SERUM AND INSULIN INDUCE A GRB2-DEPENDENT SHIFT IN AGONIST AFFINITY OF BETA-ADRENERGIC RECEPTORS

beta-Adrenergic receptors transduce catecholamine binding to activatio n of adenylylcyclase, a response counter-regulated by insulin. Insulin stimulates tyrosine phosphorylation of Tyr 350/354, which abolishes t he catecholamine response. Phosphorylation of Try 350/354 creates a Sr c homology 2 (SH2) domain on the beta(2)-adrenergic receptor and the b inding of adaptor protein Grb2 to this SH2 domain of the beta-adrenerg ic receptor takes place in an insulin-dependent manner. In membranes f rom serum-deprived S49 mouse lymphoma cells, GTP gamma S yields the we ll-known agonist specific shift in agonist affinity for beta(2)-adrene rgic: receptors. The agonist-specific shift is observed in cell membra nes either in the absence or in the presence of exogenously added puri fied Grb2. In membranes for serum-fed cells, in contrast, the addition of Grb2 induces an agonist-specific shift in receptor affinity that m imics addition of GTP gamma S to the membranes. The ability of the Grb 2 to induce an agonist-specific shift in the membranes from serum-fed cells was abolished equally effectively either by competition with pho sphopeptide harbouring the (p)YVNV motif or by disruption of the SH2 d omain of added Grb2. Challenging Chinese hamster ovary cells with insu lin (100 nM) for 30 min enabled Grb2 to induce an agonist-specific shi ft in agonist affinity for beta(2)-adrenergic receptors, suggestive of uncoupling of the receptors from G proteins. The insulin-dependent Gr b2 effect on receptor-G-protein coupling was sensitive to competition by the pYVNY phosphopeptide and to disruption of the SH2 domain of Grb 2. Tiles data provide a biochemical link between the ability of insuli n to counter-regulate catecholamine stimulation of cyclic AMP accumula tion and the phosphorylation of the P-adrenergic receptor, consequent biding of the adaptor molecule Grb2 and disruption of receptor-G-prote in coupling. CELL SIGNAL 10;8:575-582, 1998. (C) 1998 Elsevier Science Inc.