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A Primer on G Protein Signaling
Elliott RossUT-Southwestern Medical Center
Receptor G Effector
RhodopsinsAdrenergicsMuscarinicsSerotonin, DopamineHistamine, GABAb, GlutamateEiscosanoidsPAF, SphingolipidsPurinergicsPeptides(kinins, angiotenisin, opioids,endothelin, glucagon, etc.)
Glycoprotein hormonesMembrane proteins
(Smoothened, BOSS)Fungal pheromonesOdorants, tastants
Gs (>3), GolfGi (3)Gt (2), GgusGo (2)GzGq (4)G12/13
Gβ (5)Gγ (11)
Adenylyl cyclasecGMP PhosphodiesterasePhospholipase C-βChannels (K+, Ca2+, Na+)PI-3-KinaseRho GEFsRap GAPsProtein kinases (S/T and Y)
(phosphatases ?)Transporters (Mg2+, glucose ?,
biogenic amines?,)Vesicle trafficking
The MODULE
The Gq-PLC Module – An example
RHODOPSIN – a G Protein-Coupled Receptor
Extracellular
Cytoplasmic
Gα β γi1 1 2Mark Wall, Steve Sprang, et mult.
The G Protein αβγ Trimer
N. Gautam
How these proteins MAY be arranged in space
R
G
E
R1
G1
E1
R2
G2
E2
R3
G3
E3
E1
R
G
E2 E3
G1
E1
R
G2
E2
G3
E3
R1 R2
G
E
R3 R1
G1
R2
G2
E
R3
G3
But the pathwayscan branch
Gα-GDP Gα∗-GTP
GDP GTP
Pi
k1
k-1
G PROTEINS ARE TWO-STATE SWITCHES
A Gα subunit can assume active and inactive conformations.G proteins are activated when they bind GTP.“Activated” means that they can regulate an effector, either positively or negatively.
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
G proteins are two-state switches; they have active and inactive conformations.G proteins are activated when they bind GTP. “Activated” means that theycan regulate an effector either positivelyor negatively.
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
Gα subunits hydrolyze bound GTP to GDP. Hydrolysis is slow, but faster thandissociation of GTP, so a GTPase cycleis created.The “steady-state” fraction of G protein in the GTP-bound state constitutes the fractional activity of the system.
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
Receptors accelerate the release of GDPand the binding of GTP; they thus “activate”G proteins. Receptors can act catalytically (sequentially)on many G proteins, which results in signal amplification.Receptors are exchange catalysts.
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
Hydrolysis of bound GTP is slow; t1/2 ~ 10 s - 5 min. GTPase-activating proteins (GAPs)accelerate hydrolysis ~2000-fold.
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
These proteins constitute a G proteinsignaling module. Specific components can be chosen from the parts list. You getone of each in each module.
G Protein Activation Creates Two Signaling Molecules
Gα-GTP
Gβγ
Gαβγ-GDP Gαβγ-GTP
E1E2 E3
E4E5E6
Gβγ is a stable dimer from which Gα can dissociate when it binds GTP.
Activation of Gα “releases” active Gβγ
Gβγ independently regulates its own effector proteins
This means that there is also a Gβγ cycle
Pi
GDP
R
R
GTP
Gα∗-GTP
R-Gαβγ
Gαβγ-GDP
Gα-GDP
Gαβγ-GTP
Gβγ
Gβγ Cycle
G Protein Activation Creates Two Signaling Molecules
Gα-GTP
Gβγ
Gαβγ-GDP Gαβγ-GTP
E1E2 E3
E4E5E6
Activation of Gα “releases” active Gβγ
Therefore
Gβγ drives deactivation of Gαprobably not very important
G Protein Activation Creates Two Signaling Molecules
Gα-GTP
Gβγ
Gαβγ-GDP Gαβγ-GTP
E1E2 E3
E4E5E6
Activation of Gα “releases” active Gβγ
ThereforeGβγ drives deactivation of Gα
probably not very important
Therefore, too, with a lot of data and some thermodynamics thrown in,
Gβγ stabilizes the binding of GDP from GαIt’s a GDP-dissociation inhibitor, or GDI
Gβγ both inhibits G protein activation and suppresses spontaneous background noise
Functions of Gβγ
Regulates effectors when released by activated GαInhibits Gα activation (by GDI effect)
Suppresses spontaneous noiseGβγ release by one trimer may inhibit activation of another
Anchors Gα to membranesFacilitates activation of Gα by receptor
Nearly obligate Maybe by anchoring
Inhibits GAPs
G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
Nucleotide exchange is slow, but receptor catalyzes exchange by convertingthe GTP-binding site on Gα from the closed to the open configuration.
