADP-ribose (cADPR) that releases
The role of intracellular messengers is to carry inform- Ca2 + from internal stores (Module 2: Figure
ation generated at the cell surface to the internal sensors cADPR/NAADP function)
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r43
Nicotinic acidadenine dinucleotide phosphate the cell contains internal sensors to detect these intracel-
(NAADP) (Module 2: Figure cADPR/NAADP lular messengers. Typical examples of such sensors are the
function). Ca2 + -binding proteins that detect increases in Ca2 + and
relay this information to different effectors to control pro-
4. Lipid messengers:
cesses such as contraction and secretion. Some sensors are
Diacylglycerol (DAG) is a messenger in the phos- also effectors. For example, there are enzymes that respond
phoinositide signalling pathway that links receptor to messengers such as cyclic AMP and cyclic GMP that not
activation to protein kinase C and protein phos- only detect the messenger, but also carry out various ef-
phorylation (Module 2: Figure InsP3 and DAG fector functions. While some of these effectors might be re-
formation). latively simple, consisting of a single downstream effector
PtdIns3,4,5P3 is the lipid messenger that functions in system, there are more complicated effectors made up of
the PtdIns 3-kinase signalling pathway (Module 2: multiple components such as those driving processes such
Figure PtdIns 3-kinase signalling). as exocytosis, phagocytosis, actin remodelling and gene
PtdIns4,5P2 is the lipid messenger that functions as a transcription.
membrane messenger to activate many cellular pro- The activation of these sensors and effectors completes
cesses (Module 2: Figure PtdIns4,5P2 signalling). the flow of information down the cell signalling pathways
Phosphatidic acid (PA) is a messenger for the phos- (green arrows in Module 1: Figure cell signalling mech-
pholipase D signalling pathway (Module 2: Figure anism). The operation of such sensors and effectors is de-
PLD signalling). scribed in more detail in Module 4: Sensors and Effectors.
Ceramide functions in the sphingomyelin signalling
pathway (Module 2: Figure sphingomyelin sig-
OFF mechanisms
nalling).
Signalling pathways are composed of the ON mechan-
5. Protein kinase messengers. Certain protein kinases can isms that generate a flow of information into the cell
carry information to different regions of the cell: and the OFF mechanisms that switch off this internal
The mitogen-activated protein kinase (MAPK) sig- flow of information, enabling cells to recover from stim-
nalling pathway generates active kinases such as ulation (Module 1: Figure cell signalling mechanism).
extracellular-signal-regulated kinase 1/2 (ERK1/2) Module 5: OFF Mechanisms describes how the intracel-
(Module 2: Figure ERK signalling) and c-Jun N- lular messengers and their downstream effectors are in-
terminal kinase (JNK) (Module 2: Figure JNK sig- activated. The second messengers cyclic AMP and cyc-
nalling) that carry information into the cell and very lic GMP are inactivated by phosphodiesterases. Inositol
often into the nucleus. 1,4,5-trisphosphate (InsP3 ) metabolism is carried out by
both inositol trisphosphatase and inositol phosphatases.
6. Transcription factors that are activated at the cell sur- Diacylglycerol (DAG) metabolism also occurs through
face or within the cytoplasm function as messengers two enzyme systems, DAG kinase and DAG lipase.
carrying information into the nucleus: In the case of Ca2 + signalling, recovery is carried out by
Nuclear factor B (NF-B) carries information from
the cell surface into the cell (Module 2: Figure NF-B the Ca2+ pumps and exchangers that remove Ca2 + from
activation). the cytoplasm. Many of these second messengers activate
The signal transducers and activators of transcrip- downstream effectors through protein phosphorylation,
tion (STATs) transfer information from cell-surface and these activation events are reversed by corresponding
receptors into the nucleus (Module 1: Figure cy- protein phosphatases.
tokines).
Smads transfer information from the transforming
Information transfer mechanisms
growth factor (TGF-) receptor superfamily into
The function of the cell signalling pathways is to transmit
the nucleus (Module 2: Figure Smad signalling).
information from the cell periphery to the internal effect-
-Catenin functions as a messenger in the canon-
ors, such as the contractile proteins, membrane vesicles,
ical Wnt pathway (Module 2: Figure Wnt canonical
ion channels, metabolic pathways and cell cycle proteins
pathway).
that are responsible for activating cellular responses. There
GLI1 functions in the Hedgehog signalling pathway
are a number of mechanisms whereby information is trans-
(Module 2: Figure Hedgehog signalling pathway).
mitted through these pathways (Module 1: Figure signal
The function of all of these intracellular messengers is transmission mechanisms).
to transmit information to the sensors and effectors that
are responsible for the final function of the cell signalling Conformational-coupling mechanisms
pathways to activate a whole host of cellular processes. Information can be transferred from one signalling element
to the next through a process of conformational coup-
Sensors and effectors ling. If the components, which are usually proteins, are
The intracellular messengers that are produced by the dif- already associated with each other, then this transfer mech-
ferent signalling pathways function to regulate cellular anism can be very fast (mechanism 1 in Module 1: Figure
processes (Module 1: Figure cell signalling mechanism). signal transmission mechanisms). A classical example of
Just as the cell has receptors to detect external stimuli, such a conformational-coupling mechanism occurs during
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r44
Stimulus
Signal
A B transmission
1. 2.
Stimulus Stimulus
A B * A B A B *
Diffusion
Conformational-coupling
Conformational-coupling
(diffusion-dependent
(preformed complex)
complex formation)
3. 4.
A
Stimulus Stimulus
A X B * A B B *
excitationcontraction coupling in skeletal muscle, where tional change through some post-translational modifica-
the CaV 1.1 L-type channel is pre-coupled to the ryan- tion. These modifications, which function in signal trans-
odine receptor (RYR1) (Module 3: Figure L-type chan- mission, are often very specific in that they are directed
nel/RYR1 complex). Another example is the association towards particular amino acids that can by altered in many
of voltage-operated Ca2 + channels with the proteins re- different ways:
sponsible for exocytosis of synaptic vesicles (Module 4:
Figure Ca2+ -induced membrane fusion). Protein glycosylation
Conformational coupling is also used when informa- Protein phosphorylation
tion is being transferred by diffusion of signalling elements. Protein oxidation
Low-molecular-mass second messengers (e.g. Ca2 + , cyclic Protein acetylation
AMP, cyclic GMP and reactive oxygen species) or proteins Protein methylation
such as the phosphorylated extracellular-signal-regulated Sumoylation
kinase 1/2 (ERK1/2) or various activated transcription Ubiquitination
factors that translocate from the cytoplasm into the nuc-
leus carry information as they diffuse through the cell.
Protein glycosylation
In order to transmit this information, these diffusing ele-
A number of signalling proteins can be glycosylated
ments use a conformation-coupling mechanism to trans-
through the attachment of a -N-acetylglucosamine
mit information when they bind to downstream elements
(GlcNAc) residue. An O-GlcNAc transferase (OGT)
(mechanism 2 in Module 1: Figure signal transmission
uses UDP-GlcNAc, which is the end product of hex-
mechanisms).
osamine biosynthesis, to form the O-linked -N-
Post-translational modifications acetylglucosamine (O-GlcNAc) post-translational modi-
Signalling systems use a variety of post-translational pro- fication, which is then reversed by a O-GlcNAcase. The
tein modifications in order to transmit information along UDP-GlcNAc may function as a nutrient sensor because
signalling pathways (mechanism 3 in Module 1: Figure its levels fluctuate depending on the availability of meta-
signal transmission mechanisms). The basic mechan- bolites such as glucose and free fatty acids (FFAs). The
ism is for a stimulus to activate component A, which elevation of these metabolites during obesity may increase
then acts on component B to bring about a conforma- the activity of OGT resulting in the glycosylation and
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r46
416 Y Y 527
P P
Src
family SH3 SH2 Kinase Src
Fgr Src
Hck Src is the prototype of the Src protein tyrosine kinase fam-
Fyn ily (Src, Blk, Fyn, Fgr, Hck, Lck, Lyn, Yes). These tyrosine
Lck kinases function both as adaptors to assemble signalling
Lyn complexes and as a tyrosine kinase to phosphorylate com-
Yes ponents of such signalling complexes. Their structure has
domains responsible for this dual adaptor and enzymatic
Spleen tyrosine kinase (Syk) family function (Module 1: Figure non-receptor tyrosine kinases).
The kinases are attached to the membrane through the N-
Syk terminal region and this is then followed by an Src homo-
-Associated protein of 70 kDa (ZAP70) logy 3 (SH3) domain and then an Src homology 2 (SH2)
domain. The kinase region is located in the C-terminal part
Tec tyrosine kinase family of the molecule where there are two tyrosine residues (Tyr-
416 and Tyr-527) that are critical for regulating the activity
Tec of Src. The SH2 domain not only enables Src to inter-
Brutons tyrosine kinase (Btk) act with other signalling molecules, but also participates
Inducible T cell kinase (Itk) in an intramolecular interaction that regulates Src activ-
ity. The regulation of Src, which resembles that found in
C-terminal Src kinase (CSK) other members of the family, depends upon the following
Fer processes (Module 1: Figure Src activation):
Abelson tyrosine kinase (Abl) family
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r47
SH3
SH3
Target
SH3
Tyrosine Pro protein
2
phosphatase
SH2
SH2
SH2
P Tyrosine Target
P protein
kinases 4
ase
P
C-terminal Kinase
Kin
Kinase
Target
Src kinase Pro protein
3
5 Target
P protein
Abl
P
Abl
3. The activated tyrosine kinase domain can now phos- DNAX-activating protein 12 (DAP12) adaptors, which
phorylate various substrates such as Abl (Module 1: co-ordinate activation of the Ca2 + signalling pathway
Figure Abl signalling). in the developing osteoclast.
