4.03 Intracellular Traffic, Protein Sorting and Biosignaling

BIOCHEMISTRY
Intracellular Traffic, Protein Sorting and Biosginaling

4.03
PAMANTASAN NG LUNGSOD NG MAYNILA
COLLEGE OF MEDICINE 2020
DR. RIO I March 13,2017
INTRACELLULAR TRANSPORT AND PROTEIN SORTING Rough ER/membrane-bound branch

o Destined for various membranes, lysozyme and for
export via exocytosis.
BIOMEDICAL IMPORTANCE
Proteins are synthesized in polyribosomes perform
Chaperones
functions at many subcellular locations
Proteins which stabilize unfolded or partially folded
Blobel (1970): first recognized that proteins contain
intermediates allowing them time to fold properly
information (signal or coding sequence) that is important for
prevent inappropriate interaction combating formation of
them to attain their proper location (i.e. targets them
nonfunctional structures
appropriately)
Required for correct targeting of proteins to their subcellular
Certain diseases result from mutation that affect these signals
locations
Exhibit ATPase activity (binds ADP and ATP)
Signal sequences
o ADP-chaperone complex: high affinity for unfolded
Carried by proteins that direct them to their destinations
protein; when bound stimulates release of ADP with
Fundamental component of the sorting sequence
replacement by ATP
Signal sequences are usually but not always specific
o ATP-chaperone complex: releases segments that have
sequences of amino acids
folded properly
These are used to ensure that they are delivered to the
Chaperonins
appropriate membrane or cell compartment
o Form complex barrel-like structures
o Unfolded protein is sequestered away from other proteins
Targeting compound/sequence Organelle targeted giving it time and suitable conditions to fold properly
N-terminal signal peptide Endoplasmic reticulum o e.g. bacterial GroEL and heat-shock protein 60 (Hsp60)
Carboxyl-terminal KDEL (Lys- in eukaryotes
Asp-Glu-Leu) sequence in ER-
Lumen of ER
resident proteins in COP I CYTOSOLIC BRANCH
vesicles
Di-acidic sequences (e.g. Asp- Protein transport to MITOCHONDRIA
X-Glu) in membrane proteins in Golgi membranes 13 polypeptides are encoded by the mitochondrial (mt)
COP II vesicles genome and synthesized in the organelle using its own
Amino terminal sequences (20- protein synthesizing system
Mitochondrial matrix
50 residues) Matrix proteins must pass from cytosolic ribosomes through
Nuclear localization signal outer and inner mitochondrial membrane (translocation)
(NLS) (e.g. Pro -Lys -Arg-Lys- Nucleus Carry amino terminal leader sequence (20-50 AAs); contains
2 3
Val) many hydrophobic and positively charged AAs (Lys or Arg)
Peroxisomal-matrix targeting 2 translocation proteins:
Peroxisome
sequence (e.g. Ser-Lys-Leu) o TOM translocase-of-the-outer membrane
TIM translocase-of-the-inner membrane
Mannose 6-phosphate Lysosome
o
Translocation occurs posttranslationally, after matrix proteins
PROTEIN SORTING
are released from the cytosolic ribosomes.
A major sorting decision is made early in protein biosynthesis.

Note that all ribosomes have the same structure. Membrane
bound polyribosomes carry proteins that have signal
peptides, while free ribosomes do not.
Two types of ribosomes result in two branches of protein-
sorting pathways called CYTOSOLIC branch and RER
branch.
Cytosolic branch
o If protein carry specific signals, they may go to
mitochondria, nucleus or peroxisome. But if they lack Mitchondrial protein transport
signal, they remain in the cytosol.
o Preprotein: protein with targeting sequence that is
subsequently removed 1. Unfolded protein synthesized by cytosolic polyribosome and
o Preproprotein: where a second peptide is removed containing matrix-targeting sequence interacts with
cytosolic chaperone Hsp70
1D Biochemistry: Intracellular Traffic, Protein Sorting and Biosginaling (Group 2 Trans Team) 1 of 16
Bi
2. Interacts with TOM 20/22 Macromolecule export from NUCLEUS
3. Transferred to import channel TOM 40 Exportins: similar to importins; involved in export of
4. Translocation across inner mt membrane via a complex with many macromolecules
TIM 23 and 17 Cargo molecules for export carry nuclear export signals
proton-motive force across inner mt membrane is (NESs)
required for transport (electric potential or pH Karyopherins: family of importins and exportins
gradient) Translocation of mRNA molecules
Positively charged leader sequence may be helped Attached to a complex named mRNP exporter
through the membrane by the negative charge in the Ran is not involved
matrix Hydrolyzes ATP by an RNA helicase to drive
5. On the inside of inner mt membrane, it interacts with mt translocation
Hsp70 then interacts with Tim 44
6. Targeting sequence is cleaved by matrix (processing)
protease assumes its final shape (with or without Protein transport to PEROXISOME
chaperones) Its proteins are synthesized on cytosolic polyribosome and
mt Hsp70 ensures proper import into matrix and fold prior to import
prevents misfolding or aggregration Contains peroxisomal-matrix targeting sequences (PTSs)
mt Hsp60-Hsp10 system ensures proper folding o PTS 1: tripeptide located at carboxyl terminal; forms
Close apposition at contact sites between outer and complex with cytosolic receptor protein Pex5
inner membranes is necessary for traNslocation to o PTS 2: 9-AA sequence at N-terminus; forms complex
occur with Pex7
Import system can handle intact oligomers
Protein transport to NUCLEUS
Transport is bidirectional occurs through nuclear pore
complexes (NPCs)
Molecules smaller than 40 kDa pass through NPC via
diffusion
For larger molecules, special translocation mechanisms exists
Proteins carry a nuclear localization signal (NLS) e.g.
Pro2-Lys3-Arg-Lys-Val (rich is basic residues)
Peroxisomal protein transport
1. Protein assumes its folded shape prior to impot

