Recettori accoppiati a proteine G
Il neurotrasmettitore o
l’ormone si lega al recettore
Quanto veloce?
da 100 ms a 10 s
Quanto lontano?
probabilmente <1 mm
Attiva una
G proteina
Effettore:
enzima o canale
esterno
interno
b g
a
a
GDP + Pi
Risposta
GTP
b g
The alpha subunit of the heterotrimeric G protein is shown as a ribbon; the
guanine nucleotide is spacefilled. P-alpha, P-beta, and P-gamma indicate the
three phosphoryl groups in the GTP structure. As with most nucleoside
triphosphates, there is a magnesium ion associated with GTP. The "ras-like"
domain contains the catalytic residues that promote GTP hydrolysis.
PDB 1GIA
The nucleotide binding
site in each GTP-binding
switch protein consists of
loops that extend out
from a b-sheet, usually
6-stranded.
GTPgS
Inhibitory Ga
Three switch domains have been identified, that change
position when GTP substitutes for GDP on Ga. These
include residues adjacent to the terminal phosphates of
GTP and/or the Mg++ associated with them.
PDB 1GP2
PDB 1GP2
Gb - side view of b-propeller
Gb – face view of b-propeller
The b subunit of the heterotrimeric G Protein has a
b-propeller structure, formed from multiple repeats of a
sequence called the WD-repeat.
The b-propeller provides a stable structural support for
residues that bind Ga.
The alpha subunit is usually modified by a fatty acyl lipid anchor. The
gamma subunit is usually modified by an isoprenoid lipid anchor. Both lipid
anchors (zig-zag lines) permit lateral diffusion, protein-lipid, and protein-protein
interactions.
Outline the cyclic-AMP and phosphoinositide signal
transduction cascades; differentiate between activation
and inhibition of effector proteins by G protein subunits.
Turn off of the signal (when AC is activated):
1. Ga hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Ga causes it to rebind to the
inhibitory bg complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of
cAMP  AMP.
Turn off of the signal (cont.):
3. Hormone receptor desensitization occurs.
This process varies with the hormone.
 Some receptors are phosphorylated via G-proteincoupled receptor kinases (GRK).
 The phosphorylated receptor may then bind to a
protein arrestin that blocks receptor-G-protein
activation and promotes removal of the receptor from
the membrane by clathrin-mediated endocytosis.
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to proteins
via Protein Kinase A.
The Gs-alpha and Gi-alpha subunits both interact with adenylyl cyclase
isoforms. Their actions, however, are opposite: Gs stimulates and Gi inhibits the
synthesis of cyclic-AMP from ATP. The actions of these two alpha subunits may
be differentiated in the laboratory by certain bacterial toxins.
H
O
C
protein
NH2
O
+
N

O P O CH2 O
H
H
H
H
OH
OH
NH2
O
N
(CH2)3
NH
C
O
NH

O P O CH2 O
H
H
H
H
OH
OH
NH2
O
N

N
O P O CH2 N
O
O
H
H
H
H
+
NAD
OH
OH
(nicotinamide
adenine
dinucleotide)

O P O CH2
O
(CH2)3
H
NH
NH2
N
N
NH2+
+
N
H
N
O
O
C
N
H
H
OH
H
OH
H
protein
Arg
C
residue
NH2+
ADP-ribosylated
protein
NH2
nicotinamide
Cholera toxin catalyzes covalent modification of Gsa.
ADP-ribose is transferred from NAD+ to an arginine
residue at the GTPase active site of Gsa.
This ADP-ribosylation prevents Gsa from hydrolyzing
GTP. Thus Gsa becomes permanently activated.
Pertussis toxin (whooping cough disease) catalyzes
ADP-ribosylation at a cysteine residue of Gia, making
the inhibitory Ga 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.
G proteins (guanine nucleotide (GTP) -binding proteins)
G proteins, once activated, will cause the activation of several
intracellulareffectors: adenyl cyclase, cGMP phosphodiesterase, phospholipase
C, phospholipase A2, and calcium or potassium channels.
l
Gi proteins (adenylate cyclase-inhibiting) - linked to a2-adrenergic
receptor
m Gi1 protein
m Gi2 protein
m Gi3 protein
l
Go protein (Calcium or potassium channels modulators)
l
Gq protein (Phospholipase C activator) linked to a1-adrenergic
receptor
l
Gs proteins (adenylate cyclase-stimulating) - linked to b-adrenergic
receptor
Small GTP-binding proteins include (roles indicated):





initiation & elongation factors (protein synthesis).
Ras (growth factor signal cascades).
Rab (vesicle targeting and fusion).
Ran (transport of proteins into & out of the nucleus).
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.
protein-GTP (active)
Most GTP-binding
proteins depend on
helper proteins:
GAPs, GTPase Activating
Proteins, promote GTP
hydrolysis.
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
A GAP may provide an essential active site residue,
and/or promote a conformation that favors catalysis.
Ga of a heterotrimeric G protein has innate capability for
GTP hydrolysis.
However, RGS proteins, which are negative regulators
of G protein signaling, function as GAPs to 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.
The regulation of G protein signalling
Left panel RGS proteins bind to G, stimulate GTP hydrolysis, and thereby
reverse G protein activation.
Right panel, the roles of a receptor, G, and an RGS are completely
analogous to the GDSs, GDIs, and GAPs that regulate small monomeric G
proteins like Ras.
G protein Mutations Causing Disease
G protein alpha subunit in its GTP-bound form, highlighting amino acids changed by point
mutations that cause human endocrine diseases. Mutational replacements of red residues
impair GTP hydrolysis; these sites are mutated in growth hormone secreting tumors of the
pituitary. Replacement of either cyan residue produces an inactive G protein alpha
subunit, causing pseudohypoparathyroidism. Bound nucleotide is light green.