4. The active conformation of the molecule also makes the It phosphorylates and activates the Tec tyrosine kinase
SH2 and SH3 domains available to interact with a num- family.
ber of target proteins to assemble signalling complexes.
5. Src is inactivated by the C-terminal Src kinase (CSK) C-terminal Src kinase (CSK)
that phosphorylates the C-terminal Tyr-527 to rein- C-terminal Src kinase (CSK) inactivates Src by phos-
state the inactive conformation (Module 1: Figure Src phorylating Tyr-527 at its C-terminal end (Module 1: Fig-
activation). ure non-receptor tyrosine kinases). Once this residue is
phosphorylated, the C-terminal end containing the kinase
Src performs a wide range of functions:
domain bends around so that this phosphorylated group
It activates the non-receptor protein tyrosine kinase Abl interacts with the Src homology 2 (SH2) domain. Src is
(Module 1: Figure Abl signalling) inactive in the folded configuration (Module 1: Figure
It acts together with the proline-rich tyrosine kinase 2 Src activation). CSK plays a similar role in inactivating
(Pyk2) to promote formation of the osteoclast podo- Lck, which is one of the early T cell receptor transducers
some (Module 7: Figure osteoclast podosome) (Module 9: Figure TCR signalling)
It plays a role in relaying information from integrin re-
ceptors to PtdIns 3-kinase at the focal adhesion complex Fyn
(Module 6: Figure integrin signalling) In blood platelets, Fyn acts together with Lyn by binding
During osteoclastogenesis, colony-stimulating factor-1 to the collagen receptor glycoprotein VI (GPVI) where
(CSF-1) acts on the colony-stimulating factor-1 receptor they phosphorylate immunoreceptor tyrosine-based ac-
(CSF-1R) and recruits Src, which forms a complex with tivation motifs (ITAMs) on the Fc receptor (FcR)
c-Cbl and PtdIns 3-kinase (PtdIns 3-K) (Module 8: Fig- chains to provide binding sites for phospholipase C 2
ure osteoclast differentiation). Src also phosphorylates (PLC 2) (Step 2 in Module 11: Figure platelet activation).
the typical immunoreceptor tyrosine-based activation Fyn also binds to the adaptor protein SLAM-associated
motifs (ITAMs) on the Fc receptor (FcR) and protein (SAM), which mediates the action of the
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r48
signalling lymphocyte activation molecule (SLAM) fam- phosphorylates phospholipase C1 (PLC1) and Brutons
ily of co-stimulatory molecules. tyrosine kinase (Btk), which interact with each other dur-
In mast cells, Fyn is recruited to the FcRI receptor ing the activation of PLC1. Syk performs a similar func-
where it phosphorylates Gab2 and Brutons tyrosine tion in the activation of B cells (Module 2: Figure ROS
kinase (Btk), which contribute to the activation of PtdIns effects on Ca2+ signalling) and during osteoclastogenesis
3-kinase, which then phosphorylates PtdIns4,5P2 to form (Module 8: Figure osteoclast differentiation).
the lipid messenger PtdIns3,4,5P3 (Module 11: Figure
FcRI mast cell signalling). -Associated protein of 70 kDa (ZAP70)
Another important action of Fyn is to regulate the func- The -associated protein of 70 kDa (ZAP70) is a non-
tion of the classical cadherins. Stability of the cadherin/- receptor protein tyrosine kinase that functions in T cell ac-
catenin/actin complex is controlled by phosphorylation of tivation (Module 1: Figure non-receptor tyrosine kinases).
Tyr-142 on -catenin by Fyn and this regulates its inter- It is one of the key components of the T cell receptor
action with -catenin and the actin cytoskeleton (Module transducers that are drawn into the large signalling com-
6: Figure classical cadherin signalling). plex (Module 9: Figure TCR signalling). ZAP70 uses its Src
homology 2 (SH2) domains to bind to that the immunore-
Lck ceptor tyrosine-based activation motifs (ITAMs) that are
Lck is one of the main non-receptor protein tyrosine phosphorylated by Lck. Once in place, ZAP70 then phos-
kinases expressed in T cells, where it plays an early role phorylates scaffolding components such as linker for activ-
as one of the T cell receptor transducers (Module 9: Fig- ation of T cells (LAT) and SH2-domain-containing protein
ure TCR signalling). As for Src (see Step 5 in Module 1: of 76 kDa (SLP-76), which are drawn into the growing
Figure Src activation), Lck is inactive when the molecule central supramolecular activation cluster (c-SMAC) and
is folded by an intramolecular interaction between the Src function as docking sites to communicate to the different
homology 2 (SH2) domain and a phosphate group at the signalling cassettes.
C-terminal end of the molecule. In the case of T cells, Lck
is activated by the transmembrane tyrosine kinase CD45, Fer
which dephosphorylates an inhibitory phosphate group Fer is a non-receptor tyrosine kinase that plays a role in
on Tyr-505. Conversely, the molecule is inactivated when regulating the function of the classical cadherins (Module
Tyr-505 is phosphorylated by the C-terminal Src kinase 6: Figure classical cadherin signalling). It has the typical
(CSK). conserved kinase domain together with three coiled-coil
domains (CCD1CCD3) and a Src homology 2 (SH2)
Lyn domain (Module 1: Figure non-receptor tyrosine kinases).
Lyn functions in the activation of B cells, mast cells and
blood platelets. In mast cells, Lyn acts to phosphorylate Abl
the immunoreceptor tyrosine-based activation motifs (IT- The Abelson tyrosine kinase (Abl) takes its name from
AMs) on the FcRI receptor to provide binding sites to the fact that the c-abl gene was first identified as part of
recruit Syk (Module 11: Figure FcRI mast cell signalling). the Abelson murine leukaemia virus and was subsequently
Lyn also phosphorylates and activates Syk as part of the found to be a component of the human BCR-ABL onco-
cascade of signals that lead to mast cell activation. Another gene. The Abl component of the BCR-ABL oncogene is
of its functions is to phosphorylate and activate the Tec a multitasking signalling molecule capable of operating in
tyrosine kinase family. both the cytoplasm and nucleus to regulate diverse cel-
In the case of blood platelets, Lyn binds to the colla- lular processes, including actin remodelling, chemotaxis,
gen receptor glycoprotein VI (GPVI) and phosphorylates gene expression, T cell receptor signalling, DNA repair
ITAMs on the Fc receptor (FcR) chains to provide and apoptosis.
binding sites for phospholipase C2 (PLC2) (Step 2 in The ability of Abl to regulate multiple cellular processes
Module 11: Figure platelet activation). Lyn also plays a depends on its complex domain structure (Module 1: Fig-
role in B-cell antigen receptor (BCR) activation (Module ure non-receptor tyrosine kinases). The N-terminal region
9: Figure B cell activation). begins with an Src homology 3 (SH3) domain and an Src
homology 2 (SH2) domain, which are followed by the
Syk kinase domain. Like other non-receptor protein tyrosine
Syk (spleen tyrosine kinase) is a non-receptor protein kinases, there is an internal autoinhibition whereby the
tyrosine kinase (Module 1: Figure non-receptor tyrosine SH3 domain bends round to interact with the kinase do-
kinases) that plays a role in signal transduction in mast cells main. The SH3 domain is used by Abl to interact with a
and in B cells. In mast cells, phosphorylation of the im- plethora of proteins that have the PXXP motif such as 3BP-
munoreceptor tyrosine-based activation motifs (ITAMs) 1 (a Rac GTPase-activating protein), Abelson-interactor
on the FcRI receptor by Lyn provides binding sites that (Abi), ATM (ataxia telangiectasia mutated), Cbl,
recruit Syk, which is also phosphorylated and activated by DNA-dependent protein kinase (DNAPK), NR2D sub-
Lyn (Module 11: Figure FcRI mast cell signalling). The unit of theN-methyl-d-aspartate (NMDA) receptor,
activated Syk then phosphorylates the scaffolding proteins scramblase-1 and Wiskott-Aldrich syndrome protein
linker for activation of T cells (LAT) and Src homology 2 (WASP) verprolin homologous (WAVE). Many of these
domain-containing protein of 76 kDa (SLP-76) to provide binding partners are either substrates of the Abl kinase or
binding sites for other signalling components. Syk also they are upstream kinases that phosphorylate and activate
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r49
Abl. Like the SH3 domain, the SH2 domain of Abl inter- 1. Abl located in the cytoplasm is activated by Src as-
acts with a number of signalling components, such as the sociated with protein tyrosine kinase-linked recept-
Eph receptor (Module 1: Figure Eph receptor signalling), ors such as the platelet-derived growth factor re-
Cbl, Crk-associated substrate (Cas) and RNA polymerase ceptor (PDGFR). Src phosphorylates Abl, relieving the
II. autoinhibition and thus enabling the molecule to open
The middle of the molecule contains three domains con- out to begin its signalling role in actin remodelling. Abl
taining high-mobility group (HMG)-like boxes (HLB1 can bind to both G- and F-actin, but how this facilitates
HLB3) that can bind to A/T-rich DNA regions (Module actin assembly is unclear.
1: Figure non-receptor tyrosine kinases). The C-terminal 2. Abl can also be activated by integrin receptors, where it
region contains binding sites for globular (G-) and fila- can contribute to actin assembly by forming a complex
mentous (F-) actin. Interposed between these two sites with Abelson-interactor (Abi), Wiskott-Aldrich syn-
is a C-terminal domain-interacting domain (CTD-ID) drome protein (WASP) verprolin homologous (WAVE)
that is a binding site for the CTD of mammalian RNA and the actin-related protein 2/3 complex (Arp2/3 com-
polymerase II. The region containing the HLB do- plex). Such complex formation is found at focal adhe-
mains also have a number of proline-rich motifs that en- sion complexes (Module 6: Figure integrin signalling).
able Abl to interact with SH3 domain-containing pro- 3. In addition to its function in the cytoplasm, Abl also
teins such as Abelson-interactor (Abi), Crk, growth operates within the nucleus. Its nuclear function seems
factor receptor-bound protein 2 (Grb2), Nck and pro- to depend upon its interaction with the pocket pro-
line/serine/threonine phosphatase-interacting protein 1 tein retinoblastoma susceptibility gene Rb (Module 1:
(PSTPIP1). Figure Abl signalling).