2. Contains C-terminal peroxisomal-targetting sequence (PTS1)
3. Interacts with cytosolic protein Pex 5
4. Interacts with Pex 14 (receptor on peroxisomal membrane)
5. Protein-Pex14 complex passes though Pex2/10/12 complex
on the membrane and is translocated
6. Pex 5 is returned into cytosol
Nuclear protein transport Zellweger syndrome

Caused by mutations in genes of PEX family involved in
1. Cargo molecule interacts with nuclear localization signal import of proteins
(NLS) to form a complex Number of peroxisomes can vary from normal to being
2. Interacts with importin (I) virtually absent in patients
3. Interacts with Ran-GDP traverses nuclear pore complex Accumulation of VLC fatty acids, abnormal bile synthesis,
(NPC) reduction in plasmogens
4. In nucleoplasm, Ran-GDP is converted into Ran-GTP by Gives neurological symptoms and is the most sever when
guanine nuclear exchange factor (GEF) conformational compared with other diseases related to peroxisome like
change in Ran release of cargo molecule neonatal adrenoleukodystrophy and infantile refsum
5. I-Ran-GTP complex leaves nucleoplasm via NPC to return to disease (least severe)
cytoplasm
I is recycled due to action of GTPase accelerating
protein (GAP), which converts GTP to GDP
Ran-GTP is active form of complex; this also confers
directionality by its dissociation in cytoplasm
1D Biochemistry: Intracellular Transport, Protein Sorting and Biosignaling (Group 2 Trans Team) 2 of 16
ROUGH ENDOPLASMIC RETICULUM BRANCH Movement of Proteins inside cell (Fig 4)
o synthesis in membrane-bound
Proteins sorted via RER branch for various polyribosomes
membranes (ER membrane, Golgi apparatus
o insertion of new proteins from
membrane or plasma membrane) and lysozyme or for
exocytosis. ribosomes into ER membrane or
How does the cell know what proteins are to be lumen from the ribosomes or
processed into sites where they are needed? by transport out of ER
signal peptides (This is SIGNAL HYPOTHESIS in a anterograde transport - cargo in
nutshell) COP II vesicles
o ER to cis-GA
*Properties of Signal Peptides
o Enter Golgi cisternae as
Usually located at N terminal;
mature vesicles
approx. 12-35 AA
retrograde transport - cargo in
Region near N-terminus usually
COP I vesicles
carries positive (+) charge, making
o ER to GA
them attracted to phospholipid
segregation and sorting of
membrane (-)
proteins in trans-Golgi network (exit
AA at cleavage site variable;
side) be carried in either secretory
residues -1 and -3 relative to
or transport vesicles.
cleavage site are small and neutral
Met usually the terminal AA
Has central cluster (hydrophobic;
6-12 AA) and outer cluster
(hydrophilic)
Needed for COTRANSLATIONAL
TRANSLOCATION of secretory
proteins
Secretory/Exocytotic Pathway
rERGolgi appPMexternal envt

Two types of secretion:
o Regulated secretion - switched on Secretory or exocytotic pathway
and off when required; for
Lysosomal Pathway
exocytosis 1. Mannose 6-phosphate (lumabas sa quiz!) - target enzymes
proteins carried in
into lysozymes
secretory vesicles.
enzymes carry mannose 6-phosphate recognition
secretory vesicles are
markers
released
Lectins - mannose 6-phosphate receptor proteins.
o constitutive transport - occurs
- function in intracellular sorting of lysosomal
continuously; for use within cell enzymes into clathrin-coated vesicles in GA,
proteins carried in causing them to leave GA and fuse with
transport vesicles. prelysosomal compartment
Transport vesicles Inclusion Cell (I Cell) Disease faulty
fuse/insert into PM transport of enzymes into lysosomes
o Patients lack almost all lysosomal 5. Polypeptide is released into the lumen of endoplasmic
enzymes, causing lysosomes to reticulum.
contain inclusion bodies
o high levels of plasma lysosomal Posttranslational Translocation
enzymes
enzymes fail to reach their
proper intracellular destination,
and are secreted instead
PROTEIN TRANSLOCATION TO ER
rER synthesized proteins with N-

