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Chapter 15: Signal transduction
Know the terminology:
Enzyme-linked receptor
IP3+DAG
G-protein linked receptor
cAMP
nuclear hormone receptor
Ca2+
G-protein
adaptor protein
scaffolding protein
protein kinase
SH2 domain
MAPK
Ras
phosphodiesterase
phospholipase
protein phosphatase crosstalk
Chapter 15: Signal transduction
Outline:
General principles of signal transduction
Overview of:
–
–
–
–
Signaling
Receptors
Transducers
Targets
Major types of cell-surface receptors
- RTK signaling
- G-protein signaling
General Principles of Signal Transduction
• Communication usually
involves
(i) a signaling molecule,
(ii) a receptor,
(iii) intracellular signal
transducers and
(iv) targets
General Principles of Signal Transduction
2. Each cell responds to a complex profile of
signaling molecules (crosstalk)
General Principles of Signal Transduction
3. Different cells respond differently to a
particular signaling molecule
General Principles of Signal Transduction
4. Cells can remember the effects of some signals
5. Cells can adjust their sensitivity to a signal
General Principles of Signal Transduction
4. Cells can remember the effects of some signals
5. Cells can adjust their sensitivity to a signal
General Principles of Signal Transduction
6. Signal can exhibit complex responses to signal
concentration
Signaling molecules
Signaling molecules come in many chemical forms:
• Proteins: insulin, glucagon
• Steroids et al.: testosterone, estradiol, cortisol
• Amines: thyroxine, catecholamines, acetylcholine
• Gases: nitric oxide
Signaling pathways require molecules with rapid
rates of synthesis and degradation
Typically released from one cell and recognized by
another cell
Signaling molecules
Secretory signals:
• Autocrine-signals affect same cell or cell type
• Paracrine-signals affect neighbouring cell
• Endocrine-signals affect distant cells
Contact-dependent signals:
-signals are not released but affect other cells in
contact through protein-protein interactions
Autocrine signaling
Signals released by one cell affect other cells
in the immediate vicinity
Amplify a response by inducing many “like-cells”
to respond in the same way
Allows cells to exhibit a coordinated response
(a community effect)
Autocrine signaling
Paracrine signaling
Signals released by one cell affect different cells
in the immediate vicinity
Synaptic transmission resembles paracrine
stimulation but the response is limited to cells in
very close proximity
The outward propagation of the signal is limited by
-cellular uptake,
-extracellular degradation
-& binding
Endocrine signaling
Signals released by one cell affect different cells
far away
Endocrine signaling often exerts multiple effects on
the organism by affecting many different tissues
Receptors
= Proteins that bind signals
and initiate a signaling cascade
Cell membrane receptors
-integral membrane
proteins that bind an
extracellular signal and
start a signal cascade
Intracellular receptors
-nuclear hormone receptors
Nuclear hormone receptors
Examples include
-steroid hormone receptor and
-thyroid hormone receptor
-Retinoic acid receptor
-Vitamine D receptor
NHRs are transcription factors that respond to
specific ligands
Ligands alter the ability to bind to specific DNA
regulatory elements
Intracellular signal transduction
Once the receptor is activated, the signal is
propagated by proteins that act as:
•Relay proteins
•Messenger proteins
•Adaptor proteins
•Amplifier proteins
•Transducer proteins
•Bifurcation proteins
•Integrator proteins
•Latent gene regulatory proteins
Intracellular signal transduction
Activated cell membrane receptors can alter the
activity of intracellular enzymes including:
–Protein modifying enzymes
•Kinases (PKA, PKC, PKG)/ phosphatases
•acetylases/ deacetylases
–Lipid modifying enzymes
•Phospholipases (PLCβ ou γ)
•Phosphotidyl inositol kinase (PI3K)
–Nucleotide modifying enzymes
•cyclases/ phosphodiesterases
Protein kinases
Phospholipases
PLC generates DAG and
phosphoinositides, such as IP3
(inositol 1, 4, 5- triphosphate)
Targets
The final targets of signaling cascades are usually
proteins:
•Regulators of gene expression (transcription
factors, histone remodeling enzymes)
•Enzymes (metabolic enzymes)
•Structural proteins (cytoskeletal proteins)
•Effects alter activity (catalytic, DNA binding) or
the ability to interact with other proteins
(structural proteins, subcellular localization).