Gαo*-GTP
Gαo-GDP Gαc-GDP
Gαc*-GTP
RNegative heterotropic binding of R and nucleotide
Quantitatively reciprocalLigand-mediated ligand exchange
G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
Gαo*-GTP
Gαo-GDP Gαc-GDP
Gαc*-GTP
RTwo major factors drive this cycle clockwise:
Hydrolysis of GTP is favoredCellular [GTP] > [GDP]
GTP also binds much tighter than GDPAffinities have never been measured at equilibrium
(really ! )
G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
Gαo*-GTP
Gαo-GDP Gαc-GDP
Gαc*-GTP
R
If R can exchange nucleotide in less than the lifetime of the activated state,then it can catalytically (sequentially) act on multiple G proteins.
G Proteins as Four-State Systems:Closed - Open Configuration and Receptor-Catalyzed GDG/GTP Exchange
If R can exchange nucleotide in less than the lifetime of the activated state,then it can catalytically (sequentially) act on multiple G proteins.
Gα*-GTP
Gα-GDP
[R-Gα-GDP]R-Gα
Pi
GDP
GTPR
[R-Gα*-GTP]
R
The diffusion-limited rate of encounter of receptor with Gα-GDP can limit the rate of activation (“collisional coupling”).Scaffolding proteins increase the encounter rate (a lot) but limit amplification.
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
GAPsYou can’t turn off a signal upon removal of hormone any faster than you can hydrolyze GTP.
GAPs for Heterotrimeric G Proteins
ACCELERATE GTP HYDROLYSIS 2000-FOLDEFFECTORS
Phospholipase C-β : Gq GAPRho GEF p115: G13 GAP (with RGS domain)cGMP phosphodiesterase : “co-GAP” for GtGPCR kinases (RGS domain)
RGS PROTEINSMost not effectors ~30 genes in mammalsConserved RGS box, diverse functional endsFor Gi and Gq
p115’s for G12/13, maybe a new group for Gs
G-GDPReceptor
GAPG*-GTP
Regulatory Functions of G Protein GAPs
AttenuateSharpen
Turn offLowerBackground
Time
Res
pons
eG-GDP
Receptor
GAPG*-GTP
Regulatory Functions of G Protein GAPs
Time
Res
pons
e
Inhibit
Changeselectivity
Steepen
log [Agonist]
GIRK Channels in Xenopus Oocytes
Kir 3.1/3.2; m2 MAChR
Doupnik et al., PNAS 94:10461
GAPs Need Not Attenuate the Signal
Chen et al., Nature 403, 557 (2000)
Single Photon Responses of RGS9- Mice
Reconstitution of Gq-Phospholipase Signaling Pathway
M1 MuscarinicAcetylcholine Receptor
Gαqβγ Phospholipids(including PIP2)
Phospholipase C-β(± other GAP)
(purified, in detergent solution)
Slowly remove detergent
Unilamellar vesicles, ~100 nm diameter, scrambled
Measure binding and release of hormone and nucleotides, hydrolysis of GTP, hydrolysis of PIP2;both at steady state and in single catalytic cycles.
FIG. 4.concentrvesicles thmeasuredand eithe
FIG. 2. Reconstitution of Gq-mediated activation of PLC-�1 bym1AChR. m1AChR and Gq were co-reconstituted with [3H]PIP2 asdescribed under “Experimental Procedures.” The activity of addedPLC-�1 was measured in the presence of guanine nucleotide and/ormuscarinic ligands. A, GTP-dependent PLC activity; B, GTP�S-stimu-lated PLC activity. Conditions were as follows: no addition (�), 10 �M
GTP (GTP), 1 mM carbachol (Cch), carbachol plus 10 �M atropine
m1 Muscarinic Receptor-Gq-PLC8002
FIG. 3. Effect of Ca2� on Gq-stimulated PLC-�1 activim1AChR and Gq were co-reconstituted with [3H]PIP2, and the activof added PLC-�1 was measured at various free Ca2� concentratioThe data plotted are the initial rates determined from time coursesPLC activity conducted at each Ca2� concentration in the presence of�M GTP and either 1 mM carbachol (filled circles) or 10 �M atrop(empty circles). Reactions contained 0.35 nM m1AChR, 3.0 nM Gq, 0�M accessible PIP2, and 1.0 nM PLC-�1.
PLC-�1 was measured in the presence of guanine nucleotide andmuscarinic ligands. A, GTP-dependent PLC activity; B, GTP�S-stimlated PLC activity. Conditions were as follows: no addition (�), 10GTP (GTP), 1 mM carbachol (Cch), carbachol plus 10 �M atrop(C�A), and 100 nM GTP�S (�S). All samples contained 10 nM free Ca2.4 nM Gq, 0.33 nM m1AChR, 1 nM PLC-�1, and 0.56 �M accessible PIA and B show data (mean � S.D.) from the same experimentdifferent scales.
