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J. Biol. Chem., Vol. 280, Issue 19, 18950-18958, May 13, 2005

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Roles of Phosphorylation-dependent and -independent Mechanisms in the Regulation of M₁ Muscarinic Acetylcholine Receptors by G Protein-coupled Receptor Kinase 2 in Hippocampal Neurons^*

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

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

When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PH_PLC, G protein-coupled receptor kinase 2 (GRK2) can suppress M₁ muscarinic acetylcholine (mACh) receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism, most likely involving the direct binding of the RGS homology domain of GRK2 to G_q/11. To define the importance of this mechanism in comparison with classical, phosphorylation-dependent receptor regulation by GRKs, we have examined M₁ mACh receptor signaling in hippocampal neurons following depletion of GRK2 and also in the presence of non-G_q/11-binding GRK2 mutants. Depletion of neuronal GRK2 using an antisense strategy almost completely inhibited M₁ mACh receptor desensitization without enhancing acute agonist-stimulated phospholipase C activity. By stimulating neurons with a submaximal agonist concentration before (R1) and after (R2) a period of exposure to a maximal agonist concentration, an index (R2/R1) of agonist-induced desensitization of signaling could be obtained. Co-transfection of neurons with either a non-G_q/11-binding (D110A) GRK2 mutant or the catalytically inactive ^D110A,K220RGRK2 did not suppress acute M₁ mACh receptor-stimulated inositol 1,4,5-trisphosphate production. However, using the desensitization (R2/R1) protocol, it could be shown that expression of ^D110AGRK2 enhanced, whereas ^D110A,K220RGRK2 inhibited, agonist-induced M₁ mACh receptor desensitization. In Chinese hamster ovary cells, the loss of G_q/11 binding did not affect the ability of the ^D110AGRK2 mutant to phosphorylate M₁ mACh receptors, whereas expression of ^D110A,K220RGRK2 had no effect on receptor phosphorylation. These data indicate that in hippocampal neurons endogenous GRK2 is a key regulator of M₁ mACh receptor signaling and that the regulatory process involves both phosphorylation-dependent and -independent mechanisms.

Received for publication, November 9, 2004 , and in revised form, February 16, 2005.

^* This work is supported by Program Grant 062495 from the Well-come Trust of Great Britain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 44-116-252-3087; Fax: 44-116-252-5045; E-mail: jmw23{at}le.ac.uk.

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