Cell surface receptors
3 main classes of cell surface receptors:
Ion-channel linked receptors
Enzyme linked receptors
may possess intrinsic enzyme activity
or
once ligands bind, activate enzyme activity
G-protein linked receptors
are monomeric (7TM)
and activate trimeric G-protein
(GTP-binding protein) that regulate
downstream proteins
Enzyme-linked receptors
5 main classes distinguished by:
•type of effector (e.g. kinase vs. phosphatase)
•target (serine/threonine, tyrosine, histidine)
•type of linkage between receptor and enzyme
1. Receptor tyrosine kinase (RTK)
2. Tyrosine kinase linked receptor
3. Receptor serine/threonine kinase
(PKA, PKC, PKG, PK-Ca2+-CAM)
4. Receptor guanylyl cyclase
5. Histidine-kinase associated receptors
Receptor tyrosine kinases
Most common type of receptor for many common
protein hormones including EGF, PDGF, FGF, HGF,
IGF-1, VEGF, NGF.
Receptor tyrosine kinases
Receptor itself possesses intrinsic tyrosine kinase
activity
Once the ligand binds, the receptor can dimerize
and it become an active tyrosine kinase
It phosphorylates itself (autophosphorylation),
causing:
1. Increase kinase activity
2. Increased affinity for other proteins
Once bound, these docking proteins can become
phosphorylated
Page 684
Figure 19-23 Domain
organization in a variety of receptor
tyrosine kinase (RTK) subfamilies.
Structure of the 2:2:2 complex of FGF2, the
D2–D3 portion of FGFR1, and a heparin
decasaccharide.
D2
Heparin decasaccharide
D3
Ligand-dependent autophosphorylation and
docking
Ligand-dependent autophosphorylation and
docking
Page 686
Schematic diagrams of RTKs.
Page 687
Structure of the PTK domain of the insulin receptor.
PTK Domain undergoes major conformation change &
autophosphorylation (1 to 3 Tyr residues)
Relaying the signal:
Binding Modules, Adaptors, GEF, GAP
•SH2 domains mediate signal Transduction
•PTB domains bind pY-containing peptides
•SH3 domains bind Pro-rich peptides
SH2
P
T
B
GRB2
SH3
P
P
P
GEF
Page 691
Structure of the 104-residue Src SH2 domain in complex with an 11residue polypeptide containing the protein’s pYEEI target tetrapeptide.
Src homology (SH2):
•present in PTK, PLC-γ, certain GAP, etc
•SH2 domain bind specifically phosphoTyr residues in target
peptides with high affinity (hydrophobic pocket)
•recognizes sequence of target peptide on C-terminal side of pY
•does not bind phospho-Ser/phospho-Thr (much more abundant)
SH2
P
T
B
SH3
Page 692
PTB domain of Shc in complex with a 12-residue polypeptide from the
Shc binding site of a nerve growth factor (NGF) receptor.
PTB domain:
•Specifically binds phospho-Tyr target peptides
•Consensus domain NPXpY
•Recognizes the sequence on N-terminal side of pY
SH2
P
T
B
SH3
Page 693
SH3 domain from Abl protein in complex with its 10residue target Pro-rich polypeptide (APTMPPPLPP).
SH3 domain:
Molecular velcro: mediate interactions between kinases & regulatory proteins
present in great variety of proteins
GF
• receptor Tyrosine Kinases
P SH2
• non-Receptor Tyrosine Kinases,
GRB2
• adaptor proteins (ex. Grb2)
SH3 PSOS Ras
P
• structural proteins (myosin, spectrin)
P
bind Pro-rich peptides
Relaying the signal:
Binding Modules, Adaptors, GEF, GAP
•SH2 domains mediate signal Transduction
•PTB domains bind pY-containing peptides
•SH3 domains bind Pro-rich peptides
Other Binding Modules
WW domain (2 Trp residues)
Plekstrin homology domain (PH domain)
PDZ domain
Relay:
Grb2, Shc & IRS:
•adaptor proteins
•recruit Sos to the vicinity of Ras
Ras is activated by RTK via Grb2-SOS complex
Page 694
Grb2.
Complex between Ras and the
GEF-containing region of Sos.
Relay:
Grb2, Shc & IRS:
•adaptor proteins
•recruit Sos to the vicinity of Ras
Ras is activated by RTK via Grb2-SOS complex
•Sos opens Ras’s Nucleotide Binding Site
•GAP functions to turn Off Ras-mediated Signals
JUST THE TIP OF THE ICEBERG OF PROTEIN DOMAINS
USED FOR PROTEIN/PROTEIN INTERACTIONS
Docking of intracellular proteins on
phosphotyrosines
Phosphotyrosine domains are binding sites for
many different proteins with SH2 (=PTB) domains
These can be enzymes (e.g., PLC, PI3K)
or
they can act as adaptor molecules to bind other
proteins (e.g. Grb2)
Linking RTK to Ras and the MAPK cascade
Once an adaptor protein binds to the RTK (e.g.,
Grb2), it attracts another protein - Ras GEF
(guanine nucleotide exchange factor)
Ras GEF induces Ras to exchange its GDP for GTP
(activating Ras).