Abl can regulate a number of cellular processes, both 4. The inhibitory effect of Rb on nuclear Abl is relieved
in the cytoplasm and in the nucleus. It can move between by the phosphorylation of Rb by the cyclin D/cyclin-
these two compartments by virtue of having nuclear loc- dependent kinase 4 (CDK4) complex, which is a com-
alization signals (NLSs) and a nuclear export signal (NES) ponent of the cell cycle signalling pathway (Module 9:
(Module 1: Figure non-receptor tyrosine kinases). The sig- Figure cell cycle signalling mechanisms).
nalling function of Abl is described in Module 1: Figure 5. Nuclear Abl can be activated by a number of stress-
Abl signalling: ful stimuli, such as ionizing radiation acting through
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r50
ATM (ataxia telangiectasia mutated) or by DNA dam- to inhibit the ubiquitination of p53 (Step 6 in Module 1:
age acting through DNA-dependent protein kinase Figure Abl signalling).
(DNAPK).
6. Abl inhibition of mouse double minute-2 (MDM2) pre- Protein oxidation
vents the degradation of p53 by the ubiquitin ligase The redox signalling pathway generates reactive oxygen
mouse double minute-2 (MDM2) and this enhances species such as superoxide and hydrogen peroxide that
the transcription of the genes that induce apoptosis carry out their second messenger functions by oxidizing
(Module 4: Figure p53 function). specific thiol groups on specific cysteine residues in target
7. Abl can phosphorylate and activate RNA polymerase proteins (Module 2: Figure reversible and irreversible ROS
II, which contributes to gene expression (Module 1: oxidations).
Figure Abl signalling).
8. Abl can phosphorylate and activate Rad52, which con- Protein acetylation
tributes to DNA repair. Protein acetylation plays a particularly important role in
chromatin remodelling and is also responsible for reg-
ulating the activity of many other proteins particularly
Abl inhibition of mouse double minute-2 (MDM2)
transcription factors. The reversible acetylation of lysine
One function of Abl is to regulate the activity of the
residues on target proteins is carried out by an extensive
mouse double minute-2 (MDM2), which is a ubiquitin
protein acetylation toolkit (Module 1: Table protein acet-
ligase that targets the transcription factor p53 for degrad-
ylation toolkit).
ation (Module 4: Figure p53 function). A variety of cell
The histone acetyltransferases (HATs) function to acet-
stresses activate p53, which is at the heart of a signalling
ylate lysine residues on substrate proteins (Module 1: Fig-
network that controls the cell cycle network, senescence
ure protein acetylation). This acetylation reaction is re-
and apoptosis (Module 9: Figure proliferation signalling
versed by deacetylases such as the histone deacetylases
network). MDM2 plays a role in this network by virtue of
(HDACs) and the sirtuins. This reversible process of pro-
its ability to destabilize p53 by targeting it for degradation.
tein acetylation has a number of functions:
This MDM2 inhibitory action is alleviated by Abl, which
is activated by various stressful stimuli (Step 5 in Module Acetylation of histones, often acting in combination
1: Figure Abl signalling). One of the actions of Abl is to in- with nuclear receptors, opens up chromatin to make it
crease the transcriptional activity of p53. The proline-rich accessible to a whole variety of transcription factors and
regions of Abl bind to p53 and may act either by stabil- thus constitutes an ON mechanism. Such a role for acet-
izing the interaction of p53 with DNA or by masking the ylation is evident during the activation of the myocyte
lysine target sites that are ubiquitinated by MDM2. An- enhancer factor-2 (MEF2) (see Step 9 in Module 4: Fig-
other proposed action is for Abl to phosphorylate MDM2 ure MEF2 activation).
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r51
OH OH OH OH
HDACs
and SIRTs
O +
NH 3
HN CH 3 Deacetylation
C
C
Protein Protein
substrate substrate
Acetylation
HATs
O
SH S CH 3
Protein acetylation.
Reversible acetylation of lysine residues on proteins is carried out by histone acetylases (HATs) that add an acetate group (shown in red), which is
removed by histone deacetylases (HDACs) and the SIRTs. See text for further details,
The activity of the transcription factor PGC-1, which Despite the name, the target proteins acetylated by
functions as part of the AMPK signalling pathway, is HATs are not restricted to histones and this important
controlled by acetylation through SIRT1 and deacetyla- post-translational modification alters the activity of many
tion by GCN5 (Module 2: Figure AMPK control of different proteins often acting in tandem with protein
metabolism). phosphorylation. Histone acetylation is of particular im-
The protein CLOCK, which is a component of the portance in that it remodels chromatin to make it easier
circadian clock, is an acetyltransferase that acetylates for transcription factors to act at promoter sites to initiate
its partner BMAL1 to make the latter more susceptible gene transcription. The following are typical examples of
to the feedback inhibition by CRY (see Step 4 in Module such HATs:
6: Figure circadian clock molecular mechanisms).
p300
The movement of kinesin-1 along microtubules is en-
CREB binding protein (CBP)
hanced by the acetylation of the -tubulin subunits.
p300/CBP association factor (PCAF)
There is an important role for p53 acetylation in regulat-
General control of amino-acid synthesis (GCN5)
ing the transcriptional activity of p53 (Module 4: Figure
Tat interactive protein 60 (TIP60)
p53 function).
Acetylation of components such as histones, p53 and Histone deacetylases (HDACs)
ATM play a role in the G1 checkpoint signalling to The histone deacetylases (HDAC111) and sirtuins
DNA double-strand breaks (Module 9: Figure G1 (SIRT18) have been classified into five groups (Module 1:
checkpoint signalling). Table protein acetylation toolkit). These HDACs remove
Acetylation of ULK1 by TIP60 functions to control the acetyl group from the -amino group on specific acet-
autophagy (Module 11: Figure autophagy). ylated lysine residues of target proteins (Module 1: Figure
protein acetylation). The deacylation reaction is coupled
to the hydrolysis of nicotinamideadenine dinucleotide
Histone acetyltransferases (HATs) (NAD + ) to form nicotinamide (NAM) and O-acetyl-
The histone acetyltransferases (HATs) carry out the ADP-ribose (OAADPR) during which the acetyl group
transfer of an acetyl group from acetyl-CoA to the - is transferred from the protein substrate to the OAADPR.
amino group on specific lysine residues of target proteins Since NAD + is required for this reaction, this enzymatic
(Module 1: Figure protein acetylation). The enzyme ATP reaction is closely linked to the energy status of the cell as
citrate lyase (ACL) relates energy balance to HAT activity defined by the NAD + /NADH ratio and is thus a part of
through the control of the nuclear production of acetyl- the NAD signalling system (Module 2: Figure NAD-de-
CoA, which is the substrate for acetyltransferase activity. pendent signalling pathways).
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r52
One of the primary functions of protein deacylation by (Module 12: Figure hypertrophy signalling mechanisms).
HDACs is to regulate gene transcription. HDACs con- HDAC4 also has an important role in regulating neuronal
tribute to the epigenetic changes responsible for chromatin gene transcription (Module 10: Figure neuronal gene tran-
remodelling. In effect, they repress gene transcription by scription).
two mechanisms as exemplified by the actions of HDAC2
and HDAC4. By repressing gene transcription, these HDAC phosphorylation
HDACs constitute a typical OFF mechanism. The export of HDAC from the nucleus can be
HDAC activation functions in the control of many dif- induced by protein phosphorylation. For example,
ferent transcriptional processes: Ca2+ /calmodulin-dependent protein kinase IV (CaMKIV)
Inhibition of the HDACs, which enhances histone acet- phosphorylates Ser-259 and Ser-498 of HDAC5, which
ylation and chromatin remodelling, is necessary for then translocates out of the nucleus in association with
neuronal gene transcription (Module 10: Figure neur- 14-3-3 protein. An example of such a signalling mechan-
onal gene transcription) and has been shown to restore ism is evident during the activation of the myocyte en-
memory in a mouse neurodegeneration model. hancer factor-2 (MEF2) (Module 4: Figure MEF2 activa-
A cardiac histone deacetylase (HDAC) shuttle plays tion). Phosphorylation of HDAC4 and its removal from
an important role in the onset of cardiac hypertrophy the nucleus is also a critical event for the cardiac histone
(Module 12: Figure hypertrophy signalling mechan- deacetylase (HDAC) shuttle during the onset of cardiac
isms). hypertrophy (Module 12: Figure hypertrophy signalling
HDAC is part of the repressor complex that inhibits mechanisms).
the transcription of Wnt genes (Module 2: Figure Wnt
canonical pathway). HDAC oxidation
HDAC3 associates with the nuclear receptor The export of HDAC from the nucleus can be induced
co-repressor 1 (N-CoR1) to control the circadian clock through protein oxidation by various messengers of the
molecular mechanisms. redox signalling pathway. The HDACs have hyperreact-
ive cysteine residues that can be oxidized by reactive oxy-
HDAC2 gen species (ROS) or by reactive nitrogen species (RNS).