terminal signal peptides are usually
translocated to the lumen before
further sorting or possible secretion.
Translocation occurs more
frequently cotranslationally and
less frequently posttransationally.
Posttranslational pathway of protein translocation
For both pathways, the unfolded
state of the protein is necessary Proteins undergoing posttranslational
for the protein to be permitted into translocation are cytosolic (detached from
its conducting channel and into the ribosomes; free).
lumen. Chaperone proteins of the Hsp70 family bind
to the cytosolic protein to prevent it from
Cotranslational Translocation folding.
Protein then binds directly to the Sec61
translocon complex, and cytosolic
chaperones are released.
It then binds to the binding immunoglobulin
protein (BiP) in the lumen via its leading end.
The protein is moved forward gradually by
the energy released by the hydrolysis of BiP-
bound ATP to ADP upon interaction with
the membrane-bound Sec62/63 complex.
The resulting transient Bip-ADP complex
prevents the protein from escaping the
lumen.
The entire protein enters the lumen,
Cotranslational pathway of protein translocation releasing BiP which is then caused by the
Steps exchange of ADP for ATP.
1. The signal recognition particle (SRP) recognizes the

emerging signal sequence of the protein and binds to
it. PROTEIN FOLDING
2. The SRP then escorts its complex (ribosome-protein-
SRP) to the ER membrane to bind with the SRP After entering endoplasmic reticulum, newly synthesized
proteins fold with the assistance of chaperone proteins and
receptor. folding enzymes.
3. The ribosome binds to the translocon, causing the
release of SRP and the entry of the signal peptide into Chaperone proteins
the transmembrane channel. o Characteristics:
4. The signal sequence will open the translocon, Many are heat shock
resuming translation and the polypeptide chain to proteins (Hsp)
grow. Signal sequence is then cleaved by a signal prominently bind to
peptidase. hydrophobic regions of
unfolded proteins and ER-ASSOCIATED DEGRADATION (ERAD)
prevent their aggregation.
associated ATPase activity
is seen in their interaction
with unfolded proteins
o Calnexin - calcium-binding ER
membrane protein that binds MHC
antigens and monoglucosylated
glycoproteins, retaining them in ER
until glycoprotein has folded
correctly
o Calreticulin similar to calnexin but
not membrane-bound
Folding enzymes
o protein disulfide isomerase (PDI) -
promotes rapid formation and ER-Associated degradation (ERAD)
reshuffling of disulfide bonds of
proteins until the correct set for Misfolded proteins are disposed through ER-
folding is achieved associated degradation (Figure 7).
o peptidyl prolyl isomerase (PPI) - Selective retrotranslocation of misfolded luminal
accelerates folding of proline- and membrane proteins and ubiquitination
containing proteins by catalyzing happen to target them to proteasomes.
cis-trans isomerization of amino Energy partly supplied by p67, which is an AAA-
acid-proline bonds. ATPase
Possible transmembrane channels - Sec61,
Problems may arise in the proper folding of protein derlin and ERAD e3 ligas, Hrd1 and Doa10, (but
o Misfolded proteins prevent chaperone more evidence still needed to support ths claim)
proteins from being exported to intended
destination UBIQUITINATION
ER stress accumulation of errors; prolonged
interaction of misfolded proteins with chaperone
proteins; activates cell death pathways
CELL RESPONDS through Unfolded Protein
Response
Unfolded Protein Response - transmembrane ER
stress sensors sense levels of misfolded proteins and
initiate signal mechanisms:
o transient inhibition of translation
o production of more chaperones
o increased translation of proteins that
degrade misfolded proteins.
Ubiquitination
Ubiquitin - highly conserved, 76 amino-acid protruding extracytoplasmically; IV-multipass membrane
peptide used for tagging proteins for protein
destruction in proteasomes.
Cotranslational Insertion
Two types of protein degradation: 1.) ATP-
requiring (lumabas sa quiz) ubiquitination
(major pathway);and 2.) non-ATP dependent
lysosomal protease
Steps in ubiquitination (Figure 8)
o the C-terminal COO group of
ubiquitin is linked via thioester
bond to the SH group of activating
enzyme E1.
o This activated ubiquitin is
transferred to an SH group of the
conjugating enzyme E2.
o Ligase (E3) then catalyzes the
transfer of ubiquitin from E2 to Cotranslational insertion
epsilon-amino group of the target
protein. Cycles are repeated to Proteins (e.g. LDL receptors-lumabas sa
build up the polyubiquitin chain. quiz) enter ER membrane similar to
o APAT DAPAT! Minimum of four U secretory proteins (Figure 5)
molecules to tag protein for Unlike secretory proteins which are
destruction incorporated into the lumen, these receptors
o Deubiquitinating enzymes in the are retained in the membrane and become
proteasome to cleave U from the transmembrane. Through what?
target protein o highly hydrophobic halt-or stop-
transfer signal.
INCORPORATION OF PROTEINS INTO MEMBRANES The receptor to be inserted most likely exits
the translocon by a lateral gate (Figure 10).
Variations in the way proteins are inserted
Posttranslational Translocation
into membranes are caused by the different
routes of protein insertion. Similar to receptors undergoing cotranslational
four transmembrane protein types insertion, cytosolic proteins (e.g. cytochrome b5) undergoing
Routes of protein insertion these type of insertion enter ER membrane through a lateral
o cotranslational insertion gate.
o posttranslational insertion
Retention in GA followed by retrieval to ER
o retention in the Golgi apparatus
followed by retrieval to the proteins w/ KDEL (Lys-Asp-Glu-Leu) or
endoplasmic reticulum HDEL (His-Asp-Glu-Leu) sequence at their
o retrograde transport from the Golgi carboxyl terminal
apparatus. o They travel first through
anterograde vesicular transport
from ER to GA via vesicles with
coat protein II (COPII)
o In GA, they interact and transiently
stay with KDEL receptor
o They travel back to ER through
retrograde vesicular transport in
vesicles coated with COPI and then
dissociate.
Transmembrane protein types. Type I-amino head in

