G-protein-Coupled Receptor

s
G Protein Signal Cascade
Most signal molecules targeted to a cell bind at the cell sur
face to receptors embedded in the plasma membrane.
Only signal molecules able to cross t
he plasma membrane (e.g., steroid h
ormones) interact with intracellular r
eceptors.
A large family of cell surface recept
ors have a common structural motif,
7 transmembrane a-helices.
Rhodopsin was the 1st member of th
is family to have its 7-helix structur
e confirmed by X-ray crystallograph
y. R h o d o p s in PD B 1F88
 Rhodopsin is unique in that it se
nses light.
Most 7-helix receptors have do
mains facing the extracellular sid
e of the plasma membrane that re
cognize & bind particular signal
molecules (ligands).
R h o d o p s in PD B 1F88
The signal is passed from a 7-helix recept
or to an intracellular G-protein.
Seven-helix receptors are thus called GPCR, or
G-Protein-Coupled Receptors.
Approx. 800 different GPCRs are encoded in the hu
man genome.
G-protein-Coupled Receptors may dimerize or form oli
gomeric complexes within the membrane.
Ligand binding may promote oligomerization, which ma
y in turn affect activity of the receptor.
Various GPCR-interacting proteins (GIPs) modulate rec
eptor function. Effects of GIPs may include:
altered ligand affinity
receptor dimerization or oligomerization
control of receptor localization, including transfer to o
r removal from the plasma membrane
promoting close association with other signal proteins
 G-proteins are heterotrimeric, with 3 subunits a, b, g.
 A G-protein that activates cyclic-AMP formation withi
n a cell is called a stimulatory G-protein, designated
Gs with alpha subunit Gsa.
 Gs is activated, e.g., by receptors for the hormones epin
ephrine and glucagon.
The b-adrenergic receptor is the GPCR for epinephri
ne.
 hormone
signal

outside

GPCR plasma
The a subunit of membrane

a G-protein (Ga) agga cytosol

binds GTP, & ca AC
GDP bbGTP
n hydrolyze it to
GDP + Pi. GTP GDP ATP cAMP + PPi

a & g subunits have covalently attached lipid anchors that b

ind a G-protein to the plasma membrane cytosolic surface.
Adenylate Cyclase (AC) is a transmembrane protein, with c
ytosolic domains forming the catalytic site.
 hormone
signal

outside

GPCR plasma
The complex o membrane

f b & g subunit
agga cytosol
s Gb,g inhibits AC
GDP bbGTP
Ga.
GTP GDP ATP cAMP + PPi

The sequence of events by which a hormone acti

vates cAMP signaling:
1. Initially Ga has bound GDP, and a,b, & g subuni
ts are complexed together.
 hormone
signal

outside

GPCR plasma
membrane

agga cytosol
AC
GDP bbGTP

GTP GDP ATP cAMP + PPi

2. Hormone binding to a 7-helix receptor (GPCR) c

auses a conformational change in the receptor tha
t is transmitted to the G protein.
The nucleotide-binding site on Ga becomes more ac
cessible to the cytosol, where [GTP] > [GDP].
Ga releases GDP & binds GTP (GDP-GTP exchang
 hormone
signal

outside

GPCR plasma
membrane

agga cytosol
AC
GDP bbGTP

GTP GDP ATP cAMP + PPi

3. Substitution of GTP for GDP causes another conf

ormational change in Ga.
Ga-GTP dissociates from the inhibitory bg complex &
can now bind to and activate Adenylate Cyclase.
 hormone
signal

outside

GPCR plasma
membrane

agga cytosol
AC
GDP bbGTP

GTP GDP ATP cAMP + PPi

4. Adenylate Cyclase, activated by the stimulatory

Ga-GTP, catalyzes synthesis of cAMP.
5. Protein Kinase A (cAMP Dependent Protein Kin
ase) catalyzes phosphorylation of various cellular p
roteins, altering their activity.
Turn off of the signal:
1. Ga hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Ga causes it to rebind t
o the inhibitory bg complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of
cAMP  AMP.
Turn off of the signal (cont.):
3. Receptor desensitization occurs. This process
varies with the hormone.
 Some receptors are phosphorylated via specifi
c receptor kinases.
 The phosphorylated receptor may then bind to a
protein b-arrestin, that promotes removal of th
e receptor from the membrane by clathrin-medi
ated endocytosis.
4. Protein Phosphatase catalyzes removal by hyd
rolysis of phosphates that were attached to protein
s via Protein Kinase A.
Signal amplification is an important feature of si
gnal cascades:

 One hormone molecule can lead to formation

of many cAMP molecules.

