Received for publication December 31, 2007.
Revised March 20, 2008.
Accepted for publication April 2, 2008.

M₃ Muscarinic Acetylcholine Receptor-Mediated Signaling is Regulated by Distinct Mechanisms

Abstract

We have previously used RNA interference to demonstrate that GRK2 regulates endogenously expressed H1 histamine receptor in HEK-293 cells. In this report, we investigate the regulation of endogenously expressed M₃ muscarinic acetylcholine receptor (M₃ mAchR). We show that knockdown of GRK2, GRK3 or GRK6, but not GRK5, significantly increased carbachol-mediated calcium mobilization. Stable-expression of wild-type GRK2 or a kinase-dead mutant (GRK2-K220R) reduced calcium mobilization following receptor activation, while GRK2 mutants defective in Gq binding (GRK2-D110A, GRK2-R106A and GRK2-R106A/K220R) had no effect on calcium signaling, suggesting that GRK2 primarily regulates Gq following M₃ mAchR activation. The knockdown of arrestin-2 or arrestin-3 also significantly increased carbachol-mediated calcium mobilization. Knockdown of GRK2 as well as the arrestins also significantly enhanced carbachol-mediated activation of ERK1/2 while prolonged ERK1/2 activation was only observed with GRK2 or arrestin-3 knockdown. We also investigated the role of casein kinase-1 (CK1) and found that knockdown of CK1 increased calcium mobilization but not ERK activation. In summary, our data suggest that multiple proteins dynamically regulate M₃ mAchR-mediated calcium signaling while GRK2 and arrestin-3 play the primary role in regulating ERK activation.

Key words: Muscarinic cholinergic, Gq/11 family, Desensitization/uncoupling, GRKs, barrestins

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