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.
Highlights
Opioid receptors are widely expressed throughout the nervous system, and opioid drugs affect pain perception, learning and memory, epilepsy, and food intake, among other diverse functions [1]
Tyrosine residues in the putative cytoplasmic domains of G protein-coupled receptors are extremely common, and regulation of opioid receptor signaling by tyrosine kinase cascades would provide a powerful mechanism of cellular coordination
The latter is a mutant form of KIR3.2 that allows the 3.2 subunit to function as a homomultimer [17, 18], and KIR3.2(S146T) is insensitive to the effects of BDNF-activated tyrosine kinase cascades [12]
Summary
Chemicals—U69593 ((ϩ)-(5␣,7␣,8)-N-methyl-N-[7-(1-pyrrolidinyl)1-oxaspiro[4,5]dec-8-yl]benzeneacetamide) was obtained from Research Biochemicals International. Complementary DNA Clones and cRNA Synthesis—The rat -opioid receptor (KOR) was obtained from Dr David Grandy (GenBankTM/EBI accession number D16829). Plasmid templates for all constructs including KOR mutants were Oocyte Culture and Injection—Defolliculated stage IV oocytes were prepared as described [13] and incubated for 2–3 days after injection of the cRNAs in normal oocyte saline buffer (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 5 mM HEPES, pH 7.5) supplemented with sodium pyruvate (2.5 mM) and gentamycin (50 g/ml). Each oocyte was injected with 1 ng of KOR cRNA and either 0.1 ng of KIR3.1 and KIR3.2 wild-type potassium channel or 1 ng of KIR3.2(S146T) pore mutant channel cRNA. To facilitate the recording of inward Kϩ currents through the KIR3 channels, the normal oocyte saline buffer was modified to increase the KCl concentration to 96 mM Kϩ. Student’s t test was used for comparison of independent means, with values reported as two-tailed p values
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