HDAC2 is an example of a class I HDAC that is a resident In the case of cortical neurons, nitrosylation reactions at
nuclear protein that acts to inhibit gene transcription by Cys-262 and Cys-274 inactivate HDAC2 by causing it to
deacylating histones resulting in chromosome condensa- leave the nucleus. A redox-dependent pathway may also
tion, which prevents transcription factors gaining access function to export HDAC4 from the nucleus in cardiac
to their promoter sites. Just how HDAC2 is regulated cells. The two cysteine residues (Cys-667 and Cys-669) on
is unclear. As a resident nuclear protein, the activity of HDAC4 are oxidized by reactive oxygen species (ROS)
HDAC2 may be regulated by altering its expression level. to form an intramolecular disulphide bond and the result-
In neurons, the expression of HDAC2 is activated by the ing conformational change results in the oxidized HDAC4
glucocorticoid receptor (GR) that binds to the glucocor- being exported from the nucleus.
ticoid responsive element (GRE) on the promotor region
of the Hdac2 gene. HDAC2 functions to regulate neuronal
gene transcription (Module 10: Figure neuronal gene tran- Sirtuins
scription). The sirtuins are an important family of histone
Inhibitors such as sodium butyrate and suberoylanilide deacetylases (HDACs). There are seven members of
hydroxamic acid (SAHA) can reduce the activity of the the sirtuin family that have both NAD + -dependent
class 1a HDACs. These HDAC inhibitors have been suc- deacetylase (Module 1: Table protein acetylation toolkit)
cessfully used to improve some of the symptoms of neuro- and ADP-ribosylase activity. Sirtuins deacetylation func-
degenerative diseases such as Alzheimers disease (AD), tion requires the coenzyme NAD + as a co-substrate
Parkinsons disease (PD) and amytrophic lateral sclerosis to form nicotinamide (NAM) and O-acetyl-ADP-ribose
(ALS). (OAADPR); the acetyl group is transferred from the pro-
tein substrate to the OAADPR (Module 1: Figure protein
HDAC4 acetylation). Since NAD + is required for this reaction, this
HDAC4 is an example of a classIIa HDAC that regulates enzymatic reaction is closely linked to the energy status of
gene transcription by shuttling in and out of the nucleus. the cell as defined by the NAD + /NADH ratio and is thus
It binds directly to various transcription factors to repress a part of the NAD signalling system (Module 2: Figure
their activity. In the case of the Type IIa HDACs, activa- NAD-dependent signalling pathways).
tion of gene transcription depends on HDAC inactivation, A number of environmental factors, such as the avail-
which usually occurs through its translocation out of the ability of food and cell stress, can regulate sirtuin expres-
nucleus. This export from the nucleus can be induced either sion through the activity of a large number of transcrip-
by HDAC phosphorylation by protein kinases (Module 4: tion factors (c-Myc, FOXO3, p53 and E2F1) and certain
Figure MEF2 activation) or by HDAC oxidation through miRNAs (miR-34 and miR-199).
redox signalling pathways. The cardiac histone deacetylase The activity of SIRT1 is inhibited by Deleted in breast
(HDAC) shuttle is a good example of the important role cancer 1 (DBC1) and this inhibition is removed following
that the HDACs play in regulating gene transcription activation of the AMPK signalling pathway.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r53
The sirtuins can deacylate a wide range of substrates remodelling. When such methylation reactions occur
(PGC-1, p53, FOXO, NF-B, myoD and histones), within gene promoter regions, they can activate or
which reflects their multiple roles: repress gene transcription depending on their location.
During replicative senescence, Suv39h1 methylates his-
One of its important functions is to regulate p53 acet- tones to enhance formation of heterochromatin that
ylation. will silence DNA. An abbreviation system has been de-
It has also been implicated in gene silencing, senescence, veloped to describe both the location and the nature of
apoptosis and fat metabolism. the methylation reaction. The nitrogen groups of lysine
In the case of brown fat cells, SIRT1 inhibits the activity and arginine can be modified by one or more methyl
of PGC-1 by reversing the p300-dependent acetyla- groups. The lysine can be mono- , di- or trimethyl-
tion reactions (Module 8: Figure brown fat cell differ- ated (me1, me2 and me3) whereas the arginine can be
entiation). mono- or di-methylated with the added complication
SIRT1 functions in the PER regulatory loop of the that this may be either symmetrical (me2s) or asymmet-
circadian clock. rical (me2a). In the case of histone H3, the trimethyl-
The mitochondrial sirtuins have multiple roles partic- ation of arginine at position 9 (H3K9me3) represses
ularly in the mitochondria. Sirtuin 3 (SIRT3) seems transcription whereas di- and trimethylation of Arg-4
to regulate the expression of peroxisome-proliferator- (H3K4me2 and H3K4me3) promotes transcription.
activated receptor (PPAR) coactivator-1 The H3K36me3-specific histone methyltransferase
(PGC-1). Both SIRT1 and SIRT3 can be impor- WolfHirschhorn syndrome candidate 1 (WHSC1)
ted into the mitochondria where they may regulate plays an important role in development. In embryonic
mitochondrial metabolism. SIRT3 deacylates the stem cells (ES), it interacts with cell-type-specific tran-
acetyl-CoA synthetase 2 that converts acetate into scription factors such as Nanog, Sall1 and Sall4. It also
acetyl-CoA, whereas SIRT4 ribosylates and inhibits interacts with the cardiac-specific transcription factor
the glutamate dehydrogenase that converts glutamate Nkx2. Defects in WHSC1 appear to play a role in
into -ketoglutarate. Wolf-Hirschhorn syndrome (WHS).
The activity of PGC-1 is regulated by reversible acet- The transcriptional co-repressor switch independent
ylation: it is acetylated by general control of amino-acid (SIN3), which functions in chromatin remodelling, as-
synthesis (GCN5) and is deacetylated and activated by sembles a large core complex that can contain various
SIRT1 (Module 2: Figure AMPK control of metabol- methyl transferases such as the histone H3-specific en-
ism). The energy sensor AMP-activated kinase (AMPK) zyme ERG-associated protein with SET domain (ESET)
translates changes in energy stress into altered SIRT1 and the histone 3 lysine 4 (H3K4) methyl transferase
activity by regulating the intracellular level of its co- (ALL-1).
substrate nicotinamide adenine dinucleotide (NAD + )
(see step 3 in Module 2: Figure AMPK control of meta-
Sumoylation
bolism).
Sumoylation is an example of a post-translation modi-
SIRT5 influences two proteins involved in cellular meta-
fication mechanism whereby the function of a protein is
bolism: cytochrome c and carbamoyl phosphate syn-
altered by the covalent attachment of a small ubiquitin-
thetase 1 (CPS1). The latter is the rate-limiting first step
related modifier (SUMO) (Module 1: Figure sumoylation).
of the urea cycle in that helps to clear the ammonia
The addition of SUMO induces a change in the activity, sta-
generated by amino acid metabolism.
bility or location of its target proteins. The nature of these
targets and the components responsible for the sumoyla-
Protein methylation tion reaction are summarized in Module 1: Table sumoyla-
Protein function can be modified by methylation of ar- tion toolkit. There are four human SUMO proteins: the
ginine or lysine residues by enzymes such as a family of first three are widely expressed, whereas SUMO-4 is re-
protein arginine methyltransferases (PRMTs) and Smyd-2. stricted to certain cell types (kidney, spleen and nymph
Such methylation reactions are reversed by demethylases nodes). In most cases, a single SUMO molecule is added
such as the histone lysine-specific demethylase (LSD1) that to the substrate proteins, but both SUMO-3 and SUMO-4
removes the methyl group from p53. can form SUMO chains through the formation of SUMO
Methylation is used to regulate a number of different SUMO isopeptide bonds.
proteins and cellular processes: The sumoylation reaction occurs through the follow-
ing series of discrete steps as shown in Module 1: Figure
Modifying the activity of the transcriptional regulator sumoylation:
peroxisome-proliferator-activated receptor (PPAR)
coactivator-1 (PGC-1), which controls the differen- 1. The immature SUMO proteins have a variable length
tiation of brown fat cells (Module 8: Figure brown fat (211 amino acids) C-terminal region that is attached
cell differentiation). to an invariant Gly-Gly motif. This SUMO precursor
There is a role for p53 methylation in regulating gene undergoes a maturation process that entails the pro-
transcription. teolytic cleavage of the C-terminal extension to ex-
Histone methylation of lysine and arginine residues in pose these two glycine residues. This cleavage is car-
the N-terminal tail of histone H3 has a role in chromatin ried out by the sentrin-specific proteases (SENPs)
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r54
Sumo -G-G
AMP C
C
UBA2 UBA2 C
3
ATP UBC9
2
Activation
Sumoylation
Addition of the small ubiquitin-related modifier (SUMO) to protein substrates alters their signalling functions to control many cellular processes. As
described in the text, the sumoylation reaction can be divided into a series of discrete steps: maturation, activation, conjugation and deconjugation.