extracytoplasmic face; II-carboxylic head in extracytoplasmic
face; III-Type I but shorter because no signal sequence is
Retrograde transport from GA 2 Bud Coat proteins bind to Sar1-GTP. Cargo
Formation proteins become enclosed inside vesicles.
Other proteins (e.g. vesicular components to 3 Pinching off Bud pinches off, forming a completely
be recycled) with non-KDEL-containing coated vesicle. Vesicles move along the
proteins; often w/ C-terminal signal rich in cell through microtubules/actin
filaments.
basic residues.
4 Uncoating The vesicle is uncoated when bound GTP
o Same as in retention in GA
is hydrolyzed to GDP by Sar1
followed by retrieval in ER but 5 Targeting Rab molecules are attached to vesicles
WITHOUT the transient stay in and after switching of Rab-GDP to Rab-GTP.
GA due to interaction with KDEL Tethering A specific GEF Rab effector protein on
receptor target membrane bind to Rab-GTP,
tethering vesicles to target membrane.
TRANSPORT VESICLES 6 Docking v-SNARES pair with cognate t-SNARES
Transport vesicles contain proteins destined for GA or PM. in target membrane to form a four-helix
Non clathrin-containing bundles which docks vesicles and
*Clathrin initiates fusion.
-used in vesicles for exocytosis 7 Fusion When v- and t-SNARES are closely
-consists of three interlocking spirals, w/c interact to aligned, vesicle fuses with membrane and
form a lattice around the vesicle contents are released. GTP is then
- clathrin-free - COPI and II, transport and secretory hydrolyzed to GDP, and the RAB-GDP
vesicles carrying cargo from GA to PM molecules are released into cytosol. An
ATPase (NSF) and -SNAP dissociate
*Steps common to all vesicles: the four-helix bundle so that v- and t-
1. Budding SNARES can be reused.
2. Tethering 8 Recycling Rab and SNARE proteins are taken back
3. Docking to ER to be recycled.
4. Membrane fusion
MEMBRANE ASSEMBLY
*molecular switches
1. Sar formation of COPII vesicles The vesicles that were formed in the previous step will fuse
2. ARF - formation of COPI and clathrin-coated vesicles with the target membrane.
3. Rab inner portion of the vesicle - extracytosolic
side of PM
outer cytoplasmic side of fused vesicle-
remains as the cytoplasmic side of PM
Model of Transport Vesicles: anterograde transport of COPII

vesicles
Steps of anterograde transport involving COPII

vesicles
Take note of GTP and GDP, when hydrolysis
happens or simple exchanged/switching is
used. Figure 12 . Fusion of a vesicle with PM preserves orientation of
1 Initiation Sar1 is activated when GDP is any integral proteins embedded in the vesicle bilayer
exchanged for GTP via Sec12p. Sar1-
GTP is then embedded in ER membrane
as the focal point for bud formation.
SIGNAL TRANSDUCTION
2. Formation of second messengers via effector proteins
OVERVIEW Effector Proteins signaling proteins distal to but
activated by the agonist-bound receptor
Communication is a basic need in biological processes,
specifically from the induction of embryonic development to o Examples (in G protein mediated pathway)
the integration of physiological responses to environmental - Adenylyl cyclase
changes - Phospholipase
Complex mechanisms are involved to control which signals - Phosphodiesterase
are to be released at what time and what responses should - Ion channels
be elicited as an interpretation of the signal Second Messengers
This communication between cells requires mainly o Generated upon receptor activation and they are
extracellular signal molecules that may either travel over amplified and diffused away from their source to
long or short distances. spread the signal to other parts of the cell
These signals then bind to receptor proteins, which may be o Pass the signal on and bind to other signaling
found on the cell surface or intracellularly. proteins or effector proteins.
The binding activates the receptor and consequently activates o Confer specificity and diversity
one or more molecules (intracellular signaling molecules). The - Same signaling molecule different
processed signals are distributed to effector proteins which response
implement the appropriate change of cell behavior. o Two Types:
This cascade of events involving the recognition of a signal, - Water-soluble (cyclic AMP and Ca2+) diffuse
transmission of the signal and activation of intracellular in the cytosol
events, and response, is called signal transduction. - Lipid soluble (diacylglycerol) diffuse in the
plasma membrane
Major Steps:
1. Recognition of the ligand by the receptor
4 types of ligands or extracellular signaling molecules
o Amines: epinephrine
o Peptides and proteins: angiotensin II and insulin
o Steroids: aldosterone, estrogens, and retinoic
acid
o Small molecules: amino acids, nucleotides, ions,
and gases
Ligands may be agonists or antagonists.
o Agonist - a chemical messenger that binds to a
receptor and elicits a response
o Antagonist - competes for a receptor with a
chemical messenger that is normally present in
the body; deactivates the signal transduction
Ligands may be physiological (naturally occurring) or Second messengers confer specifity.
pharmacological (synthetic molecule)
Two types of receptors:
o Cell surface receptors (for peptides and amines) 3. Transmission of the signal to the appropriate
o Intracellular receptors (for steroid and thyroid intracellular signaling proteins
hormones) help relay the signal into the cell by generating small
intracellular mediators or activating the next effector
protein in the pathway
Scaffold proteins
o bind together groups of interacting signaling
proteins into a complex
o close proximity of the signaling proteins results to
interaction of the components at high local
concentrations and to facilitate fast, efficient and
selective activation
o avoids unwanted cross-talk with other signaling
pathways
Types of cell receptors
Altering the receptor
Features of signal transduction pathway

