esterno interno GTP GDP + P i    Il neurotrasmettitore o l’ormone si lega al recettore Attiva una G proteina    Risposta Effettore: enzima o canale Recettori accoppiati a proteine G Quanto veloce? da 100 ms a 10 s Quanto lontano? probabilmente <1  m

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.

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. The nucleotide binding site in each GTP-binding switch protein consists of loops that extend out from a  -sheet, usually 6-stranded.

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 .

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. G  hydrolyzes GTP to GDP + P i. (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.

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.

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.

Cholera toxin catalyzes covalent modification of G s . ADP-ribose is transferred from NAD + to an arginine residue at the GTPase active site of G s . This ADP-ribosylation prevents G s   from hydrolyzing GTP. Thus G s  becomes permanently activated. Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of G i , 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.

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

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 ) A ll 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.

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.

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 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