CH1131 Biomolecular

Engineering

Week 5 (Sep 08, 15)

Cellular Communication and Signaling

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 Learning Objectives

Mechanism of Intracellular Signaling by GPCR

Mechanism of Intracellular Signaling by RTK

http://www.youtube.com/watch?v=V_0EcUr_txk (GPCR)

http://www.youtube.com/watch?v=0nA2xhNiAow&feature=related (cAMP)

http://www.youtube.com/watch?v=ObrsQl-vPA4 (RTK)

http://www.youtube.com/watch?v=FkkK5lTmBYQ (Insulin and RTK)

http://www.youtube.com/watch?v=OvvXgzf58MQ&NR=1&feature=endscre
en (Protein Synthesis and RTK)

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 Intra-Cellular Signaling: learning objectives
Outline general characteristics of signal
transduction systems

Describe mechanism of G protein coupled-

receptor activated second messagers

Outline structure and function of G proteins

and their interacting receptors

Outline protein kinase cascade leading to

glucose release

Structure of Transmembrane Describe receptor tyrosine kinase activation

Receptor: Rhodopsin
and Ras-MAP kinase cascade

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 Cell Signaling: background

Cells communicate to each other to monitor

surrounding conditions and respond to stimuli
appropriately

Cell Signaling is the process in which cells work

together and coordinate their activities

Cell Signaling facilitates cell-cell communication and

allows an organism to function as a coherent system

Understanding of cell signaling can tie together

independent cellular activities, including regulation of
cell cycle and cancer development

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 Cell Signaling: basic elements
Cells communicate through
extracellular messenger (sender and
receiver cells)

Receiver cells need transmembrane

receptors

1st signaling route: cytoplasmic

domain of receptor interacts with
effector to release second messengers

2nd signaling route: cytoplasmic

domain of receptor recruits other
signaling proteins

Common outcome: activation of

downstream signaling proteins
involving kinases and phosphatases

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Components and Flow of Cell Signaling
 Cell Signaling: signal transduction pathway
Importance of kinases and
phosphatases:
- main components of signal transduction
pathway
- over 500 kinases and 100 phosphatases
in human genome
-kinase phosphorylates its substrate
- kinase can be substrate of another
kinase

Effects of phosphorylation:
- activate or inactivate an enzyme
- increase or decrease protein-protein
interactions
- activate intracellular transport of protein
- activate protein degradation
Signal Transduction Pathway

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 Cell Signaling: activation of signal transduction

Outcome of Signal Transduction

- change in gene expression

- alteration of activity of metabolic
enzymes
- reorganization of cytoskeleton
- change in cell motility
- change in ion permeability
- activation of DNA synthesis
- death of cell

Activation of Signal Transduction

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 Cell Signaling: termination of signal transduction

Signaling transduction needs to be

switched off once appropriate
response is generated

Extracellular messengers can be

destroyed by enzymes

Extracellular messengers can be

internalized together with receptors
by endocytosis

Signal transduction can be turned

off by phosphatases

Termination of Signal Transduction 8

 Cell Signaling: messengers and receptors
Extracellular Messengers
- amino acids as
neurotransmitters and
hormones
- steroids (cholesterol) as
regulators for sexual
differentiation and
carbohydrate metabolism
- proteins (when secreted) as
regulators of cell cycle, immune
response and cell death
Receptors
- G-protein coupled receptors (with 7
transmembrane helices), they translocate
messenger binding to activation of GTP-
binding proteins (G proteins)
- G proteins involved in vesicle budding, MT
dynamics and protein synthesis
- Receptor protein-tyrosine kinases (RTKs),
messenger binding leads to receptor
dimerization and activation of kinase
(cytoplasmic side of RTK)
- RTKs involved in cell cycle
- ligand-gated channels involved in nerve
impulse
- steroid hormone receptors bind to steroid
hormones (diffused) in cytoplasm and
activate transcription factor in nucleus
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 Cell Signaling: G protein-coupled receptors
Named from interaction with G proteins
Single largest protein superfamily
Natural ligands include hormones,
neurotransmitters, odorants
Consists of 7 transmembrane -helices
G proteins bind to cytoplasmic loop

Ligand binding leads to new loop

conformation
New conformation displays higher
Structure of GPCR Complex affinity to G protein, forming G protein-
coupled receptors (GPCR)
GPCR activates signaling transduction