Active Ras then activates MAPKKK,
which phosphorylates and activates MAPKK,
which phosphorylates and activates MAPK,
which phosphorylates many proteins,
including transcription factors.
Ras GTPase Cycle
Ras-GTP
H2O
GTP
Hydrolysis
Exchange
Pi
GDP
Ras-GDP
Ras GTPase Cycle
Ras-GTP
GTP
GTPase Activating
Proteins
(GAPs)
Guanine Nucleotide
Exchange Factors
(GEFs)
GDP
H2O
Pi
Ras-GDP
-GAPs discovered biochemically
-GEFs discovered genetically- first in yeast and then drosophila
Ras belongs to the larger family of small GTPbinding switch
Small GTP-binding proteins:
Š initiation & elongation factors (protein synthesis).
Š Ras (growth factor signal cascades).
Š Rab (vesicle targeting and fusion).
Š ARF (forming vesicle coatomer coats).
Š Ran (transport of proteins into & out of the nucleus).
Š Rho (regulation of actin cytoskeleton)
All GTP-binding proteins differ in conformation
depending on whether GDP or GTP is present at
their nucleotide binding site.
Generally, GTP binding induces the active state.
Most GTP-binding
proteins depend on
helper proteins:
GAPs, GTPase
Activating Proteins,
promote GTP hydrolysis.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
A GAP may provide an essential active site residue, while
promoting the correct positioning of the glutamine
residue of the switch II domain.
Frequently a (+) charged arginine residue of a GAP
inserts into the active site and helps to stabilize the
transition state by interacting with (-) charged O atoms
of the terminal phosphate of GTP during hydrolysis.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
Ga of a heterotrimeric G protein has innate capability
for GTP hydrolysis.
It has the essential arginine residue normally provided
by a GAP for small GTP-binding proteins.
However, RGS proteins, which are negative regulators
of G protein signaling, stimulate GTP hydrolysis by Ga.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
GEFs, Guanine Nucleotide Exchange Factors, promote
GDP/GTP exchange.
The activated receptor (GPCR) serves as GEF for a
heterotrimeric G protein.
Mutant Ras in Tumors Can’t Hydrolyze GTP and
Accumulates in the Active GTP-Bound State
Ras-GTP
GTP
X
Exchange
GDP
Ras-GDP
Ras Superfamily
Ras
Rho
H-Ras
N-Ras
K-Ras
RhoA
RhoB
RhoC
TC21
RhoG
RhoE
Rap1
Rap2
R-Ras
RalA
RalB
Rab
Arf
Rab1-N
Arf1-6
Ran
Ran
CDC42
Rac1
Rac2
Growth/
Cytoskeleton
Differentiation
Vesicle sorting
NuclearTranslocation
Functions of Ras Proteins
1) Promote Cell Proliferation
-fibroblasts, epithelial cells, lymphocytes
-mediate actions of growth factors
2) Promote Cell Differentiation
-neuronal progenitor cells (PC12)
-mediate action of neurotrophins
3) Contribute to Differentiated Cell Functions
-CNS neurons
-mediate effects of calcium signaling
Multiple Regulators of Ras Function
Tyrosine Kinases
SOS
GRB2
Phospholipase C
Ras-GRP
DAG
Ca2+
Calmodulin
Ras-GRF
Ras
Each GEF has motif that connects it to distinct upstream signals but similar catalytic
domain that allows it to activate Ras
Multiple Effectors of Ras Function
Ras
Ral-GEF
PI3Kinase
Raf
Ral
PDK1
Akt
Mek
Erk
Exocyst
Src
RalBP
Page 697
Structure of the Ras binding domain of Raf (RafRBD; orange)
in complex with Rap1A·GDPNP (=homolog of Ras light blue).
Page 700
Structure of Src·ADPNP lacking its
N-terminal domain and with Tyr 527
phosphorylated.
MAP kinase Pathway
Activation of Ras
Activation of MAPK cascade
Page 696
The Ras-activated
MAP kinase cascade
Page 698
MAP kinase cascades in mammalian cells.
Scaffolding proteins help organize MAPKs
Page 699
Scaffold proteins that modulate mammalian MAP
kinase cascades. (a) JIP-1(JNK-interacting protein).
Page 699
Scaffold proteins that modulate mammalian
MAP kinase cascades. (b) MEKK1.