(Module 1: Table sumoylation toolkit). The SUMO is lication and repair, mitochondrial fission and fusion, ion
now in a form to enter the sumoylation reaction, which channels and the axonal transport of proteins. In carrying
is a reversible protein modification that depends on out these functions, sumoylation can alter substrate pro-
the formation of an isopeptide bond between the C- tein activity in different ways. The addition of a SUMO
terminal glycine and the -amino group on a specific group can bring about a conformational change in the
lysine residue in the substrate. substrate or it can alter its interaction with other pro-
2. The first step in the sumoylation reaction is the ac- teins. In addition, the SUMO group can provide additional
tivation of SUMO by the E1 activating enzyme AOS1- binding sites to interact with other downstream effector
UBA2 which uses the energy of ATP to form a thioester proteins, which have a SUMO-interacting/binding motif
bond between the C-terminal carboxy group of SUMO (SIM/SBM). The enzymes that function in the conjugation
and a Cys-173 residue on UBA2 (Module 1: Figure process also have this SIM/SBM motif. The following ex-
sumoylation). amples illustrate how sumoylation can regulate a variety
3. The conjugation process begins with the transfer of of cell signalling components and cellular responses:
SUMO from UBA2 to the E2 conjugating enzyme
UBC9. Sumoylation causes RanGAP1 to translocate from the
4. The final conjugation step, which is carried out by vari- cytoplasm to the nucleus by interacting with the nuc-
ous SUMO E3 ligases such as the PIAS family and leoporin RanBP2, which also functions as a SUMO E3
RanBP2 (Module 1: Table sumoylation toolkit), results ligase.
in the transfer of SUMO from UBC9 to its various The stability of sarco/endo-plasmic reticulum
substrates. These substrates have consensus SUMO ac- Ca2+ -ATPase (SERCA) isoform SERCA2a in car-
ceptor sites such as KxE or KxExxpSP, where is diac cells is enhanced following sumoylation by
an aliphatic branched amino acid and x is any amino SUMO1. The proteasomal degradation of SERCA2a is
acid (Module 1: Figure sumoylation). reduced following the addition of SUMO1 to lysines
5. The same SENPs that function in maturation are re- 480 and 585. A decline in the level of SUMO1 seems to
sponsible for reversing sumoylation by removing the be responsible for the decrease in SERCA2a that occurs
SUMO groups from the substrate proteins. during congestive heart failure (CHF). SUMO-1 gene
transfer has proved to be an effective therapy in animal
Sumoylation of substrate proteins contribute to the con- models of CHF.
trol of a number of cellular processes such as protein trans- Sumoylation regulates synaptic function by controlling
location across the nucleus, gene transcription, DNA rep- the endocytosis of kainate receptors. When the subunit
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r55
E2 conjugating enzyme UBC9 Transfers SUMO from UBA2 to UBC9 (Step 3 in Module 1: Figure sumoylation)
E3 ligases There are a number of ligases that transfer SUMO from UBC9 to downstream substrates (Step
4 in Module 1: Figure sumoylation)
PIAS family Protein inhibitor of activated STAT (PIAS) family that contain the SP-RING motif
PIAS1
PIAS3
PIASx
PIASx
PIASx
RanBP2 A nuclear pore protein
MMS21 This SP-RING ligase (also known as NSE2) functions in DNA repair
SUMO substrates Characterized by having KxE or KxExxpSP SUMO-acceptor sites
RanGAP1
PML
Sp100
IB
p53
HSF1 Heat shock factor-1
SNIP1 Smad nucleus interaction protein-1
MEF2
GluR6 is Sumoylated following receptor activation, it is substrate protein. In some cases, only a single ubiquitin
internalized and degraded. is added (monoubiquitination) whereas in others there
The promyelocytic leukaemia (PML) protein is are multiple additions (polyubiquitination) with ubiquitin
Sumoylated when it functions to assemble the PML molecules being added to each other to form a chain. This
nuclear bodies (PML NBs). post-translational modification alters the conformation of
Translocation of the transcriptional co-repressor the target proteins to enable them to participate in vari-
C-terminal binding protein (CtBP) from the cytoplasm ous signalling pathways to control cellular responses. The
into the nucleus is controlled by sumoylation. cell signalling response is terminated by removal of the
The transcriptional activity of p53 may be repressed by ubiquitin groups by deubiquitinating enzymes (DUBs).
a process of p53 sumoylation. The ubiquitinproteasome system carries out the other
The half-life of axin, which is a negative regulator of the major function of protein ubiquitination. In this case, the
canonical Wnt/-catenin pathway, is prolonged follow- addition of ubiquitin to protein substrates marks them
ing sumoylation. out for degradation by the proteasome (see lower panel in
Module 1: Figure protein ubiquitination). Before the pro-
tein enters the proteasome, DUBs strip off the attached
Ubiquitination ubiquitin that are then recycled for further rounds of pro-
Ubiquitination is a protein modification mechanism that tein degradation. Such protein degradation can markedly
depends on the covalent addition of the highly conserved influence cellular responses when the substrates are com-
76-residue polypeptide protein ubiquitin to specific tar- ponents of cell signalling pathways.
get proteins (Module 1: Figure protein ubiquitination).
This process of protein ubiquitination has two import- Ubiquitin signalling system
ant functions. First, it is used in a ubiquitin signalling The ubiquitin signalling system is highly versatile in that
system whereby the reversible ubiquitination of certain it controls the activity of a number of cellular responses
cell protein substrates, which are components of cell sig- (see top panel in Module 1: Figure protein ubiquitination).
nalling pathways, functions to control a number of cellu- This signalling system is based on the reversible ubiquitin-
lar responses (see top panel in Module 1: Figure protein ation of protein substrates, which are components of cell
ubiquitination). A set of activating, conjugating and lig- signalling pathways. The following examples illustrate the
ase enzymes are responsible for adding ubiquitin to the multiple functions of this signalling system:
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r56
DUBs
Cellular responses
Substrate Gene transcription
Ubiquitin
(Foxo4, Myc)
Cell cycle control
Substrate Polyubiquitination Checkpoint regulation
DNA repair
Substrate Growth factor
endocytosis
Ubiquitin NF-B signalling
ligases Monoubiquitination
Cellular responses
Gene transcription (p53)
GPCR down-regulation
Ubiquitin Substrate Cell cycle control
Substrate
ligases Mitosis
Cell signalling (Wnt and
Proteasome Notch signalling)
DUBs
Protein
Ubiquitin degradation
ubiquitin signalling and gene transcription where the reversible ubiquitin signalling system functions
ubiquitin signalling and cell cycle regulation in cell cycle control and particularly in checkpoint sig-
ubiquitin signalling and cell signal transduction nalling:
ubiquitin signalling and endocytosis
The reversible ubiquitination of Cdc20 functions in
Ubiquitin signalling and cell signal transduction spindle-assembly checkpoint signalling (Module 9: Fig-
The reversible ubiquitination of various signalling com- ure spindle assembly checkpoint).
ponents contributes to the processing of information in A ubiquitin signalling system based on the Fanconi an-
the following cell signalling pathways: aemia/BRCA pathway functions in both DNA repair
and cell cycle control (Module 9: Figure Fanconi an-
In the tumour necrosis factor (TNF) signalling path-
emia pathway).
way, ubiquitination contributes to the formation of a
macromolecular cell signalling complex (Module 2: Fig-
ure NF-B activation). Ubiquitin signalling and endocytosis
In the Toll receptor signalling pathway, the reversible The ubiquitin signalling system plays a role in the processes
ubiquitination of TRAF6 functions to assemble a signal of endocytosis and is particularly important with regard
transducing complex (Module 2: Figure Toll receptor to the process of down-regulating various receptors.
signalling).
The Cbl down-regulation of signalling components
Ubiquitin signalling and cell cycle regulation is evident during the endocytosis of protein tyrosine
There are many cell cycle stages when ubiquitination func- kinase-linked receptors (PTKRs). For example, the
tions to regulate either progression or cell cycle arrest at epidermal growth factor receptor (EGFR) and the Met
specific checkpoints that are activated during DNA dam- receptor, are down-regulated through the action of the
age. The ubiquitinproteasome system functions during adaptor protein Cbl, which binds to the activated re-
the cyclin E control of G1 progression and DNA synthesis ceptor and initiates its degradation through a ubiquitin-
and during chromosome separation at anaphase (Module ation process (Module 1: Figure receptor down-regula-
9: Figure chromosome separation). These are examples tion).
where ubiquitin-dependent protein degradation has a sig- The receptor down-regulation of G protein-coupled
nalling function. By contrast, the following are examples receptors (GPCRs) depends upon the reversible
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r58
Ub
Ub Ub
E1
Ub
5
Ub
1 DUBs
Proteasome
E1 Ub Substrate Ub Protein
Ub Ub degradation
Ub 4
2 3
E2 E3
E2 Ub
E1
Substrate
Ub; Ubiquitin
E1; Ubiquitin-activating enzyme
E2; Ubiquitin-conjugating enzyme
E3; Ubiquitin-protein ligase
DUBs; Deubiquitinating enzymes
during the cyclin E control of G1 progression and DNA of signalling components (Module 1: Figure receptor
synthesis. This onset of DNA synthesis is also depend- down-regulation).
ent on the degradation of the cyclin E/CDK2 complex The ubiguitin ligase Nedd4-2 regulates the membrane
by SCFFbw7 . Chromosome separation at anaphase is expression of the epithelial Na+ channels (ENaC).
controlled by the degradation of Emi2 by the ubiquit- Inactivation of Notch signalling depends on the pro-
ination complex SCF (Module 9: Figure chromosome teasomal degradation of phosphorylated NICD after its
separation). ubiquitinylation by the nuclear ubiquitin ligase SEL-10
The process of chromosome separation at anaphase de- (Step 9 in Module 2: Figure Notch signalling).
pends on the anaphase-promoting complex (APC) that A number of ubiquitin ligases [Itch , Deltex , Mind
ubiquitinates and thus degrades securin to release the bomb (Mib) and Neutralized (Neur)] participate in
enzyme separase that is responsible for hydrolysing co- the modulation of Notch signalling (Module 2: Figure
hesin molecules (Module 9: Figure chromosome separ- Notch modulation).
ation). The ubiquitin ligase Cullin 3 plays a key role in the
Operation of the spindle-assembly checkpoint sig- regulation of stress-sensing transcription factor nuclear
nalling pathway depends on the ubiquitination and ac- factor erythroid 2 related factor 2 (NRF-2) (Module 4:
tivation of the Cdc20 protein (Module 9: Figure spindle Figure NRF-2 antioxidant function).
assembly checkpoint). HMG-CoA reductase degradation protein 1 (HRD1)
In the Wnt signalling pathways, phosphorylated - is an E3 ligase that carries out the hydrolysis of the
catenin is ubiquitinated by the SCF-TrCP ubiquitin lig- transcription factor ATF6 that is responsible for ER
ase and is then degraded under resting conditions (Steps stress signalling (Module 2: Figure ER stress signalling).
2 and 3 in Module 2: Figure Wnt canonical pathway).
