Insulin stimulates sequestration of β-adrenergic receptors and enhanced association of β-adrenergic receptors with Grb2 via tyrosine 350

G-protein-linked receptors, such as the β₂-adrenergic receptor, are substrates for growth factor receptors with intrinsic tyrosine kinase activity (Karoor, V., Baltensperger, K., Paul, H., Czech, M.P., and Malbon C. C. (1995) J. Biol. Chem. 270, 25305-25308). In the present work, the counter- regulatory action of insulin on catecholamine action is shown to stimulate enhanced sequestration of β₂-adrenergic receptors in either DDT₁MF-2 smooth muscle cells or Chinese hamster ovary cells stably expressing β₂- adrenergic receptors. Both insulin and insulin-like growth factor-1 stimulate internalization of β-adrenergic receptors, contributing to the counter- regulatory effects of these growth factors on catecholamine action. In combination with β-adrenergic agonists, insulin stimulates internalization of 50-60% of the complement of β-adrenergic receptors. Insulin administration in vitro and in vivo stimulates phosphorylation of Tyr-350 of the β-adrenergic receptor, creating an Src homology 2 domain available for binding of the adaptor molecule Grb2. The association of Grb2 with β- adrenergic receptors was established using antibodies to Grb2 as well as a Grb2-glutathione S-transferase fusion protein. Insulin treatment of cells provokes binding of Grb2 to β₂-adrenergic receptors. Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homology 3 domain of Grb2. Both these interactions as well as internalization of the β-adrenergic receptor are shown to be enhanced by insulin, β-agonist, or both. The Tyr-350 → Phe mutant form of the β₂- adrenergic receptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 in response to insulin, fails to display internalization of β₂- adrenergic receptor in response to insulin, and is no longer subject to the counter-regulatory effects of insulin on cyclic AMP accumulation. These data are the first to demonstrate the ability of a growth factor insulin to counter-regulate G-protein-linked receptor, the β-adrenergic receptor, via a new mechanism, i.e. internalization.