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Originally published In Press as doi:10.1074/jbc.M111217200 on February 20, 2002

J. Biol. Chem., Vol. 277, Issue 18, 15523-15529, May 3, 2002
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Endogenous G Protein-coupled Receptor Kinase 6 Regulates M3 Muscarinic Acetylcholine Receptor Phosphorylation and Desensitization in Human SH-SY5Y Neuroblastoma Cells*

Jonathon M. WilletsDagger , R. A. John Challiss, and Stefan R. Nahorski

From the Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN, United Kingdom

We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M3 muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M3 mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 (K215RGRK6). K215RGRK6 expression inhibited methacholine-stimulated M3 mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5'-O-(3-[35S]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M3 mACh receptor-Galpha q/11 uncoupling was attenuated by 50% in cells expressing K215RGRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase K215RGRK5 had no effect on M3 mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [3H]inositol phosphate accumulations were more sustained in cells expressing K215RGRK6 compared with control and K215RGRK5-expressing cells, whereas K215RGRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF<UP><SUB><RM><IT>4</IT></RM></SUB><SUP><RM><IT>−</IT></RM></SUP></UP>. The ability of K215RGRK6 to inhibit agonist-mediated M3 mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M3 mACh receptor desensitization in the SH-SY5Y cell line.

* This work was supported by Wellcome Trust Grant 062495.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Cell Physiology and Pharmacology, University of Leicester, Maurice Shock Medical Sciences Bldg., University Rd., Leicester, LE1 9HN, UK. Tel.: 0116-252-3075; Fax: 0116-252-5045; E-mail: jmw23@le.ac.uk.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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