Akt mediates sequestration of the β₂-adrenergic receptor in response to insulin

The counterregulation of catecholamine action by insulin includes insulin-stimulated sequestration of the β₂-adrenergic receptor. Herein we examined the signaling downstream of insulin receptor activation, focusing upon the role of 1-phosphatidylinositol 3-kinase and the serine-threonine protein kinase Akt (also known as protein kinase B) in the internalization of β₂-adrenergic receptors. Inhibition of 1-phosphatidylinositol 3-kinase by LY294002 blocks insulin-induced sequestration of the β₂-adrenergic receptor, implicating Akt in downstream signaling to the β₂-adrenergic receptor. Phosphorylation studies of the C-terminal cytoplasmic domain of the β₂-adrenergic receptor by Akt in vitro identified Ser³⁴⁵ and Ser³⁴⁶ within a consensus motif for Akt phosphorylation. Double mutation (i.e. S345A/S346A) within this motif abolishes insulin counterregulation of β-adrenergic stimulation of cyclic AMP accumulation as well as insulin-stimulated sequestration. Furthermore, expression of constitutively activated Akt (T308D/S473D) mimics insulin action on cyclic AMP responses and β₂-adrenergic receptor internalization. Expression of the dominant-negative version of Akt (K179A/T308A/S473A), in contrast, abolishes both insulin counterregulation of the cyclic AMP response as well as insulin-stimulated sequestration of the β₂-adrenergic receptor. The action of the serine-threonine protein kinase Akt in insulin counterregulation mirrors the central role of protein kinase A in β-agonist-induced desensitization.