Phosphorylation of tyrosyl residues 350/354 of the β-adrenergic receptor is obligatory for counterregulatory effects of insulin

Insulin stimulates a loss of function and increased phosphotyrosine content of the β₂-adrenergic receptor in intact cells, raising the possibility that the β₂-receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the β₂-adrenergic receptor by the insulin receptor in vitro and peptide mapping of the β₂-adrenergic receptor phosphorylated in vivo in cells stimulated by insulin reveal tyrosyl residues 350/354 and 364 in the cytoplasmic, C-terminal region of the β₂-adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate β-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes β- adrenergic stimulation itself.