Beta-adrenergic receptors transduce catecholamine binding to activation of adenylylcyclase, a response counter-regulated by insulin. Insulin stimulates tyrosine phosphorylation of Tyr 350/354, which abolishes the catecholamine response. Phosphorylation of Try 350/354 creates a Src homology 2 (SH2) domain on the beta2-adrenergic receptor and the binding of adaptor protein Grb2 to this SH2 domain of the beta-adrenergic receptor takes place in an insulin-dependent manner. In membranes from serum-deprived S49 mouse lymphoma cells, GTPgammaS yields the well-known agonist-specific shift in agonist affinity for beta2-adrenergic receptors. The agonist-specific shift is observed in cell membranes either in the absence or in the presence of exogenously added purified Grb2. In membranes for serum-fed cells, in contrast, the addition of Grb2 induces an agonist-specific shift in receptor affinity that mimics addition of GTPgammaS to the membranes. The ability of the Grb2 to induce an agonist-specific shift in the membranes from serum-fed cells was abolished equally effectively either by competition with phosphopeptide harbouring the (p)YVNV motif or by disruption of the SH2 domain of added Grb2. Challenging Chinese hamster ovary cells with insulin (100 nM) for 30 min enabled Grb2 to induce an agonist-specific shift in agonist affinity for beta2-adrenergic receptors, suggestive of uncoupling of the receptors from G proteins. The insulin-dependent Grb2 effect on receptor-G-protein coupling was sensitive to competition by the pYVNY phosphopeptide and to disruption of the SH2 domain of Grb2. These data provide a biochemical link between the ability of insulin to counter-regulate catecholamine stimulation of cyclic AMP accumulation and the phosphorylation of the beta-adrenergic receptor, consequent biding of the adaptor molecule Grb2 and disruption of receptor-G-protein coupling.