Insulin signalling
•
•
•
•
•
•
Two extracellular alpha chains
each with an insulin-binding site,
linked to two transmembrane beta chains, each with a cytosolic
tyrosine kinase domain.
Following insulin binding to the alpha chains, the tyrosine
kinase domain of each beta chain catalyzes
autophosphorylation of tyrosine residues in the adjacent kinase
domain.
The tyrosine kinase domains also catalyze the
phosphorylation of proteins called insulin-receptor substrates
(IRSs).
Some of the effects of insulin binding are mediated through the
second messenger system of PIP3 which regulates several
serine-threonine protein kinases. Binding of insulin in some cell
types (e.g., muscle) leads to stimulation of phoshatase
cascades leading to inactivation of glycogen phoshorylase in
the glycogen degradation pathways
Insulin formation of PIP3
•
•
•
•
Binding of insulin to its receptor activates the protein tyrosine kinase activity of the
receptor, leading to the phosphorylation of insulin-receptor substrates (IRSs).
The phosphorylated IRSs interact with the phosphotidylinositide 3-kinase (PI kinase) at the
plasma membrane, where the enzyme catalyzes the phophorylation of PIP2 to PIP3.
PIP3 acts as a second messenger, carrying the message from extracellular insulin to certain
intracellular protein kinases.
Recent research has found that many of the effects of insulin on cells are mediated
through PIP2. Whereas the inositol-phospholipid signalling system leads to hydrolysis of PIP2,
insulin activates phophorylation of PIP2 to PIP3.
INSULIN SIGNALLING
Page 719
Figure 19-64 Insulin signal
transduction.
CAP: Cbl-associated protein
C3G: a G-nucleotide exchange factor
CrkII: a SH2/SH3 adapter protein
PDK1: phophoinositide-dependent kinase mTOR: target of rapamycin
Insulin
Transduction
PI3K
Page 694
Structure of an insulin receptor substrate protein.
Protein Tyr Phosphatases
•SHP-2
Protein Ser/Thr Phosphatases
•PP1
•PP2A
•PP2B (=calcineurin / target of immunosuppressant drugs)
•PP2C
Page 706
Protein Tyrosine Phosphatase
SHP-2.
PP2A
•Structurally variable
•Functionally diverse
•Catalytic subunit ©)
•Scaffold subunit (A)(PR65)
•Four different regulatory subunits (B, B’, B’’,
B’’’), bind to A & C subunits
A subunit of PP2A.
Page 707
Calcineurin. (a) human
FKBP12·FK506–CaN.
Calcineurin. (b) human
CaN with CaNA yellow, its
autoinhibitory segment red,
and CaNB cyan.
Abl-Akt
Page 703
Structure of the Abl PTK domain in complex with
a truncated derivative of gleevec (anticancer drug).
Integrins-Pakcytoskeleton
Non-Receptor-Tyrosine kinases
Domain organization of the
major NRTK subfamilies.
Page 701
Model of Src activation.
Page 702
The JAK-STAT pathway for the intracellular
relaying of cytokine signals.
JAKSTAT
JakSTAT
GPCR
= G-protein coupled Receptors
G-protein linked receptors
Ligand: Diverse ligands, such as epinephrine
Receptor: Integral membrane protein with 7TM
(7 transmembrane domains)
G-protein: trimeric protein (α, β, γ) attached to
the cell membrane by lipid anchors
Effectors: Target proteins that show altered
activity when they interact with activated Gprotein subunits (α, or βγ)
G Protein Signal Cascade
A hormone (e.g., epinephrine or
glucagon) that activates
formation of cAMP or IP3, binds
at the cell surface to a receptor
with 7 transmembrane αhelices.
Rhodopsin was the first member
of the family of 7-helix
receptors to have its structure
determined by X-ray
crystallography.
Rhodopsin
PDB 1F88
Cytosolic domains of 7-helix receptors interact
with G-proteins (=heterotrimeric GTP-binding
protein).
A G-protein has 3 subunits, designated α, β, γ.
7-Helix receptors that interact with G-proteins
are called GPCR, or G-Protein-Coupled Receptors.
Various proteins interact with GPCRs to modulate
their activity. Effects of these interactions
include:
Šaltered ligand affinity
Šreceptor dimerization that may enhance or
alter activity
Šaltered receptor localization
Ligand-induced receptor clustering may also
regulate receptor function.