Phospholipase C Regulation in Receptor-Gq-PLC Vesicles
- GTP - - GTP GTP- - CCh +At CCh +At [Ca2+] (nM)
0 100 200 300
Phos
phol
ipas
e A
ctiv
ity (
pmol
IP3/
min
)
[GAP] (nM)
GTP
ase
(mol
/ min
/m
ol G
q)
0
10
20
30
40
50
60
RGS4PLC-β1
0.1 1 10 100 1000
M1AChR - Gq Vesicles
S. Mukhopadhyay
What kind of mechanistic information
can you get out of a system like this?
G. Berstein
Synergistic Action of Receptors and GAPs
Carbachol-Stimulated GTP Binding to Gq
G. Biddlecome
G. Biddlecome
PLC Activation Displays a Lag When Initiated by Carbachol
Gα*-GTP
Gα-GDP
[R-Gα-GDP]R-Gα
R-Gα*-GTP-G
R-Gα-GDP-GR-Gα-G
Pi
GDP
GTPR
[R-Gα*-GTP]
R
Quench-Flow Assay of GTP Hydrolysis Rate1. Receptor-G protein vesicles, GAP, agonist, GTP
Incubate to steady-state 2. Equilibrate with [γ-32P]GTP3. Add excess unlabeled GTP, antagonist: t = 04. Terminate with H3PO4 at time t
VesiclesGAPGTP
Agonist
[γ-32P]GTP Cold GTP
A B CBuffer
0.0 0.2 0.40
150
175
200
0.0 0.2 0.40
150
200
250
300
0 2 4 60
100
150
200
250
0 2 4 60
100
200
300
400
Time (s)
GTP
Hyd
r oly
zed
(fm
ol)
Hydrolysis of Gαq-Bound GTP
t1/2=25 ms t1/2=57 msRGS4
PLC-β1
S. Mukhopadhyay
Time (s)0 2 4 6 8 10
[ α-32
P]G
DP
Bou
nd (f
mol
)
0
5
10
15
20
25
30o
GDP Dissociation from Gq
t1/2 = 460 ms
S. Mukhopadhyay
Gα-GDP
[R-Gα-GDP]R-Gα
R-Gα*-GTP-G
R-Gα-GDP-GR-Gα-G
Pi
GDP
GTPR
[R-Gα*-GTP]
R
2 s-1
2 s-1
20 s-1105 M-1s-1
Gα*-GTP
Receptor-G Protein-GAP Complex
Kinetically limited by receptor-G protein binding or other receptor eventSlow: t1/2 ~ 20 s; agonist dependent, GTP-independent
Scaffolded in cells ? Association with GAP is fast
Stability during steady-state turnoverrequires agonist and GTP (not GTPγS)
Complex dissociates slowly upon removal of agonistt1/2 30-90 sec by quenching assay kdissoc for Gα-GTP ~ 0.05 - 0.1 s-1 τ ~ 10-20 s
One more aspect to maintaining a relatively stable R - G - GAP module:
Only a receptor that can bind G-GTP tightly enough to traverse the cycle can signal; these receptors will be kinetically tuned by the GAP.
Signals from receptors that bind less tightly will simply be inhibited.
So what’s wrong with this picture?Good:Predicts Km and Vmax at
steady-statePredicts reasonable amount of
Gα*-GTPHas physiologically fast rates
Not so good:Amplitude of Pi release in single-
turnover hydrolysis ~8X higherthan the predicted steady-state amount
Likely explanation: During rapid turnover, the binding site on Gαnever relaxes to the closed configuration because receptor is always bound. And GAPs amplify the ability of receptors to drive activation. And a bunch of other stuff.
Gα-GDP
[R-Gα-GDP]R-Gα
R-Gα*-GTP-G
R-Gα-GDP-GR-Gα-G
Pi
GDP
GTPR
[R-Gα*-GTP]
R
2 s-1
2 s-1
20 s-1105 M-1s-1
Gα*-GTP
G*-GTP
G-GDP
G*-GTP-E*
E
GR + H R*-HPiGTP
GDP
GAP
So here’s a module.
PhosphorylationDephosphorylation
PalmitoylationDe…
EndocytosisDegradationArrestin bindingOther proteins…Scaffold assemblyOther GDP/GTP exchange
catalystsOther GDIs
There are lots of extra-modular regulatory inputs --feedback, off-pathway, and/or cell type-dependent ...
What is extramodular ???All this stuff, plus the products of the effector and the incoming signal.