1. Specificity signal molecule fits binding site on its
complementary receptors; other signals dont fit.
2. Amplification when enzymes activate other enzymes,
the number of affected molecules increase in a
geometrical cascade
Scaffold proteins 3. Desensitization/Adaptation Receptor activation
triggers a feedback circuit that shuts off receptor or
Anchoring proteins removes it from the cell surface
o tether signaling proteins at particular subcellular 4. Integration - When two signals have opposite effects on
locations metabolic characteristic, the regulatory outcome results
from the integrated input from both receptors.
4. Modulation of the effector via phosphorylation and COMMUNICATION OF THE CELL FROM THE CELL
GTP binding MEMBRANE TO THE OUTSIDE ENVIRONMENT
Phosphorylation
o Protein Kinase Cell Interaction with Extracellular Matrix
o Protein Phosphatase
GTP Binding Integrins are adhesion receptors mediating the interaction
of the cells cytoskeleton to the extracellular matrix
especially its protein components such collagen, elastin,
fibronectin and laminin.
It is also responsible for cell to cell adhesion
The activated integrin allows the interaction of the cell
cytoskeleton with protein found in ECM.
This interaction enters the core of the cell where
phosphorylation may be used to provide signal from
inside the cell out into the ECM.
Intracellular Signal Transduction
Signaling molecules can act over long or short distances and

Molecular switches can require cell-to-cell contact or very close cellular proximity.
5. Cell response Contact-Independent Signaling

changes in the permeability, transport properties or o involves release of secreted molecules from the
electrical states of the plasma membrane sender cells. The secreted molecules diffuse
change in cell's metabolism and secretory and through extracellular space to the target cells where
contractile activity they interact with receptor proteins resulting in an
alteration of rate of proliferation, differentiation and activation of intracellular signaling and altered
gene expression function. This permits intercellular communication
between cells separated by large distance.
6. Termination of response
altering the receptor Paracrine Signaling
inactivating intracellular signaling proteins o Molecules that are released and act locally.
Paracrine signals are released by one type of cell
o Phosphatases
and act on another type; they are usually taken up
o feedback mechanisms
by target cells or rapidly degraded within minutes by
enzymes.
o Paracrine signaling occurs on the immediate or the function of the cell. Important elements of this
local environment thus they are also called Local signaling include a variety of proteins, such as
Mediators. integrins, involved in cell to cell adhesion and
o They are diverse type of signaling molecules like regulation of cell shape and motility
histamine, ATP and adenosine through passive
diffusion.
Autocrine signaling
o Involves the release of a molecule that affects the
same cell or other cells of the same type.
o It is important in tissue growth, organ development,
immune and inflammatory response.
Synaptic signaling
o Occurs in very short distances when neurons
transmit electrical signals along their axon and
release neurotransmitters at synapses that affect the
function of other neurons or cells that are distant
from the neuron cell body.
o The physical relationship between the nerve
terminal and the target cell ensures that the
neurotransmitter is delivered to a specific cell.
Endocrine signals are hormones that are secreted
into the blood and are widely dispersed in the body.
o This requires high affinity and selectivity of cell
receptors because hormones cannot easily
dissociate
o Contact-Independent Signaling Endocrine signals
are hormones that are secreted into the blood and
are widely dispersed in the body.
dissociate
Endocrine signals
o Hormones that are secreted into the blood and are
widely dispersed in the body.
dissociate
Contact-dependent signaling COMMUNICATION WITHIN THE CELL FROM THE PLASMA