G-protein linked receptors and G-proteins
Receptor
G-protein
Interaction between receptor and G-protein
Once the ligand binds, the activated receptor
recruits a G-protein
Nucleotide exchange occurs (GTP replaces GDP)
and the trimer dissociates into 2 parts:
-α subunit
-βγ subunit
Both parts can regulate downstream pathways
Variety of G-proteins
•
•
•
•
Gs are stimulatory
Gi/0 are inhibitory
Gq act on PLC
G12/13 act on ion channels
– 22 α subunits
– 5 β subunits
– 12 γ subunits
Gs proteins are stimulatory
Upon dissociation, a Gs protein stimulates an
effector enzyme, such as
-adenylate cyclase,
-phospholipase Cβ
or
-ion channels (K+ or Ca2+)
Adenylate cyclase converts ATP to cAMP
Elevated cAMP stimulates cAMP-dependent
protein kinase (PKA) by inducing the release of
inhibitory subunits
A G-protein that
is part of a
pathway that
stimulates
Adenylate Cyclase
is called Gs & its
α subunit Gsα.
hormone
signal
outside
GPCR
plasma
membrane
α γ
GDP β
GTP
GDP
γ + α
AC
GTP
β
cytosol
ATP cAMP + PPi
The α subunit of a G-protein (Gα) binds GTP, & can
hydrolyze it to GDP + Pi.
α & γ subunits have covalently attached lipid
anchors that bind a G-protein to the plasma
membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein,
with cytosolic domains forming the catalytic site.
hormone
signal
outside
The complex of
β & γ subunits
Gβ,γ inhibits Gα.
GPCR
plasma
membrane
α γ
GDP β
GTP
GDP
γ + α
AC
GTP
β
cytosol
ATP cAMP + PPi
The sequence of events by which a hormone
activates cAMP signaling:
1. Initially Gα has bound GDP, and α, β, & γ
subunits are complexed together.
hormone
signal
outside
GPCR
plasma
membrane
α γ
GDP β
GTP
GDP
γ
β
+
α
AC
GTP
cytosol
ATP cAMP + PP i
2. Hormone binding to a 7-helix receptor (GPCR)
causes a conformational change in the receptor that
is transmitted to the G protein.
The nucleotide-binding site on Gα becomes more
accessible to the cytosol, where [GTP] > [GDP].
Gα releases GDP & binds GTP (GDP-GTP exchange).
hormone
signal
outside
GPCR
plasma
membrane
α γ
GDP β
GTP
GDP
γ + α
AC
GTP
β
cytosol
ATP cAMP + PPi
3. Substitution of GTP for GDP causes another
conformational change in Gα.
Gα-GTP dissociates from the inhibitory βγ complex &
can now bind to and activate Adenylate Cyclase.
hormone
signal
outside
GPCR
plasma
membrane
α γ
GDP β
GTP
GDP
γ + α
AC
GTP
β
cytosol
ATP cAMP + PPi
4. Adenylate Cyclase, activated by Gα-GTP,
catalyzes synthesis of cAMP.
5. Protein Kinase A (cAMP Dependent Protein
Kinase) catalyzes phosphorylation of various
cellular proteins, altering their activity.
G-protein dissociation
GTP hydrolysis ends signaling and induces
trimerization
The stimulatory Gsα, when it binds GTP, activates
Adenylate cyclase.
An inhibitory Giα, when it binds GTP, inhibits
Adenylate cyclase.
Different effectors & their receptors induce Giα
to exchange GDP for GTP than those that
activate Gsα.
In some cells, the complex of Gβ,γ that is released
when Gα binds GTP is itself an effector that
binds to and activates other proteins.
Variety of G-proteins
•
•
•
•
Gs are stimulatory
Gi/0 are inhibitory
Gq act on PLC
G12/13 act on ion channels
– 22 α subunits
– 5 β subunits
– 12 γ subunits
VARIETY OF G-PROTEINS: Gα, -β, -γ
ADP-Ribosylation
Š Cholera toxin catalyzes covalent modification of Gsα. ADPribose is transferred from NAD+ to an arginine residue at
the GTPase active site of Gsα.
This ADP-ribosylation prevents Gsα from hydrolyzing GTP.
Thus Gsα becomes permanently activated.
Š Pertussis toxin (whooping cough disease) catalyzes ADPribosylation at a cysteine residue of Giα, making the
inhibitory Gα incapable of exchanging GDP for GTP. Thus
the inhibitory pathway is blocked.
ADP-ribosylation is a general mechanism by which activity of
many proteins is regulated, in eukaryotes (including mammals)
as well as in prokaryotes.
Page 680
Figure 19-19
Mechanism of action of cholera toxin
.
