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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
protein
O
C
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
N
H
H
OH
H
OH
H
protein
Arg
C
residue
NH2+
ADP-ribosylated
C
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.