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Cell Signaling: activation of G proteins
Ligand binding on GPCR triggers G
protein binding and replacement of
GDP by GTP on G

GTP-bound G has decreased affinity

for G leading for their release from
G

Free GTP-bound G binds to effector

For effector protein such as adenylyl

cyclase, second messenger cAMP is
generated

cAMP and other second messengers

activate more signaling proteins

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Cell Signaling: activation of G proteins
After interaction with effector, G
hydrolyzes GTP and becomes
GDP- G

GDP- G detaches from effector and

re-associates with G subunit

When in dissociated form, G

subunit can couple to distinct
effectors to activate respective
signal transduction

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Cell Signaling: termination of GPCR activation
To prevent overstimulation, receptors, G
proteins and effectors need to return to
inactive state

1st step: cytoplasmic domain of GPCR is

phosphorylated

2nd step: phosphorylated GPCR leads to

binding of arrestins

Binding of arrestins prevent further

activation of G protein

G protein inactivated by GTP hydrolysis

Arrestins bound to phosphorylated GPCR

interact with clathrin, leading to
endocytosis and recycling of receptors
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 Cell Signaling: specificity of GPCR activation
Wide variety of ligands require wide variety and yet specific cell
response through GPCR activation

Receptors have different isoforms

Different isoforms have different affinities to ligand and interact with

different G proteins

G proteins have different isoforms for each subunits, resulting in

greater variety of heterotrimeric complex

Receptors isoforms may have tissue specificities, thus the same

extracellular messenger can activate diverse pathways in different
cells

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Cell Signaling: GPCR and blood glucose level
Glucose oxidation provides energy in
ATP

Glucose can be stored in glycogen

Glycogen breakdown to glucose

controlled by hormones

Glucagon, released when blood

glucose levels are low, stimulates
glycogen phosphorylase to break
down glycogen

Insulin stimulates glycogen synthase

to convert glucose in excess to
glycogen
Glucose Storage or Utilization 15
Cell Signaling: GPCR and blood glucose level
Glucagon binds to GPCR

GPCR activates heterotrimeric G

protein

Heterotrimeric G protein activated

effector adenylyl cyclase

Adenylyl cyclase is a two-part

transmembrane enzyme

Active site in cytoplasmic part and

converts ATP to cAMP
(monophosphate)
Adenylyl Cyclase and cAMP

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Cell Signaling: GPCR and blood glucose level
cAMP binds to regulatory site
(R) of cAMP-dependent
protein kinase (PKA)
R site normally inhibits
catalytic site (C) of PKA
cAMP bound R site releases
C site and activates PKA

Activated PKA
phosphorylates and activates
phospghorylase kinase
Active phosphorylase kinase
phosphorylates and activates
glycogen phosphorylase
Active glycogen
phosphorylase removes
glucose from glycogen
Successive removal of
glucose from glycogen
increases blood glucose level
Liver Cell Response to Glucagon 17
 Cell Signaling: GPCR and PKA
Synergistically, active PKA
phosphorylates and
inactivates glycogen
synthase, thus preventing
conversion of glucose to
glycogen

Active PKA also translocates

to nucleus to phosphorylate
and activate transcription
factor (CREB)
CREB activates gene
expression of enzymes in
glucose metabolism

cAMP signal transduction is

turned off by phophatases
cAMP phosphodiesterase
destroys cAMP from cells

Liver Cell Response to Glucagon 18

 Cell Signaling: summary of GPCR
G protein-coupled receptors (with 7 transmembrane helices), they
translocate messenger binding to activation of GTP-binding proteins
(G proteins)

G proteins consist of three subunits: , and (heterotrimeric G

proteins)

Ligand binding on GPCR triggers G protein binding and replacement

of GDP by GTP on G

Free GTP-bound G binds to effector activates downstream signal

transduction

GPCR activation is reversed by inhibitor binding and receptor

recycling

G protein activation is reversed by GTP hydrolysis

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 Cell Signaling:
receptor protein-tyrosine kinases (RTKs)
Protein-tyrosine kinases phosphorylate
specific tyrosine residues on target proteins

These kinases are divided into

transmembrane receptor protein-tyrosine
kinases (RTKs) and cytoplasmic protein-
tyrosine kinases

