G-protein-linked receptors and intrinsic tyrosine-kinase growth receptors represent two prominent modalities in cell signaling. Cross-regulation among members of both receptor superfamilies has been reported, including the counter-regulatory effects of insulin on beta-adrenergic catecholamine action. Cells stimulated by insulin show loss of function and increased phosphotyrosine content of beta 2-adrenergic receptors. Phosphorylation of tyrosyl residues 350/354 of beta 2-adrenergic receptors is obligatory for counter-regulation by insulin (Karoor, V., Baltensperger, K., Paul, H., Czech, M., and Malbon, C. C. (1995) J. Biol. Chem. 270, 25305-25308), suggesting the hypothesis that G-protein-linked receptors themselves may act as substrates for the insulin receptor and other growth factor receptors. This hypothesis was evaluated directly using recombinant human insulin receptor, hamster beta 2-adrenergic receptor, and an vitro reconstitution and phosphorylation assay. Insulin is shown to stimulate insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor. Phosphoamino acid analysis establishes that insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor in vitro is confined to phosphotyrosine. High pressure liquid chromatography and two-dimensional mapping reveal insulin receptor-catalyzed phosphorylation of the beta 2-adrenergic receptor at residues Tyr132/Tyr141, Tyr350/Tyr354, and Tyr364, known sites of phosphorylation in response to insulin in vivo. Insulin-like growth factor-I receptor as well as the insulin receptor displays the capacity to phosphorylate the beta 2-adrenergic receptor in vitro, establishing a new paradigm, i.e. G-protein-linked receptors acting as substrates for intrinsic tyrosine kinase growth factor receptors.