MEMBRANE TO THE INTRACELLULAR COMPARTMENT
Cell-to-cell communication can occur via the gap
junctions that form between adjacent cells and via
interaction of proteins expressed on the surface of the
sender cell with a receptor protein expressed on the
surface of the target cell.
o Gap junctions are specialized junctions that allow
intracellular signaling molecules to diffuse from the
cytoplasm of one cell to an adjacent cell. The
permeability of gap junctions is regulated by
cytosolic calcium, hydrogen ions and cAMP and by
the membrane potential. Gap junctions also allow
cells to be electrically coupled, which is vitally
important for the coordinated activity of cardiac and
smooth muscles.
o Interaction of proteins expressed on the surface of Intracellular Receptors
the sender cell with a receptor protein expressed on Receptors located inside the cell rather than on its cell
the surface of the target cell. This interaction membrane. The ligands that bind to them are usually
changes the conformation of the receptor protein intracellular second messengers like inositol
and triggers a cascade of intracellular signaling trisphosphate and extracellular lipophilic hormones like
events in the target cell that results to altered steroid hormones.
Cell Surface Receptors Hence, a catalytic receptor is an integral membrane
protein possessing both enzymatic, catalytic and receptor
Receptors that are embedded in the membranes of cells. cations.
They act in cell signaling by receiving (binding Its most distinguishable characteristics are:
to) extracellular molecules. They are specialized integral o Intracellular domains have protein kinase, protein
membrane proteins that allow communication between phosphatase, protease or nucleotide
the cell and the extracellular space. phosphodiesterase activity
The extracellular molecules may o Regulate long-term cell functions on a time scale of
be hormones, neurotransmitters, cytokines, growth minutes to hours
factors, cell adhesion molecules, or nutrients. o Antagonist are usually large, secreted proteins that
They react with the receptor to induce changes in function as paracrine/endocrine growth or
the metabolism and activity of a cell. In the process differentiation factor.
of signal transduction, ligand binding affects a cascading
chemical change through the cell membrane. Enzyme-linked receptors based on activity
a. Receptor Tyrosine Kinase (RTK) the most
common receptor; receptors for epidermal growth
Ligand-gated Ion Channel Receptor factor (EGF), platelet derived growth factor (PDGF),
insulin and other polypeptide growth factor
b. Serine/Threonine Kinase - e.g. TGF- mutation
Groups of transmembrane ion channel proteins which open
to allow ions such as sodium, potassium, calcium and involved in some familial colon cancer cases
c. Guanylate Cyclase Activity - e.g. Atrial Natriuretic
chloride to pass through the membrane in response to the
binding of a chemical messenger, such as Factor (ANF); involved in blood pressure regulation
Receptor Tyrosine Kinase (RTK)
a neurotransmitter.
Neurotransmitters
o Chemical messengers
o Enable neurotransmission Single pass transmembrane proteins
o They transmit signals across a chemical synapse, Bind polypeptide ligands that induce oligomerization
such as a neuromuscular junction, from one neuron to Play important role in cellular growth, differentiation,
another "target" neuron, muscle cell, or gland cell. metabolism, and motility
Many neurotransmitters are synthesized from simple Includes:
and plentiful precursors such as amino acids, which o epidermal growth factor receptor (EGFR)
are readily available from the diet and only require a
small number of biosynthetic steps for conversion.
o A neurotransmitter can influence the function of a
neuron through a remarkable number of mechanisms.
In its direct actions in influencing a neurons electrical
excitability, however, a neurotransmitter acts in only
one of two ways: excitatory or inhibitory.
Excitatory Neurotransmitters
Influences trans-membrane ion flow to increase the
probability that the cell with which it comes in contact will
produce an action potential.
Acetylcholine, glutamate, ATP and serotonin
Inhibitory Neurotransmitters
Influences trans-membrane ion flow to decrease the
probability that the cell with which it comes in contact will
produce an action potential.
Gamma-aminobutyric acid (GABA) and glycine.
Effect of permeating ions on the cells

Cations depolarizes the cell
Anions hyperpolarizes the cell
Enzyme-linked Receptors (Catalytic Receptors)
Where the binding of an

