Prolonged Kappa Opioid Receptor Phosphorylation Mediated by G-protein Receptor Kinase Underlies Sustained Analgesic Tolerance

Jay P. McLaughlin, Lisa C. Myers, Paul E. Zarek, Marc G. Caron, Robert J. Lefkowitz, Traci A. Czyzyk, John E. Pintar, Charles Chavkin

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Abstract

Kappa opioid receptor (KOR) desensitization was previously shown to follow agonist-dependent phosphorylation of serine 369 by G-protein receptor kinase (GRK) and β-arrestin binding in transfected cells. To study the in vivo effects induced by phosphorylation of KOR(S369), C57B1/6 mice were administered single or repeated doses of the KOR agonist, U50,488, and isolated brain glycoprotein was probed with an antibody, KOR-P, that specifically recognized phosphoserine 369 KOR. Western blot analysis using KOR-P antibody showed that labeling intensity increased after either single or repeated treatment of mice with U50,488 by 59 ± 22% and 101 ± 29%, respectively. In contrast, there was no change in labeling intensity by nonphosphoselective KOR antibodies following acute or chronic in vivo treatment with kappa agonist. Moreover, mice lacking GRK3 showed no increase in KOR-P labeling and developed significantly less analgesic tolerance following treatment with kappa agonist. The result suggests that tolerance to kappa agonists includes phosphorylation of serine 369 within KOR by GRK3. Recovery of analgesic potency and reduction of elevated KOR-P labeling in wild-type mice both required 2 weeks to return to base line. Consistent with these results, in vitro phosphorylation by GRK3 of KOR isolated from tolerant mice resulted in 46 ± 7% less 32P incorporation than in KOR isolated from untreated mice. In addition, in vitro 32P incorporation returned to base line levels only in KOR isolated from tolerant mice allowed to recover for 2 weeks. The coincident reversal of analgesic tolerance and slow return to a basal phosphorylation state matched the regeneration rate of functional kappa receptors following irreversible antagonism and suggested that receptor replacement rather than dephosphorylation was required to restore sensitivity.

Original languageEnglish (US)
Pages (from-to)1810-1818
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number3
DOIs
StatePublished - Jan 16 2004

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    McLaughlin, J. P., Myers, L. C., Zarek, P. E., Caron, M. G., Lefkowitz, R. J., Czyzyk, T. A., Pintar, J. E., & Chavkin, C. (2004). Prolonged Kappa Opioid Receptor Phosphorylation Mediated by G-protein Receptor Kinase Underlies Sustained Analgesic Tolerance. Journal of Biological Chemistry, 279(3), 1810-1818. https://doi.org/10.1074/jbc.M305796200