One of the mechanisms for the down-regulation of cell Ubiquitin ligases
signalling depends upon a process of signalsome de- The E3 ubiquitin ligases play a critical role in recog-
gradation as exemplified by the Cbl down-regulation nizing protein substrates that are to be degraded by the
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r60
There are approximately 15 mammalian calpains which Dipeptidyl peptidase type 4 (DPP-4)
are divided into typical calpains (nine members) and atyp- A protease that functions in the metabolism of various
ical calpains (six members). The sensitivity to Ca2 + of hormones such as growth hormone-releasing hormone
the typical CAPNs depends on EF-hand motifs located (GHRH) and glucagon-like peptide 1 (GLP-1).
in the C-terminal region. The atypical CAPNs lack these
EF-hands and it is not clear how they respond to Ca2 + . Insulin-degrading enzyme (IDE)
Most cells constitutively express two calpains called - The insulin-degrading enzyme (IDE) is part of the amyloid
calpain and m-calpain (calpain 1 and calpain 2). The former cascade hypothesis. It is released from the microglia to
is a heterodimer formed between CAPN1 and a smaller hydrolyse amyloids to reduce their deleterious effects
CAPN4 subunit and is sensitive to Ca2 + concentrations on neuronal survival (Module 12: Figure amyloid cascade
of about 50 M. The -calpain, which is also a heterodimer hypothesis).
formed by CAPN2 and CAPN4, requires higher levels of
Matrix metalloproteinases (MMPs)
Ca2 + (about 200 M) for its activation. Ten Ca2 + ions are
The matrix metalloproteinases (MMPs) are a large fam-
bound to the penta-EF-hand domains that are distributed
ily of zinc-containing endopeptidases that cleave compon-
throughout the molecule.
ents of the extracellular matrix (ECM), growth factors (e.g.
The intrinsically unstructured protein calpastatin is
transforming growth factor ) and cell adhesion compon-
a potent inhibitor of the calpains. Calpastatin acts at
ents such as the cadherins and integrins, and they can re-
very low doses and plays an important role in protect-
lease the apoptotic ligand Fas (Module 1: Table MMPs and
ing cells when calpains are over-activated when Ca2 +
their inhibitors) . They play a particularly important role
concentrations are abnormally high, as occurs during
in remodelling the ECM during tissue development such
glutamate-induced excitotoxic cell death of neurons or
as angiogenesis.
during ischaemia/repurfusion injury in cardiac cells.
These different proteases have a multidomain organiz-
Calpains are thought to function in cell signalling by
ation that contains a number of similar domains (Module
cleaving various substrates, but these are still to be clearly
1: Figure MMP structure). The N-terminal region begins
defined. Despite the lack of information on the physiolo-
with a secretion sequence (S) followed by a prodomain,
gical role of calpains, they have been linked to several hu-
which is cleaved during the activation of the enzyme by
man diseases. Mutations in calpain 3 have been linked to
plasmin or by other members of the MMP family. There is
limb girdle muscular dystrophy type 2A. Mutations in the
a conserved catalytic domain that is connected through a
atypical calpain 10 have been linked to Type 2 diabetes.
hinge region (H) to a haemopexin-like domain, which con-
sists of four twisted -sheets to form a propeller structure
similar to that found in the serum protein haemopexin.
Caspases This haemopexin-like domain acts together with the hinge
Caspases are a family of cysteinyl aspartate-specific region to recognize substrates and to unravel their struc-
proteases, which function in the caspase cascade respons- ture so that they become accessible to the catalytic domain.
ible for apoptosis (Module 11: Figure apoptosis). Caspases The matrilysins lack the hinge region and the haemopexin-
3, 7 and 9 are potently inhibited by X-chromosome-linked like domain. The membrane-type MMPs have C-terminal
inhibitor of apoptosis protein (XIAP). specialization such as a transmembrane domain (TMD) or
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r61
a glycosylphosphatidylinositol residue that anchors them MMP-2 and MMP-9 play a critical role in the late stages
to the membrane. The gelatinases contain fibronectin do- of cancer cells, which secrete matrix proteinases that de-
mains located within the catalytic domain. grade the extracellular matrix and thus greatly enhance
The matrix metalloproteinases (MMPs) are secreted as the metastatic potential by enabling tumour cells to in-
a proMMP, and the prodomain has to be cleaved before vade other tissues.
the enzyme can function. This cleavage is carried out by Alterations in the expression of the MMPs and their inhib-
enzymes such as plasmin, by certain of soluble MMPs or itors are a major contributor to diabetic nephropathy.
by one of the membrane-type MMPs (shown in green in MMPs have been implicated in the development of
Module 1: Figure MMP activation and function), which rheumatoid arthritis and osteoarthritis.
cleave off the prodomain. The activated MMPs degrade
components of the extracellular matrix (ECM), as shown Tissue inhibitors of metalloproteinases (TIMPs)
in the figure, but can also act on other substrates such The tissue inhibitors of metalloproteinases (TIMPs)
as growth factors, cadherins and integrins. This hydro- are specific inhibitors of the matrix metalloproteinases
lytic activity is inhibited by soluble tissue inhibitors of (MMPs) (Module 1: Table MMPs and their inhibitors).
metalloproteinases (TIMPs) or by the membrane-bound Reversion-inducing cysteine-rich protein with Kazal
reversion-inducing cysteine-rich protein with Kazal mo- motifs (RECK)
tifs (RECK). The reversion-inducing cysteine-rich protein with Kazal
The MMPs have been implicated in a large number of motifs (RECK) is a membrane-anchored inhibitor of some
disease states: of the matrix metalloproteinases (MMPs) such as MMP-2,
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r62
Matrilysins
MMP-7, -26 S Prodomain Catalytic
Collagenases H
MMP-1, -8, -13, -18 Prodomain Hemopexin
S Catalytic
MMP-3, -10
Stromelysins
Gelatinases
H
MMP-2, -9 S Prodomain Catalytic Fn Fn Fn Hemopexin
Glycosylphosphatidylinositol
anchor
H
MMP-17, -25 S Prodomain Catalytic Hemopexin
PI
MMP-9 and MMP-14 (Module 1: Table MMPs and their DNA methyltransferase
inhibitors) This family of enzymes catalyses the transfer of a methyl
group from S-adenosyl methionine (SAM) to cytosine
Separase residues on DNA.
Separation of chromosome at anaphase is controlled by
DNA methyltransferase 1 (DNMT1)
separase, which is a caspase-related protease that cleaves
The primary function of DNA methyltransferase 1
the cohesin molecules that hold chromosomes together on
(DNMT1) is to maintain the DNA methylation patterns
the mitotic spindle (Module 9: Figure chromosome separ-
established by the DNMT3 de novo methyltransferases.
ation).
DNMT1 is the somatic isoform and there is an oocyte
isoform (DNMT1o), which is found in the cytoplasm of
Urokinase-type plasminogen activator (uPA)
the oocyte and then translocates to the cell nucleus during
One of the functions of urokinase-type plasminogen ac-
development.DNMT1 associates with the transcriptional
tivator (uPA) is to hydrolyse the hepatocyte growth factor
repressor methyl-CpG-binding protein 2 (MeCP2) that
(HGF) precursor.
functions in gene silencing (Module 4: Figure MeCP2 ac-
tivation).
DNA methylation
tRNA aspartic acid methyl transferase 1 (TRDMT1)
Methylation of DNA is the basis of the epigenetic mech-
This RNA cytosine methyltransferase was originally
anism responsible for gene silencing. The methyl group is
thought to be a DNA methyltransferase and was known
usually attached to the N5 position on cytosine (C) that is
as DNA methyltransferase 2 (DNMT2). Subsequently, it
linked to a guanine (G) through a phosphodiester (p) that
was found to methylate the aspartic acid tRNA.
forms the CpG dinucleotide complex. Such CpGs often
occur as clusters where they are referred to as CpG islands. DNA methyltransferase 3 (DNMT3)
CpG dinucleotides are often located in the 5 regulatory re- The DNA methyltransferase 3 (DNMT3) family consists
gions of many genes where they function in gene silencing of three members: DNMT3a, DNMT3b and DNMT3L.
once they have been methylated by DNA methyltrans- DNMT3a and DNMT3b are responsible for setting up
ferases. the DNA methylation patterns during development and
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r63
Extracellular
matrix
MMP
MMP
Pro
Timp MMP
Pro
MMP Reck
are thus known as de novo DNA methyltransferases. stimulation, this rapid desensitization/resensitization se-
DNMTL was included in the family on the basis of its quence occurs without a change in the number of sur-
homology with other DNA methyltransferases, but it face receptors. There are two types of desensitization:
lacks enzyme activity. However, it facilitates the de novo homologous desensitization and heterologous desensitiz-
methyltransferases by enhancing their DNA binding and ation.
enzyme activities.
In embryonic stem cells, DNMT 3a and DNMT 3b are
silenced by the miR-290-295 cluster (Module 8: Figure ES Homologous desensitization
cell miRNAs). In homologous desensitization, only those receptors that
are being activated undergo desensitization, which is car-
ried out by the combined actions of G protein receptor
Desensitization of cell signalling kinases (GRKs) and a family of proteins called arrestins. A
There are a number of diverse mechanisms for desens- good example of this occurs during the formation of cyclic
itizing cell signalling mechanisms. These can operate at GMP in phototransduction when rhodopsin is phos-
many levels along a signalling pathway, but most atten- phorylated by rhodopsin kinase (GRK1) (Step 2 in Module
tion has focused on receptor desensitization and receptor 10: Figure phototransduction). In the case of -adrenergic
down-regulation. receptors, the phosphorylation of the activated receptor is
carried out by -adrenergic receptor kinase 1 (ARK1).