extracellular ligand causes enzymatic activity on the
intracellular side.
o platelet-derived growth factor receptors
o fibroblast growth factor receptors (FGFRs)
o vascular endothelial growth factor receptors
o Met (hepatocyte growth factor/scatter factor
[HGF/SF] receptor)
o Ephs (ephrin receptors)
o insulin receptor
Structure:
1. Extracellular binding domain
2. Single transmembrane helix
3. Cytoplasmic region containing the enzyme domain
(tyrosine kinase)
Pathway:
1. Binding of ligand to both monomers causes
dimerization (producing cross-linked dimers) Mutation of RTKs
2. Tyrosine kinase activation via cross-phosphorylation
(beta subunit of one phosphorylates the other) Usually causes cancer/uncontrolled cell growth since
3. Activated tyrosine kinases act as attachments for RTKs are mostly associated with growth factors
relay proteins containing SH2 domain.
a. ErbB1 = bladder, breast, kidney, non-small-cell lung
Insulin Receptor a. Cetuximab = antibody preventing ligand-
A2 B2 heterotetramer (instead of a single-chain induced activation of EGF-binding
receptor) b. ErbB2 = codes for HER2
Pathway: a. HER2 usually complexes with other HERs
1. Insulin binds to two alpha subunits for effect, but can also have an effect by
2. Two alpha subunits (from two insulin receptor dimers) itself
closes in to each other b. Trastuzumab = binds to extracellular
3. Two beta subunits will also approach each other domain of HER2 receptor ; results to arrest
4. Beta subunits will cross-phosphorylate, activating the during G1 phase
insulin receptor c. ErbB3 = breast, colon, prostate, stomach
5. Phosphorylated tyrosine residue acts as attachment d. ErbB4 = ovarian granulosa
for Insulin Receptor Substrate-1 (IRS-1)
6. Phospohrylated IRS-1 acts as attachment for
Phosphoinositide 3-kinase (PI 3-kinase) ***Fourth type: Tyrosine Associated Receptors
7. PI 3-kinase migrates to cell membrane and
phosphorylates the membrane lipid PIP2 to PIP3 Do not have intrinsic kinase activity
8. PIP3 activates PIP3-dependent protein kinase (PDK-1) Need to associate with those that have kinase activity
(*not in diagram) (Src family and Janus family (JAK))
9. PDK-1 activates protein kinase b (Akt) Undergo JAK (Janus Kinase) STAT (signal
10. The non-membrane bound Akt freely migrates to: transducer and activator of transcription) signaling
a. Activate glycogen synthase pathway
b. Recruit glucose transporters to membrane Ligands: cytokines (interferons and interleukins),
erythropoietin
Pathway:
1. Ligand binding
2. Receptor dimerization activates JAK phosphorylation
of receptor
3. STAT attaches to receptor
4. JAK once again phosphorylates, this time the STATs
5. Phosphorylated STATs dimerizes
6. STAT dimer travels to nucleus for effect
Drugs/Toxins:
G-PROTEIN COUPLED RECEPTORS
1. Cholera toxin
membrane receptors that bind ligands that are light a. From Vibrio cholera
sensitive compounds, hormones, pheromones, b. Keeps Gas (s) GTP-bound
neurotransmitters, etc c. Increases cAMP levels
d. Increases Cl, Na, H2O efflux in intestinal
Components: lumen
1. Extracellular Ligand Receptor 2. Pertussis toxin
2. Alpha helix that crosses the membrane 7 times a. From Bordetella pertussis
3. G Protein b. Keeps Gai (i) ADP-ribosylated
4. *GTP/GDP c. Increases cAMP levels
d. Increases mucus secretion
G-Protein Sub-units
1. Beta Sub-unit SECOND MESSENGERS
2. Gamma Sub-unit Small intracellular mediators that are generated
3. Alpha Sub-unit in large numbers as a response to receptor
a. As (s) = activates adenylyl cyclase activation
b. Ai (i) = inhibits adenylyl cyclase Spreads signals to other parts of the cell by
c. Aq (q) = activates phospholipase C diffusing away from the source by binding to or
d. At (t) = activates phosphodiesterase altering the conformation of selected signaling
and/or effector proteins
Cyclic AMP (cAMP)
Pathway: It is a secondary messenger derived from ATP
1. Ligand binding to GPCR catalyzed by the enzyme adenylyl cyclase
2. GPCR undergoes conformational change It is produced when membrane receptors like G-
3. Alpha subunit (with GDP) will exchange GDP for GTP protein coupled receptors are activated.
with the help of Guanine Nucleotide Exchange Factor Its production is also inhibited by by agonists of
(GEF) adenylate cyclase inhibitory Gi protein coupled
4. Alpha subunit (now a monomer) will dissociate from receptors which activate phosphodiesterase
the beta and gamma subunit (now a dimer) which degrades cAMP.
5. Alpha subunit with the attached GTP will proceed to cAMP relays the signals from the GPCR to
activate a specific enzyme/protein (commonly protein kinase A, which is a cAMP dependent
adenylyl cyclase, phosphodiesterase, or enzyme that phosphorylates and activates
phospholipase C) enzymes like glycogen phosphorylase and
6. ***will continue until GDP is hydrolysed to GDP via G transcription factors that regulate gene
protein GTPase and GTPase-accelarating proteins expression.
(GAPs) -> will result to alpha subunit associating
again with beta and gamma subunit
The -adrenergic pathway is a cAMP dependent
pathway ( as shown above) and here is a
summary of steps of the pathway:
(Lifted from 1D 2017 trans)
1. The binding of epinephrine to a site on
the receptor deep within the membrane
i. Inactive Gs dissociates
from adenylyl cyclase,
rendering the cyclase
inactive
ii. Gs reassociates with the
and subunits
iii. Gs is again available to
interact with a hormone-
bound receptor
5. cAMP activates Protein Kinase A (PKA)
a. catalyzes the phosphorylation of
inactive phosphorylase kinase b
yields the active form
6. Phosphorylation of cellular proteins by PKA
causes the cellular response to epinephrine
7. cAMP is degraded, reversing the activation
of PKA.
a. cAMP has a short half life in this
pathway
b. It is quickly degraded by
a. Promotes a conformational change
phosphodiesterase
in the receptors intracellular
domain that affects its interaction
Some hormones act by inhibiting adenylyl cyclase,
with the second protein in the
lowering cAMP levels and suppressing protein
signal-transduction pathway, a
phosphorylation.
heterotrimeric GTP-binding
o The binding of somatostatin to its receptor
stimulatory protein, on the cytosolic
leads to an activation of an inhibitory G
side of the plasma membrane
protein (Gi) counterbalances the effects of
b. When GTP is boind to Gs, Gs
glucagon
stimulates the production of cAMP
o In adipose tissue, prostaglandin E1 (PGE1)
by adenylyl cyclase in the plasma
inhibits adenylyl cyclase, thus lowering
membrane.
[cAMP] and slowing the mobilization of lipid
i. The function of Gs as a
reserved triggered by epinephrine and
molecular switch
glucagon.
resembles Ras receptor
Olfactory transduction is an example of a cAMP
(another class of G
dependent pathway
proteins)
c. Gs ( subunit) occupied by GTP= o The odorant molecule is the odorant
Gs active molecule, which will stimulate the production
of cAMP
i. Can activate adenylyl
o Which will activate different ion channels on
cyclase
d. Gs with GDP bound= GS inactive the membrane
2. Binding of epinephrine enables the receptor o Which will depolarize the olfactory cell that
will create an action potential that will be
to catalyze the displacement of bound GDP
transmitted to the brain.
to GTP converting Gs to its active form
a. As this occurs, the and subunits
of Gs dissociate from subunit.
3. Gsa, with its bound GTP, moves in the plane
of the membrane from the receptor to a
nearby molecule of adenylyl cyclase.
a. Gs is held to the membrane by a
covalently attached palmitoyl group
4. Adenylyl cyclase catalyzes the synthesis of
cAMP from ATP: The association of active
Gs with adenylyl cyclase stimulates the
cyclase to catalyse cAMP synthesis
cytosolic cAMP
a. This stimulation by Gs is self- Cyclic GMP (cGMP)
limiting It is a secondary messenger derived from GTP and it
b. Gs is GTPase that turns itself off acts much like cAMP in which that It activates
by converting its bound GRP to different protein kinases in response to binding of
GDP peptide hormones to the external cell surface.
Guanylate cyclase synthesizes cGMP from GTP.
Guanylyl cyclase has two isoforms: Two intracellular second messengers produced
in the hormone sensitive phosphadityl inositol
1. Membrane-spanning guanylyl system.
cyclases (Activated by their Activation of Gq activates the PIP2-specific PLC
extracellular ligands) (Phospholipase C), which catalyzes the production
of IP3 and DAG from the membrane phospholipid,
Phosphatidylinositol 4,5-biphosphate, or PIP2
a. Atrial Natriuretic Factor (ANF)
- Receptors found in cells of renal
collecting ducts and smooth muscle
Both contribute to the activation of protein kinase C
of blood vessels
+ (PKC)
o increased renal excretion of Na and, o Inositol triphosphate a water soluble
Consequently, of water, driven by the compound, diffuses from the plasma
change in osmotic pressure membrane to the ER, where it binds to
o causes relaxation (vasodilation) of the blood specific IP3-gated Ca2+ channels, causing
vessel (blood flow; blood pressure) them to open and increase cytosolic Ca2+
b. Guanylin 2+
o Receptors in intestinal epithelial cells o Diacylglycerol cooperates with Ca in
- activating PKC
(Regulates Cl secretion in the intestine)
Protein Kinase C phosphorylates
o Target of a type of bacterial endotoxin
different cellular proteins which produce
(produced by E.coli) that triggers diarrhea
the cellular response to the hormone
-
(Cl secretion reabsorption of water)
2. Soluble NO-activated guanylyl cyclase
o Found in many tissues (smooth
muscle of the heart and blood
vessels)
2+
o stimulates ion pumps that expel Ca from
the cytosol
reduces forcefulness of contraction
o Nitric oxide (NO) cross plasma membrane
w/o carrier
The visual photo transduction pathway is mediated by
cGMP.
1. Light stimulation of rhodopsin leads to
activation of G-protein, transducing
2. Activated G-protein activates cGMP
PDE
3. PDE hydrolyzes cGMP reducing its
concentration
4. This leads to the closure of Na channels
that will lead to the depolarization of the
cell which will create an action potential
that will be propagated to the brain.
Inositol 1,4,5-triphosphate (IP3) and Diacylglycerol (DAG)
Calcium (Ca2+)
One of the major secondary messenger
- Related to IP3 and DAG as second messenger
- It also binds to Calmodulin, a protein mediator of many
2+
Ca -stimulated enzymatic reactions
2+
o When Calmodulin binds to Ca , it undergoes a
change in conformation, and activates the
2+
Ca /calmodulin-dependent protein
kinases (CaM kinases) phosphorylates
target enzymes, regulating their activities.
REFERENCES
2017 1D Trans
2019 1A Trans
Alberts B, Johnson A, Lewis J, et al.
Molecular Biology of the Cell. 4th edition. New
York: Garland Science; 2002