Cholera Toxin
composition = AB5
B:103AA / A: 240AA
Activation: clivage en A1 (195AA) + A2(45AA) reliés par -S-SA pénètre dans cellule (endocytose) / dirigé dans ER: liaison par séquence KDEL de A2
Effet: ADP-ribosylation de Arg de Gsα
ADP-ribosylation de
Cys sur Giα
ADP-RIBOSILATION BY
PERTUSSIS TOXIN
ADP
ribosylation
oO
−
H
o
NH2 O
C
protein
o 2
NH
+N
N
o CH2
O
CH2 O
o
o P O
H
H
H
H
OH
OH
NH2
NH2
o
O
N
N
o
O
NH2
NH
− o P o CH2
O P O CH2 O
o
H
H
H
H
OH
OH
NH
o
NH2
O
2
N
NN
N
N
N
N
N
N
P
o
CH2
oO P O CH
2 O
o
o
O
H
H
H
H
+
NAD
OH
OH
o P
O
−
o
O
protein
(CH2)3
NH
Arg
C
residue
NH2
NH
NH
C
−
(nicotinamide
adenine
dinucleotide)
C 2)3
(CH
NH2+
o
O
NH2 O
H
o
C
C
CH2
CH2
H
H
OH
N
N
o
O
+
NH2
N
N
H
H
OH
ADP-ribosylated
protein
NH2
+
N
N nicotinamide
H
VARIETY OF G-PROTEINS: Gα, -β, -γ
Structure of G proteins:
PDB 1GIA
The nucleotide binding site
in Gα consists of loops that
extend out from the edge
of a 6-stranded β-sheet. Three switch domains have
been identified, that
change position when GTP
substitutes for GDP on Gα.
GTPγS
Inhibitory Gα
These domains include residues adjacent to the
terminal phosphate of GTP and/or the Mg++
associated with the two terminal phosphates.
O
O
GTP hydrolysis
N
N
H
H
H O
O
H
O
O
O
O
O
O
−
O PP O
CH2
O P
P O
O P
P O
O C
O
O
O
H
O−
O−
O−
O
O
O
H
OH
N
N
NH
N
N
N
NH2
NH2
H
H
OH
GTP hydrolysis occurs by nucleophilic attack of a
water molecule on the terminal phosphate of GTP.
Switch domain II of Gα includes a conserved
glutamine residue that helps to position the attacking
water molecule adjacent to GTP at the active site.
PDB 1GP2
PDB 1GP2
Gβ - side view of β-propeller
Gβ – face view of β-propeller
The β subunit of the heterotrimeric G Protein has a
β-propeller structure, formed from multiple repeats
of a sequence called the WD-repeat.
The β-propeller provides a stable structural
support for residues that bind Gα.
Adenylate Cyclase catalyzes: ATP
cAMP + PPi
Binding of certain hormones
(e.g., epinephrine) to the outer
surface of a cell activates
Adenylate Cyclase to form
cAMP within the cell.
Cyclic AMP is thus considered
to be a second messenger.
NH
2
NH2
cAMP
N
N
N
N
N
N
N
N
H2
5'C 4'
O
O
O
O
O
O
H
H 3'
PP O
O
O
O-
H
1'
2' H
OH
Protein Kinase A (cAMP-Dependent Protein Kinase)
transfers Pi from ATP to OH of a Ser or Thr in a
particular 5-amino acid sequence.
Protein Kinase A in the resting state is a complex of:
• 2 catalytic subunits (C)
• 2 regulatory subunits (R) :
R2C2
Each regulatory subunit (R) of Protein Kinase A
contains a pseudosubstrate sequence, like the
substrate domain of a target protein but with Ala
substituting for the Ser/Thr.
The pseudosubstrate domain of (R), which lacks a
hydroxyl that can be phosphorylated, binds to
the active site of (C), blocking its activity.
When each (R) binds 2 cAMP, a conformational
change causes (R) to release (C).
Each catalytic subunit can then catalyze
phosphorylation of Ser or Thr on target proteins.
R2C2 + 4 cAMP Æ R2cAMP4 + 2 C
R2C2 + 4 cAMP Æ R2cAMP4 + 2 C
AKAPs, A-Kinase anchoring proteins, bind to the regulatory
subunits (R) of Protein Kinase A.
AKAPs localize Protein Kinase A to specific regions of a cell.
PKIs, Protein Kinase Inhibitors, modulate activity of the
catalytic subunit (C).
PKA activation by cAMP
PKA activates gene expression
CREB =
cAMP responsive
Elements Binding
Protein
EXAMPLE _ Glycogen Metbolism
Gs & Gi Pathways
Inactivation of PKA pathway
The G-protein -PKA pathway is inactivated by:
–Receptor desensitization (phophorylation by PKA)
–GTP hydrolysis in G-protein (GTPase of a-subunit)
–cAMP hydrolysis by phosphodiesterase
–PKA inhibition
–Phosphatase action on PKA targets
–Activation of an antagonistic pathway (Gi)
Turn off of the signal:
1. Gα hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Gα causes it to rebind
to the inhibitory βγ complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of
cAMP Æ AMP.