Receptor protein-tyrosine kinases These kinases are involved in cell growth,

cell cycle and mutants lead to cancer

Receptor protein-tyrosine kinases (RTKs)

- Extracellular messenger binding to RTKs leads
to receptor dimerization
- dimerization leads to auto phosphorylation and
activation of kinase
http://www.youtube.com/watch?v=oDjDUUhGVsI
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http://www.youtube.com/watch?v=ObrsQl-vPA4
 Cell Signaling: activation of RTKs

Receptor dimerization upon ligand

binding
- ligand-mediated
- receptor-mediated

Dimerization allows trans-

autophosphorylation
- phosphorylation opens up kinase
domain
- ATP is able to enter and kinase is
active

Autophosphorylation allows binding

of cellular signaling proteins
- phosphorylation-dependent protein-
protein interaction

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Activation of RTKs
Cell Signaling: RTK-interacting proteins

SH2 Domain
- Src-homology 2 domain
- first found in oncogenic viruses
- sequence loosely conserved but
structurally well conserved
- mediate phosphorylation-dependent
protein-protein interactions

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 Cell Signaling: RTK downstream signaling
Adaptor Proteins
- function as linkers to join 2
signaling proteins in the signaling
complex
Grb2 - usually contain SH2 and SH3
domains
- SH2 binds to phosphorylated
tyrosine
Adaptor Protein in RTK Signaling - SH3 binds to proline rich domains

In Ras signaling, Grbs brings

Sos (activator of Ras) closer to
activated RTKs.

Adaptor Protein
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- Grb2
Cell Signaling: RTK signaling and virus infection
15 min 36 min

HBX

HBX contains proline-rich region HepG2

Mutant
HBX

HBX disrupts cell adhesion

Proline-rich region in HBX binds to through SH3-binding
SH3-containing cytoskeletal proteins
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Cell Signaling: RTK-activated Ras signaling
Molecular Switch of Ras Protein
Mutant Ras frequently
found in cancers
Ras is GTPase (GTP binding
and GTP hydrolysis)
Ras is attached to plasma
membrane
Molecular switch: active
GTP-bound and inactive
GDP-bound
GTP-Ras from action of
Exchange Proteins (GEF)
GDP-Ras from GTP
hydrolysis (in-built or
assisted by GTPase-
activating protein, GAP)
Constitutive mutants: active
RasV12, inactive RasN17
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 Cell Signaling:
RTK-activated Ras-MAP kinase signaling
1. Ligand binding to RTK

2. Binding triggers RTK

autophosphorylation

3. Autophosphorylation recruits
adaptor protein (Grb2), which
recruits signaling protein Sos

4. Sos, a GEF, activates Ras by

replacing GDP on Ras with
GTP

Ligand Binding and Ras Activation

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 Cell Signaling:
RTK-activated Ras-MAP kinase signaling
4. Activation of Ras by GEF

5. Activated Ras recruits Raf to

membrane, followed by its
phosphorylation and activation
(MAPKKK)

6. Activated Raf phosphorylates

and activates MEK (MAPKK)

7. Activated MEK phosphorylates

and activates ERK (MAPK)

Activation of MAP Kinase Cascade

* MAP kinase = mitogen-activated protein kinase 27

 Cell Signaling:
RTK-activated Ras-MAP kinase signaling
7. Activation of MAP Kinase (ERK)

8. Activated ERK translocates into

nucleus to phosphorylate and
activates transcription factors
(TF)

9. Activated TFs have higher DNA

binding affinity and enhance
target gene expression

10. One target gene is MAP Kinase

MAP Kinase Activation and
Transcription Activation
phosphatase (MKP-1) which is
able to remove phosphote
groups from ERK, thus
inactivates MAP kinase cascade
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 Cell Signaling: summary of RTK signaling
Protein-tyrosine kinases phosphorylate specific tyrosine residues on target
proteins

These kinases are divided into transmembrane receptor protein-tyrosine

kinases (RTKs) and cytoplasmic protein-tyrosine kinases

Extracellular messenger binding to RTKs leads to receptor dimerization and

auto phosphorylation and binding of downstream signaling proteins

Ras, one of the key signaling protein, is activated by RTK

Ras is a molecular switch, active as GTP-Ras and inactive as GDP-Ras

Activated Ras activates MAP kinase cascade resulting in transcription

activation

RTK signaling can be terminated by phosphatases and receptor recycling

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