Receptor desensitization The phosphorylated residues on the GPCRs provide bind-
Receptor desensitization has been studied in some detail in ing sites for accessory proteins called arrestins that in-
the case of G protein-coupled receptors (GPCRs), which hibit further receptor activity by preventing the receptor
often desensitize rapidly following agonist-induced activ- from binding the heterotrimeric G proteins (Module 2:
ation. The initial rapid formation of second messengers Figure heterotrimeric G protein signalling). The following
such as cyclic AMP can be reduced within minutes. After sequence of events illustrates how this GRK-arrestin regu-
removal of the agonist, the receptor can recover its former latory system operates in homologous receptor desensitiz-
sensitivity equally quickly. For short periods of agonist ation (Module 1: Figure homologous desensitization):
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r64
1 P Arrestins P
Arrestins P
P P
P P
UB
UB
GDP 5 7
2 3
GDP
GTP P
GRK 6
h
Arrestins at
GTP P
MDM2
Cl AP-2 P
4 Arrestins
P
UB
UB
Signalling ERK1/2 CSK II
pathways
8
Arrestins
UB
P
10
Internalization
a th
Recycling Cl AP
-2
P
9 Arrestins
P
UB
UB
Lysosomal
degradation
1. When a G protein coupled receptor (GPCR) detects a receptors, receptor down-regulation is usually a slower
stimulus it undergoes a conformation change that en- process that depends upon the removal of receptors from
ables it to function as a GTP exchange factor (GEF) for the cell surface by endocytosis. The activated receptors
heterotrimeric G proteins such that the GDP on the are concentrated into clathrin-coated pits, which are then
G subunit is exchanged for GTP. The active GGTP pinched off from the surface by the cytoplasmic GTPase
complex is then able to stimulate a number of signalling dynamin.
pathways (see Module 1: Table G protein-coupled re- Receptor down-regulation has been described for many
ceptors). different receptor types. The sequence of events that oc-
2. The change in the conformation of the active receptor curs for G protein-coupled receptors (GPCRs) is shown
opens up sites that are phosphorylated by one of the in Module 1: Figure homologous desensitization:
G protein receptor kinases (GRKs).
3. Arrestin then binds to this phosphorylated receptor and 5. The Steps 14 are described in the section on the re-
results in receptor desensitization in that the bound ar- versible process of receptor desensitization. Here we
restin physically prevents the receptor from activating deal with the process of receptor down-regulation
further G proteins. that begins with a protein phosphatase removing the
4. Before the arrestin can bind to the activated receptor phosphate moiety from arrestin.
it has to be phosphorylated and this is carried out by 6. The dephosphorylated arrestin is then ubiquitinated
various kinases such as ERK1/2, which is a component by the E3 ubiquitin ligase mouse double minute-2
of the ERK pathway, or casein kinase II (CSKII). (MDM2) that sets the stage for the onset of endo-
cytosis.
This process of receptor desensitization is rapid and it 7. The receptor complex enters the clathrin-coated pit by
can be reversed equally rapidly. However, if the stimulus is arrestin binding to clathrin using adaptor protein-2
particular strong or persistent, receptor down-regulation (AP2) as an adaptor protein.
occurs through a process of endocytosis as the receptors 8. The membrane and associated receptor complex are
enter clathrin-coated pits. internalized through a process driven by the GTPase
dynamin.
Receptor down-regulation 9. The endocytic vesicle can then be directed to separ-
In contrast with receptor desensitization, which is often ate pathways. In one pathway, it can enter a lyso-
fast and there is no reduction in the number of cell-surface somal degradation pathway resulting in receptor
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r65
proteolysis. Alternatively, the arrestin can be deu- GRK6 is expressed widely. It is particularly evident in
biquitinated and the arrestin is returned to the cyto- immune cells. Ablation of GRK6 or -arrestin 2 can reduce
loplasm. The receptor is also dephosphorylated. the chemotactic response of lymphocytes to CXCL12 that
10. The receptor is then recycled back to the plasma acts through CXCR4 receptors.
membrane and can once again participate in cell sig- GRK46 lack a -binding domain, but can associate
nalling. with the membrane through either PtdIns4,5P2 -binding
domains or through a palmitate lipid modification.
Receptor down-regulation is not restricted to G GRK7 is an iodopsin kinase.Levels of GRK2 and GRK6
protein-coupled receptors (GPCRs), but also applies to are low in lymphocytes from patients with rheumatoid
the protein tyrosine kinase-linked receptors (PTKRs). The arthritis.Mice that are heterozygous for GRK2, were
Cbl down-regulation of signalling components is an ex- found to be more sensitive to autosomal encephalomyel-
ample of how such PTKRs are down-regulated. itis, which is a model for multiple sclerosis.Up-regulation
of both GRK2 and GRK3 have been described in patients
Heterologous desensitization suffering from depression. One suggestion is that the in-
Heterologous desensitization occurs through a mechanism creased levels of these two GRKs may result in desensit-
that depends on the phosphorylation of G protein-coupled ization of dopamine receptors.There also is the suggestion
receptors by various kinases that are activated by other sig- that the abnormal expression of neuronal Ca2+ sensor-1
nalling pathways. A classic example is the ability of pro- (NCS-1), which has been noted in the prefrontal cor-
tein kinase A (PKA) to inactivate the 2 adrenoceptor by tex of patients with schizophrenia and bipolar dosorders,
phosphorylating a single residue in the third cytoplasmic may desensitize D2 dopamine receptors by acting through
loop. This phosphorylation does not require the receptor GRK2.
to be activated, as occurs for homologous desensitization.
It thus provides a mechanism for cross-talk between dif-
Arrestins
ferent receptor systems. Activation of one receptor, which
Arrestins are multifunctional adaptor proteins that act to-
generates cyclic AMP and activates PKA, can thus phos-
gether with the G protein receptor kinases (GRKs) to
phorylate and inactivate other receptor types.
regulate the activity of G protein-coupled receptors (GP-
CRs). There are four arrestin isoforms. Arrestin 1 and
G protein receptor kinases (GRKs)
arrestin 4 are found only in rods and cones where they
The family of G protein receptor kinases (GRKs) func-
regulate the activity of rhodopsin.The other two isoforms,
tion in the homologous desensitization of G protein-
-arrestin 1 (arrestin 2) and -arrestin 2 (arrestin 3), are
coupled receptors (Module 1: Figure homologous desens-
expressed more widely and function to regulate the activ-
itization).
ity of GPCRs. These two isoforms are fairly similar but
GRK1 is also known as rhodopsin kinase and functions there also are examples were they act specifically on certain
in the inactivation of rhodopsin (see Step 2 in Module receptors. For example, -arrestin 2 is specific for 2 ad-
10: Figure phototransduction). renoceptor whereas -arrestin 1 is specific for proteinase-
GRK2 is expressed widely in cells and normally resides activated receptor 1 (PAR1). With regard to receptor de-
in the cytoplasm. During receptor activation, it interacts sensitization, -arrestins have two main functions. First,
with the G protein subunit that puts it in position to they function in homologous desensitization by associat-
phosphorylate receptors in a number of cell types: ing with active receptors to inhibit G protein activation
GRK2 and -arrestin 1 interact to control -adrenergic (Steps 14 in Module 1: Figure homologous desensitiza-
responses in cardiac cells. tion). Secondly, they function in receptor down-regulation
The GRK2 in lymphocytes functions to control the by linking the receptor complex to AP2 and clathrin dur-
CCR5 receptor that responds to CCL4 and CCL5 ing the process of endocytosis (Steps 58 in Module 1: Fig-
(Modules 1: Figure chemokines). ure homologous desensitization). The arrestins function as
Many brain areas express GRK2 that controls the sens- clathrin-associated sorting proteins (CLASPs) by binding
itivity of the many G protein-coupled receptors that to calthrin and AP2 to guide GPCRs into the coated pits
modulate neural activity. ready for internalization (Module 4: Figure cargo sorting
signals).
GRK3, which has properties similar to GRK2, is ex- In addition to this role in receptor desensitization, ar-
pressed in airway smooth muscle cells where it modulates restins can also function as scaffolding/targeting protein
the activity of the acetylcholine-dependent contractions to assemble a variety of signalling components. This sig-
(Module 7: Figure bronchiole-arteriole contraction). nalling role is particularly evident in the way the GPCRs
GRK4 is expressed mainly in the testis, but is also found can activate signalling pathways normally associated with
in kidney tubules. Polymorphic variants of GRK4 result protein tyrosine kinase-linked receptors. For example,
in constitutive activation and this causes a decrease in Na + the arrestins can assemble components of the different
secretion and hypertension. mitogen activated protein kinase (MAPK) signalling path-
GRK5, which is expressed widely, modulates the sens- ways such as the ERK pathway and the JNK3 pathway.
itivity of acetylcholine receptors that controls the con- The omega-3 fatty acid receptor GPR120 uses arrestin-2 to
traction of airway smooth muscle cells (Module 7: Figure inhibit various inflammatory responses (Module 2: Figure
bronchiole-arteriole contraction). omega-3 fatty acids).
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r66
-Adrenergic receptor kinase 1 (ARK1) Phosphorylation of two tyrosine residues (Tyr-368 and
-Adrenergic receptor kinase 1 (ARK1), which is also Tyr-371) in the L region plays a critical role in switching
known as GRK2, is one of the G protein receptor kinases on the ubiquitination activity of Cbl.
(GRKs) that functions in receptor desensitization. 5. The displacement of Spry2 frees up the RING domain,
which then binds to the E2 conjugating enzyme.
Cbl down-regulation of signalling components 6. The E2/RING complex then ubiquitinates a variety of
One of the main functions of the adaptor protein Cbl is to residues dispersed throughout the receptor and also on
function as an E3 ubiquitin ligase with a particularly im- Spry2.
portant role in the down-regulation of many cell signalling 7. Once the receptor is ubiquitinated, it is directed to-
components. The ubiquitinproteasome system is one of wards coated pits through a process that is facilitated by
the major protein degradation pathways in cells (Module the adaptor Cbl-interacting protein of 85 kDa (CIN85).
