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1. Quanto lontano? probabilmente <1 mm Quanto veloce? da 100 ms a 10 s Il neurotrasmettitore o l’ormone si lega al recettore Attiva una G proteina Effettore: enzima o canale esterno interno a a g g GDP + Pi b b Risposta GTP Recettori accoppiati a proteine G

4. 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.

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

6. The bsubunit 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.

7. 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.

12. Outline the cyclic-AMP and phosphoinositide signal transduction cascades; differentiate between activation and inhibition of effector proteins by G protein subunits.

14. 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 bgcomplex. Adenylate Cyclase is no longer activated. 2. Phosphodiesterasecatalyzes hydrolysis of cAMPAMP.

15. 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.

17. 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.

20. 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 Gsafrom 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.

26. 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. lGi proteins (adenylate cyclase-inhibiting) - linked to a2-adrenergic receptor mGi1 protein mGi2 protein mGi3 protein lGo protein (Calcium or potassium channels modulators) lGq protein (Phospholipase C activator) linked to a1-adrenergic receptor lGs proteins (adenylate cyclase-stimulating) - linked to b-adrenergic receptor

27. 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.

28. Most GTP-binding proteins depend on helper proteins: GAPs, GTPase Activating Proteins, promote GTP hydrolysis. 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.

29. GEFs, Guanine Nucleotide Exchange Factors, promote GDP/GTP exchange. The activated receptor (GPCR) serves as GEF for a heterotrimeric G protein.

30. 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.

32. 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.