Site Mutation in the Rat μ-Opioid Receptor Demonstrates the Involvement of Calcium/Calmodulin-Dependent Protein Kinase II in Agonist-Mediated Desensitization

Authors


Address correspondence and reprint requests to Dr. V. Höllt at the Department of Pharmacology and Toxicology, Otto-von-Guericke University, 39120 Magdeburg, Leipziger Str. 44, Germany.

Abstract

Abstract: The rat μ-opioid receptor (rMOR1), expressed in human embryonic kidney 293 (HEK293) cells, shows a desensitization to the inhibitory effect of the μ agonist DAMGO on adenylate cyclase activity within 4 h of DAMGO preincubation. To investigate the role of calcium/calmodulin-dependent protein kinase II (CaM kinase II) on μ-opioid receptor desensitization, we coexpressed rMOR1 and constitutively active CaM kinase II in HEK293 cells. This coexpression led to a faster time course of agonist-induced desensitization of the μ-opioid receptor. The increase of desensitization could not be observed with a μ-opioid receptor mutant (S261A/S266A) that lacks two putative CaM kinase II phosphorylation sites in the third intracellular loop. In addition, injection of CaM kinase II in Xenopus oocytes led only to desensitization of expressed rMOR1, but not of an S261A/S266A receptor mutant. These results suggest that phosphorylation of Ser261 and Ser266 by CaM kinase II is involved in the desensitization of the μ-opioid receptor.

Abbreviations used: CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; cAMP, cyclic AMP; DAMGO, [d-Ala2, MePhe4,Gly5-ol]enkephalin; GIRK, G protein-gated, inwardly rectifying K+ channel; HEK293, human embryonic kidney 293; HK, high K+; rMOR, rat μ-opioid receptor.

Ancillary