Insulin stimulates sequestration of beta-adrenergic receptors and enhancedassociation of beta-adrenergic receptors with Grb2 via tyrosine 350

G-protein-linked receptors, such as the beta(2)-adrenergic receptor, are su bstrates for growth factor receptors with intrinsic tyrosine kinase activit y (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-r egulatory action of insulin on catecholamine action is shown to stimulate e nhanced sequestration of beta(2)-adrenergic receptors in either DDT1MF-2 sm ooth muscle cells or Chinese hamster ovary cells stably expressing beta(2)- adrenergic receptors, Both insulin and insulin-like growth factor-1 stimula te internalization of beta-adrenergic receptors, contributing to the counte r-regulatory effects of these growth factors on catecholamine action, In co mbination with beta-adrenergic agonists, insulin stimulates internalization of 50-60% of the complement of beta-adrenergic receptors, Insulin administ ration in vitro and in vivo stimulates phosphorylation of Tyr-350 of the be ta-adrenergic receptor, creating an Src homology 2 domain available for bin ding of the adaptor molecule Grb2, The association of Grb2 with beta-adrene rgic receptors was established using antibodies to Grb2 as well as a Grb2-g lutathione S-transferase fusion protein. Insulin treatment of cells provoke s binding of Grb2 to beta(2)-adrenergic receptors, Insulin also stimulates association of phosphatidylinositol 3-kinase and dynamin, via the Src homol ogy 3 domain of Grb2, Both these interactions as well as internalization of the beta-adrenergic receptor are shown to be enhanced by insulin, beta-ago nist, or both, The Tyr-350 --> Phe mutant form of the beta(2)-adrenergic re ceptor, lacking the site for tyrosine phosphorylation, fails to bind Grb2 i n response to insulin, fails to display internalization of beta(2)-adrenerg ic receptor in response to insulin, and is no longer subject to the counter -regulatory effects of insulin on cyclic AMP accumulation. These data are t he first to demonstrate the ability of a growth factor insulin to counter-r egulate G-protein-Linked receptor, the beta-adrenergic receptor, via a new mechanism, Le. internalization.