Turn off of the signal (cont.):
3. Hormone Receptor desensitization occurs.
This process varies with the hormone.
Š Some receptors are phosphorylated via Gprotein-coupled receptor kinases.
Š The phosphorylated receptor may then bind to
a protein arrestin that blocks receptor-Gprotein activation & promotes removal of the
receptor from the membrane by clathrinmediated endocytosis.
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to
proteins via Protein Kinase A.
Phosphodiesterase enzymes catalyze: cAMP
cAMP + H2O Æ AMP
N
N
N
N
The phosphodiesterase that cleaves
cAMP is activated by phosphorylation
catalyzed by Protein Kinase A.
Thus cAMP stimulates its own
degradation, leading to rapid turnoff
of a cAMP signal.
NH2
NH2
N
N
H2
5' C
C 4'
O
O
O
O
O
O
H
H 3'
PP
O
O
O
O-
N
N
H
1'
2' H
OH
G-protein coupled Receptor
ARRESTIN
Model for interaction of β-loop with
Arrestin
Novel roles of endocytosis and scaffolding in signal propagation
H
Kholodenko B.N. (2002) Trends Cell Biol. 12: 173.
Signal amplification is an important feature of
signal cascades:
Š One hormone molecule can lead to formation of
many cAMP molecules.
Š Each catalytic subunit of Protein Kinase A
catalyzes phosphorylation of many proteins
during the life-time of the cAMP.
PLCβ & PKC
G-proteins and phospholipases
Some G-proteins activate
PLCβ (phospholipase Cβ),
triggering formation of
inositol triphosphate (IP3)
and diacylglycerol (DAG)
DAG, IP3, Ca2+ and signal transduction
DAG:
•substrate for production of eicosanoids, potent
signaling molecules including arachadonic acid
•activates PKC
IP3:
induces release of Ca2+ from ER stores via IP3sensitive Ca-channels
Ca2+:
Elevated Ca2+ can activates PKC and CamK.
Phosphatidylinositol Signal Cascades
O
O
C OH
R2
C
O
O
O
C R
O
H2C
O
R1 C
C R
O C
CH
HO
H2C
C R
O
P O
O
P
O−
O
1
OH
HO
2
phosphatidylinositol
H
H
OH
3
H
OH
OH
H
6
OH
H
4
OH
OH
5
H
OH
OH
Some hormones activate a signal cascade based
on the membrane lipid phosphatidylinositol.
Page 707
IP3 as second messenger - Phosphatidylinositides
Molecular formula of the
phosphatidylinositides.
Page 714
Flow chart of reactions in the synthesis of
phosphoinositides in mammalian cells.
Page 714
Domain organization of the 3 classes of PI3Ks.
OO
O
O
O
OO C C R2OH
HH
2C
R2
2C R
R
HO
RO
R11 CC
C O
OO
O
CH
C
CH
H2CC O
H2C
O
R
P
O
P O
O
O− −
O
O1
OH
OH H
HO
2
2
H
PIP
2
phosphatidylH
phosphatidylinositolinositol-4,5-bis-Ph
4,5-bisphosphate
H
6
1
OHH
OH
3
OH
H
6
OPO32−
OH
OPO3H
OH
H OH 5
H
4
H3
OH
H
5
H
2−
4 3
OPO
H
OPO3H
OH
Kinases sequentially catalyze transfer of Pi from ATP
to OH groups at positions 5 & 4 of the inositol ring,
to yield phosphatidylinositol-4,5-bisphosphate (PIP2).
PIP2 is cleaved by the enzyme Phospholipase C.
Page 709
A phospholipase is named according to the bond
that it cleaves on a glycerophospholipid.
Different isoforms of PLC
O
O
C R
O
H2C
have different regulatory
domains, & thus respond to RO1 C
C R
O C
CH
different signals.
H2C
O
C R
One form of PLC is
activated by a G-protein, cleavage by
Phospholipase C
Gq.
O
O
C
C
O
O
R2
O
P O
O
O−
O
OH
2
H
PIP2
phosphatidylinositol4,5-bisphosphate
H
1
6
H
OH
OH
5
H
OPO3
3
H
4
OPO3
OPO32−
H
OPO32−
A GPCR (receptor) is activated.
GTP exchanges for GDP.
Gqa-GTP activates Phospholipase C.