 Each catalytic subunit of Protein Kinase A cat

alyzes phosphorylation of many proteins durin
g the life-time of the cAMP.

View an animation of a G-protein signal cascade

.
 The stimulatory Gsa, when it binds GTP, acti
vates Adenylate cyclase.
 An inhibitory Gia, when it binds GTP, inhibit
s Adenylate cyclase.
Different effectors & their receptors induce Gia to
exchange GDP for GTP than those that activate
Gsa.
In some cells, the complex of Gb,g that is release
d when Ga binds GTP is itself an effector that bin
ds to and activates other proteins.
 protein-GTP (active)
Most GTP-binding prote
GDP
ins depend on helper
proteins: GEF GAP
GAPs, GTPase Activati GTP Pi
ng Proteins, promote G
TP hydrolysis. protein-GDP (inactive)

A GAP may provide an essential active site residue,

while promoting the correct positioning of the gluta
mine residue of the switch II domain.
Frequently a (+) charged arginine residue of a GA
P inserts into the active site and helps to stabilize th
e transition state by interacting with (-) charged O a
toms of the terminal phosphate of GTP during hydro
lysis.
 protein-GTP (active)

GDP

GEF GAP

GTP Pi

protein-GDP (inactive)

 Ga of a heterotrimeric G protein has innate capa

bility for GTP hydrolysis.
It has the essential arginine residue normally pro
vided by a GAP for small GTP-binding proteins.
 However, RGS proteins, which are negative reg
ulators of G protein signaling, stimulate GTP hyd
rolysis by Ga.
 protein-GTP (active)

GDP

GEF GAP
GEFs, Guanine Nucleoti GTP Pi
de Exchange Factors, pr
omote GDP/GTP excha protein-GDP (inactive)
nge.
 An activated receptor (GPCR) normally serves a
s GEF for a heterotrimeric G-protein.
 Alternatively, AGS (Activator of G-protein Signalin
g) proteins may activate some heterotrimeric G-pr
oteins, independent of a receptor.
Some AGS proteins have GEF activity.
Phosphatidylinositol Signal Cascad
es
O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H
1 6
OH OH
H OH
2 H 5
OH
phosphatidyl- H H
3 4
inositol H OH

Some hormones activate a signal cascade based

on the membrane lipid phosphatidylinositol.
 O

O H2C O C R2

R1 C O CH O

H2C O P O

O- H
1 6
OH OPO32-
H OH
2 OH H 5

H H
PIP2 3 4
phosphatidylinositol- H OPO32-
4,5-bisphosphate

Kinases sequentially catalyze transfer of Pi from AT

P to OH groups at positions 5 & 4 of the inositol ring,
to yield phosphatidylinositol-4,5-bisphosphate (P
IP2).
PIP is cleaved by the enzyme Phospholipase C.
 O
Different isoforms O H2C O C R2
of Phospholipase R1 C O CH O
C have different re H2C O P O
gulatory domains,
cleavage by O- H
& thus respond to 1 6
Phospholipase C OH OPO32-
different signals. H OH
5
2 OH H
A G-protein, Gq ac H H
PIP2 3 4
tivates one form of phosphatidylinositol- H OPO32-
Phospholipase C. 4,5-bisphosphate

When a particular GPCR (receptor) is activated, GTP

exchanges for GDP. Gqa-GTP activates Phospholip
ase C.
Ca++, which is required for activity of Phospholipase C
, interacts with (-) charged residues & with Pi moieties
 OPO32- H
1 6
OH OPO32- O
H OH
2 H 5 O H2C O C R2
OH
H H R1 C O CH
3 4
H OPO32- H2C OH
IP3
inositol-1,4,5-trisphosphate diacylglycerol

Cleavage of PIP2, catalyzed by Phospholipase C, yiel

ds 2 second messengers:
 inositol-1,4,5-trisphosphate (IP3)
 diacylglycerol (DG).
Diacylglycerol, with Ca++, activates Protein Kinase
C, which catalyzes phosphorylation of several cellula
r proteins, altering their activity.
 Ca++ calmodulin

IP3 Ca++-release channel

++
endoplasmic
View an ani Ca reticulum
mation.
Ca++-ATPase
ATP ++ ADP + Pi
Ca

IP3 activates Ca++-release channels in ER membran

es.
Ca++ stored in the ER is released to the cytosol, w
here it may bind calmodulin, or help activate Protein
Kinase C.
Signal turn-off includes removal of Ca++ from the c