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esterno
interno
GTP
GDP + Pi
Il neurotrasmettitore o l’ormone si lega al recettore
Attiva unaG proteina
Risposta
Effettore: enzima o canale
Recettori accoppiati a proteine GQuanto veloce?da 100 ms a 10 s
Quanto lontano?probabilmente <1 m
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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.
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Three switch domains have been identified, that change position when GTP substitutes for GDP on G. These
include residues adjacent to the terminal phosphates of GTP and/or the Mg++ associated with them.
Inhibitory G
GTPS
PDB 1GIA
The nucleotide binding site in each GTP-binding switch protein consists of loops that extend out from a -sheet, usually 6-stranded.
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The subunit of the heterotrimeric G Protein has a -propeller structure, formed from multiple repeats of a sequence called the WD-repeat.
The -propeller provides a stable structural support for residues that bind G.
G - side view of -propeller
PDB 1GP2
G – face view of -propeller
PDB 1GP2
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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.
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Outline the cyclic-AMP and phosphoinositide signal transduction cascades; differentiate between activation and inhibition of effector proteins by G protein subunits.
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Turn off of the signal (when AC is activated):
1. G hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on G causes it to rebind to the inhibitory complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of cAMP AMP.
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Turn off of the signal (cont.):
3. Hormone receptor desensitization occurs. This process varies with the hormone.
Some receptors are phosphorylated via G-protein-coupled 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.
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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.
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CH2
HHOH OH
H HOOP
O
HHOH OH
H HOCH2
N
N
N
NH2
OP
O
O
N O
(CH2)3
NH
C NH2+
protein
NH
O
H
CNH2
O
CH2
H
N
HOH OH
H HOOP
O
HHOH OH
H HOCH2
N
N
N
NH2
OP
O
O
O
N O
H
CNH2
O
NH
+
+
(CH2)3
NH
C NH2+
protein
NH2
NAD+
nicotinamideArg
residue
ADP-ribosylated protein
(nicotinamideadeninedinucleotide)
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Cholera toxin catalyzes covalent modification of Gs. ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gs.
This ADP-ribosylation prevents Gsfrom hydrolyzing GTP. Thus Gs becomes permanently activated.
Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of Gi, making the inhibitory G 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.
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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.
Gi proteins (adenylate cyclase-inhibiting) - linked to 2-adrenergic receptor Gi1 protein Gi2 protein Gi3 protein Go protein (Calcium or potassium channels modulators)
Gq protein (Phospholipase C activator) linked to 1-adrenergic receptor
Gs proteins (adenylate cyclase-stimulating) - linked to -adrenergic receptor
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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.
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A GAP may provide an essential active site residue, and/or promote a conformation that favors catalysis.
G 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 G.
Most GTP-binding proteins depend on helper proteins:
GAPs, GTPase Activating Proteins, promote GTP hydrolysis.
protein-GTP (active) GDP GEF GAP GTP Pi protein-GDP (inactive)
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GEFs, Guanine Nucleotide Exchange Factors, promote GDP/GTP exchange.
The activated receptor (GPCR) serves as GEF for a heterotrimeric G protein.
protein-GTP (active) GDP GEF GAP GTP Pi protein-GDP (inactive)
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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.
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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.
G protein Mutations Causing Disease