Ca++, which is required for activity of Phospholipase C,
interacts with negatively charged residues & with
phosphate moieties of IP3 at the active site.
H
OPO32− OPO3
1
OH
OPO3
H
2
OH
H
3
H
6
OH
H
4
O
O
2−
OPO3
OPO3
5
H
OPO32−
IP3
inositol-1,4,5-trisphosphate
H2C
C
O
O
R1 C
C
HO
O
R
C
C OH
R2
O
R
CH
C
H2C
C OH
OH
diacylglycerol
Cleavage of PIP2, by PLC, yields two 2nd messengers:
inositol-1,4,5-trisphosphate (IP3) & diacylglycerol (DG).
Diacylglycerol, with Ca++, activates Protein Kinase C,
which catalyzes phosphorylation of several cellular
proteins, altering their activity.
Page 708
Role of PIP2 in intracellular signaling.
Page 709
Domain organization of the four classes of
phosphoinositide-specific PLCs.
Page 713
Activation of PKC.
Ca++
Ca++-release channel
IP3
Ca
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
++ ADP + Pi
IP3 activates Ca++-release channels in ER membranes.
Ca++ stored in the ER is released to the cytosol,
where it may bind calmodulin, or help activate Protein
Kinase C.
Signal turn-off includes removal of Ca++ from the
cytosol via Ca++-ATPase pumps, & degradation of IP3.
OPO32− OPO3
H
OH
OPO3
H
OH
OH
H
OPO32−
OPO3
H
H
H
IP3
OH
OPO32−
(3 steps)
OH
H
OH
H
OH
OH
H
+ 3 Pi
H
H
H
OH
inositol
Sequential dephosphorylation of IP3 by enzymecatalyzed hydrolysis yields inositol, a substrate for
synthesis of PI.
IP3 may instead be phosphorylated via specific
kinases, to IP4, IP5 or IP6. Some of these have signal
roles.
E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some
cells activates plasma membrane Ca++ channels.
O
O
R
O1
C R
O
C
H2C
C
CH
C
C
H2C
phosphatidylinositol3-phosphate
O
O
O
C
R C
R2
O
O
O
O
O
O PP O
O−
O
OH
2
H
1
H
6
OH
OH
H
5
H
OPO32− OPO3
H
3
H
4
OH
The kinases that convert PI (phosphatidylinositol) to
PIP2 (PI-4,5-P2) transfer Pi from ATP to OH at
positions 4 & 5 of the inositol ring.
PI 3-Kinases instead catalyze phosphorylation of
phosphatidylinositol at the 3 position of inositol ring.
O
O
H2C
O
O C
C OH
R2
O
C R
CH
O
RO
C
O C
C R
1
O
C R
O P
P O
H2C
O
O
O−
phosphatidylinositol3-phosphate
OH
2
H
1
H
6
OH
OH
H
5
2−
H
OPO3 OPO3
H
3
H
4
OH
PI-3-P, PI-3,4-P2, PI-3,4,5-P3, & PI-4,5-P2 have
signaling roles.
These are ligands for particular pleckstrin
homology (PH) and FYVE protein domains that
bind proteins to membrane surfaces. PKB (Protein Kinase B, also called Akt) becomes
activated when it is recruited from the cytosol to
the plasma membrane surface by binding to
products of PI-3 Kinase, e.g., PI-3,4,5-P3.
Š Other kinases at the cytosolic surface of the
plasma membrane then catalyze phosphorylation
of PKB, activating it.
Š Activated PKB catalyzes phosphorylation of Ser
or Thr residues of many proteins, with diverse
effects on metabolism, cell growth, and
apoptosis.
Š Downstream metabolic effects of PKB include
stimulation of glycogen synthesis, stimulation
of glycolysis, and inhibition of gluconeogenesis.
Signal cascades may be mediated by complexes of
proteins that assemble at the cytosolic surface of
the plasma membrane, frequently in areas of
distinct lipid composition called lipid rafts.
Signal proteins may be recruited into such
complexes by
• insertion of their lipid anchors in the plasma
membrane,
• interaction with membrane-associated
scaffolding proteins,
• or
• interaction of their pleckstrin homology
domains with transiently formed PI derivatives.
Interactions between G-proteins and RTKs
Summary on Enzyme-linked receptors
Enzyme-linked receptors generate variable
cellular responses
Multiplicity of players (receptors, kinases etc)
arise from gene duplication and divergence
Recognize the critical role of phosphorylation/
dephosphorylation control as molecular switches
Adaptor molecules allow construction of protein
signaling cascades with variable outputs
JAK-STAT
JAK-STAT
cGMP Pathway