Mutation of tyrosine-141 inhibits insulin-promoted tyrosine phosphorylation and increased responsiveness of the human β2-adrenergic receptor

Abstract

The ability of insulin to promote phosphorylation of the human β2-adrenergic receptor (β2AR) was assessed in Chinese hamster fibroblasts transfected with β2AR cDNA. Phosphotyrosine residues were detected in purified β2AR using a polyclonal anti-phosphotyrosine antibody and by phosphoamino acid analysis following metabolic labelling with inorganic 32P. Treatment of the cells with insulin induced a 2.4-fold increase in the phosphotyrosine content of the receptor. The insulin-promoted phosphorylation of the β2AR was accompanied by an increase in the β-adrenergic-stimulated adenyl cyclase activity. Substitution of a phenylalanine residue for tyrosine-141 completely prevented both the increased tyrosine phosphorylation and the enhanced responsiveness of the β2AR promoted by insulin treatment. Mutation of three other tyrosines located in the cytoplasmic domain of the receptor, tyrosine-366, tyrosine-350 and tyrosine-354, did not abolish the insulin-promoted tyrosine phosphorylation. Taken together, these results suggest that insulin promotes phosphorylation of the β2AR on tyrosine-141 and that such phosphorylation leads to a supersensitization of the receptor.