Tyrosine phosphorylation of the κ-opioid receptor regulates agonist efficacy

To explore the role of highly conserved tyrosine residues in the putative cytoplasmic domains of the seven transmembrane G protein coupled opioid receptors, we expressed the rat kappa -opioid receptor (KOR) in Xenopus oocytes and then activated the intrinsic insulin receptor tyrosine kinase, KOR activation by the agonist U69593 produced a strong increase in potassium current through coexpressed G protein-gated inwardly rectifying potassium channels (K(IR)3), Brief pretreatment with insulin caused a 60% potentiation of the KOR-activated response. The insulin-induced increase in kappa -opioid response was blocked by the tyrosine kinase inhibitor genistein. In contrast, insulin had no effect on the basal activity of K(IR)3, suggesting that KOR is the target of the tyrosine kinase cascade. Mutation of tyrosine residues to phenylalanines in either the first or second intracellular loop of KOR to produce KOR(Y87F) and KOR(Y157F) had no effect on either the potency or maximal effect of U69593, However, neither KOR(Y87F)- nor KOR(Y157F)-mediated responses were potentiated by insulin treatment. Insulin pretreatment shifted the dose-response curve for U69593 activation of KOR by increasing the maximal response without changing the EC50 value for U69593. These results suggest that insulin increases the efficacy of KOR activation by phosphorylating two tyrosine residues in the first and second intracellular loops of the receptor. Thus, tyrosine phosphorylation may provide an important mechanism for modulation of G protein-coupled receptor signaling.