1: Figure ubiquitinproteasome system). Cbl uses this A proline-rich sequence located close to the C-terminus
system to down-regulate a variety of signalling proteins. binds to CIN85, which functions to target receptor
The best example of this process is the Cbl-dependent complexes to the clathrin-coated vesicles by binding to
down-regulation of protein tyrosine kinase-linked recept- endophilin that associate with clathrin adaptor protein-
ors (PTKRs) such as the epidermal growth factor receptor 2 (AP2).
(EGFR), hepatocyte growth factor receptors (HGFRs), 8. The clathrin-coated pits are taken in to form vesicles
colony-stimulating factor-1 receptor (CSF-1R), neuro- which fuse to form multivesicular bodies where the
trophin and vascular endothelial growth factor (VEGF) receptors are sorted for either recycling back to the
(Module 1: Figure receptor down-regulation). However, plasma membrane or sending to the lysosome for de-
this mechanism is not restricted to PTKRs, because Cbl struction. The Cbl-dependent ubiquitination determ-
can also down-regulate other receptors (e.g. FcRI, 5 in- ines this decision by targeting the receptors for lyso-
tegrin subunit and components of the T cell receptor). Cbl somal degradation.
is also able to down-regulate many other downstream sig-
nalling components, such as various non-receptor protein
tyrosine kinases (e.g. Src, Syk, Hck, Fgr, Lyn and c-Abl), Spatial and temporal aspects of cell signalling
Bim, Sprouty 2 (SPRY2) and signal transducer and activ- Cell signalling pathways are highly organized with re-
ator of transcription 5 (STAT 5). There are some substrates gard to both space and time. With regard to the spatial
that are ubiquitinated by Cbl, but are not degraded, such aspects, the basic principle is that signalling components
as the p85 regulatory subunit of PtdIns 3-kinase, Vav, Crk- are linked together through modular proteinprotein do-
like (CrkL) and phospholipase C1 (PLC1). The descrip- mains, which are often held in close apposition using a
tion of Cbl structure and regulation reveals the existence variety of structural devices such as protein scaffolds, lipid
of numerous domains (Module 6: Figure Cbl structure) rafts and caveolae. These organized complexes of signalling
that enable it to interact with proteins to carry out its role components maximize the flow of information between
in terminating cell signalling. Its role in degradation oc- signalling components.
curs through a series of steps as illustrated by its action on This spatial organization of signalling molecules can
PTKRs (Module 1: Figure receptor down-regulation): lead to highly localized signalling events, and these are
referred to as the elementary events of signalling. They
1. The growth factor brings together two receptor sub- have been particularly well characterized for the Ca2 + sig-
units, and their tyrosine kinase regions phosphorylate nalling pathway because they can be visualized in real time.
each other to provide the phosphorylated binding sites Such localized elementary events can have a localized ac-
that draw in various signalling components (for details tion or they can be recruited to create more global signals.
see Module 1: Figure PDGFR activation ). Such globalization phenomena are not necessarily restric-
2. One of these components is the adaptor protein called ted to individual cells, but they can spread from cell to cell
growth factor receptor-bound protein 2 (Grb2). through gap junctions. Such intercellular communication
3. The cytosolic Cbl attaches to the activated receptor, the can co-ordinate the activity of cell communities.
tyrosine kinase-binding (TKB) domain attaches to one The temporal aspects of signalling concern the way in-
of the phosphorylated residues (e.g. phosphorylated formation is organized in the time domain. Many biolo-
Tyr-1045 on the EGFR), whereas the proline-rich re- gical processes are rhythmical. Of particular importance
gion (Pro) attaches to growth factor receptor-bound are the cellular oscillators that set up oscillating intra-
protein (Grb2) (Module 1: Figure receptor down-reg- cellular signals that can operate over an enormous range
ulation). of frequencies to drive many different cellular processes.
4. Once Cbl is attached, it is activated by phosphoryla- Membrane oscillators set up rapid membrane potential os-
tion by a variety of tyrosine kinases and in particular by cillations that can drive neural processing of information
non-receptor protein tyrosine kinases, such as Src, Hck, and pacemaker activity in contractile systems such as the
Fyn and Syk. These phosphorylation events enable Cbl heart and smooth muscle. Cytosolic oscillators (second to
to activate its ubiquitin ligase activity that is located in minute range) set up oscillations in intracellular Ca2 + to
the RING finger domain, which is inhibited by Sprouty control many cellular processes. An important feature of
2 (Spry2). The phosphorylation of Spry2 causes its dis- such oscillations is the way information can be encoded
placement from the RING domain to the TKB domain. and decoded depending on the frequency, amplitude or
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r67
Growth factor
1
7
Src
h
Tyr kinase
Tyr kinase
Tyr kinase
Clat
4 UB
-2
UB AP
TKB
TKB
Cla
Spry2
P UB th th
P
Cla
Endophilin
P P UB
Tyr kinase
P P P
UB
Grb2 P P P P 6 P
2
P UB
b2
P
rb
TKB
P
Gr
G
shape of the individual transients. At the other end of the 2: Figure PtdIns 3-kinase signalling) to control glycogen
temporal scale is the circadian clock, which is a transcrip- synthesis.
tional oscillator that is responsible for driving the 24 h An important feature of signalsomes is that they are
diurnal rhythm. constantly being remodelled. Phenotypic and genotypic
The organization of signalling systems in both time and remodelling of the signalsome can alter the nature of the
space is described in Module 6: Spatial and Temporal output signal (Module 1: Figure remodelling the signal-
Aspects of Signalling. some). Such remodelling of cell signalling systems can have
both beneficial and pathological consequences. An import-
Signalsome ant role for such remodelling is to maintain signalsome
The genome contains a very large repertoire of signalling stability and also to adjust the properties of the signalsome
components, from which each cell type assembles a unique to cope with changing demands on the cell. There is in-
set of components that will be referred to as a signalsome. creasing evidence that signalling systems can regulate the
During the process of differentiation at the end of devel- transcription of their own signalling components. There
opment, each specialized cell selects out those components are examples of such phenotypic remodelling being either
that are particularly suited to provide the signalsome most beneficial or pathological.
appropriate to control its unique functions. Many cells ex- The following modules deal with different aspects of
press combinations of the different signalling pathways to these cell-specific signalsomes:
provide the cell-specific signalsomes that are necessary to
regulate their particular functions. For example, skeletal Module 7: Cellular Processes describes how different
muscle (Module 7: Figure skeletal muscle E-C coupling) signalsomes function to control specific mammalian
selects out one of the Ca2 + signalling modules (i.e. mod- cell types (Module 7: Table cell inventory). The cell-
ule 4 in Module 2: Figure Ca2+ modules) to control con- specific signalsomes function to process information
traction, the cyclic AMP signalling pathway (Module 2: into a form that activates different effectors to give
Figure cyclic AMP signalling) to control glycogen break- changes in specific cellular responses such as cell growth,
down and the PtdIns 3-kinase signalling pathway (Module contraction, secretion and metabolism.
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Cell Signalling Biology Michael J. Berridge r Module 1 r Introduction 1 r68
Elevated
output signals
Normal
output signals
Reduced
output signals
Phenotypic
remodelling
Remodelled Normal Remodelled
signalsome signalsome signalsome
Remodelled Remodelled
signalsome Genotypic signalsome
remodelling
Module 8: Development describes the role of signalling cells are able to adapt to changing demands by remod-
pathways in controlling development during which dif- elling cellular signalsomes. Expression of the signalling
ferent cell types emerge. An important aspect of cell components that make up the cell-specific signalsomes
differentiation is the selection and expression of those are under constant review. The remodelling process is
signalling components that are required to control adult usually carefully orchestrated, but there are many ex-
cell functions. amples of signalsome remodelling defects that result in
Module 9: Cell Cycle and Proliferation describes the a whole range of different diseases.
control mechanisms that operate during the cell cycle to
control cell division.
Module 10: Neuronal Signalling describes the function References
of different neuronal cells, which all have very differ- Steroids
ent signalsomes to carry out their particular functions Lisurek, M. and Bernhardt, R. (2004) Modulation of aldosterone and
within the neural circuits in the brain. cortisol synthesis on the molecular level. Mol. Cell. Endocrinol.
215:149159.
Module 11: Cell Stress, Inflammatory Responses and Connell, J.M.C. and Davies, E. (2005) The new biology of aldosterone.
Cell Death describes how each cell signalsome contains J. Endocrinol. 186:120.
systems that can cope with a variety of cell stresses that
result in responses such as inflammatory responses,
Growth factors
autophagy, senescence and apoptosis. Benvenuti, S. and Comoglio, P.M. (2007) The Met receptor tyrosine
Module 12: Signalling Defects and Disease describes kinase in invasion and metastasis. J. Cell. Physiol. 213:316325.
how alterations in the signalsome result in various dis- Conover, C.A. (2008) Insulin-like growth factor-binding proteins and
bone metabolism. Am. J. Endocrinol. Metab. 294:E10E14.
ease states. A remarkable feature of cell signalling sys- Ornitz, D.M. (2000) FGFs, heparin sulphate and FGFRs: complex
tems is their plasticity. Signalsomes are not fixed in interactions essential for development. BioEssays 22:108112.
stone, but are highly plastic, and the properties of their Zachow, R. and Uzumcu, M. (2007) The hepatocyte growth factor
system as a regulator of female and male gonodal function.
output signals can be adjusted through both phenotypic J. Endocrin. 195:359371.
and genotypic remodelling of the signalsome (Module 1:
Figure remodelling the signalsome). It will be argued
Gut hormones
that the operation of the signalsome is under constant re- Kojima, M. and Kangawa, K. (2005) Ghrelin: structure and function.
view and contributes to signalsome stability. However, Physiol. Rev. 85:495522.
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