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Tyrosine phosphorylation of the kappa -opioid receptor regulates agonist efficacy
S M Appleyard1, J P McLaughlin, C Chavkin
1Department of Pharmacology and the Neurobiology Program, University of Washington, Seattle, Washington 98195-7280, USA.
Abstract:
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 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. However, neither KOR(Y87F)- nor KOR(Y157F)-mediated responses were potentiated by insulin treatment. Insulin pretreatment shifted the dose-response curve for activation of KOR by increasing the maximal response without changing the EC(50) value for. 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.
Insights
Insulin enhances kappa-opioid receptor (KOR) signaling by phosphorylating specific tyrosine residues. This tyrosine phosphorylation increases the receptor
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- G protein-coupled receptors (GPCRs) mediate diverse cellular responses.
- Opioid receptors, a class of GPCRs, are crucial targets for pain management.
- The role of tyrosine phosphorylation in GPCR regulation is not fully understood.
Purpose of the Study:
- To investigate the involvement of tyrosine residues in the cytoplasmic domains of kappa-opioid receptors (KOR).
- To determine if insulin receptor tyrosine kinase activity modulates KOR signaling.
- To elucidate the mechanism by which insulin affects KOR function.
Main Methods:
- Expression of rat kappa-opioid receptor (KOR) in Xenopus oocytes.
- Activation of KOR using an agonist and measurement of potassium currents through coexpressed G protein-gated inwardly rectifying potassium channels (K(IR)3).
- Treatment with insulin and tyrosine kinase inhibitor (genistein) to assess modulation of KOR activity.
- Site-directed mutagenesis of tyrosine residues (Y87F, Y157F) in KOR to study their role in insulin potentiation.
Main Results:
- Insulin pretreatment significantly potentiated the KOR-activated potassium current.
- Insulin's potentiation effect was blocked by the tyrosine kinase inhibitor genistein, implicating tyrosine kinase activity.
- Mutating tyrosine residues Y87 and Y157 in KOR abolished insulin-induced potentiation.
- Insulin increased the maximal response of KOR activation without altering its potency (EC50).
Conclusions:
- Insulin enhances kappa-opioid receptor (KOR) signaling efficacy through tyrosine phosphorylation.
- Specific tyrosine residues (Y87 and Y157) in the intracellular loops of KOR are critical for insulin-mediated potentiation.
- Tyrosine phosphorylation represents a novel regulatory mechanism for G protein-coupled receptor signaling.