4.03 Intracellular Traffic, Protein Sorting and Biosignaling

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4.03 Intracellular Traffic, Protein Sorting and Biosignaling

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BIOCHEMISTRY

Intracellular Traffic, Protein Sorting and Biosginaling

INTRACELLULAR TRANSPORT AND PROTEIN SORTING Rough ER/membrane-bound branch

A major sorting decision is made early in protein biosynthesis.

Peroxisomal protein transport

1. Protein assumes its folded shape prior to impot

Nuclear protein transport Zellweger syndrome

rERGolgi appPMexternal envt

rER synthesized proteins with N-

1. The signal recognition particle (SRP) recognizes the

Transmembrane protein types. Type I-amino head in

Model of Transport Vesicles: anterograde transport of COPII

Steps of anterograde transport involving COPII

Types of cell receptors

Altering the receptor

Features of signal transduction pathway

Intracellular Signal Transduction

Signaling molecules can act over long or short distances and

5. Cell response Contact-Independent Signaling

Contact-dependent signaling COMMUNICATION WITHIN THE CELL FROM THE PLASMA

Effect of permeating ions on the cells

Enzyme-linked Receptors (Catalytic Receptors)

Where the binding of an

Inositol 1,4,5-triphosphate (IP3) and Diacylglycerol (DAG)

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