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

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania

We have used RNA interference previously to demonstrate that G protein-coupled receptor kinase 2 (GRK2) regulates endogenously expressed H1 histamine receptor in human embryonic kidney 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 after receptor activation, whereas GRK2 mutants defective in G_q binding (GRK2-D110A, GRK2-R106A, and GRK2-R106A/K220R) had no effect on calcium signaling, suggesting that GRK2 primarily regulates G_q after M₃ mAChR activation. The knockdown of arrestin-2 or arrestin-3 also significantly increased carbachol-mediated calcium mobilization. Knockdown of GRK2 and the arrestins also significantly enhanced carbachol-mediated activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), whereas 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, whereas GRK2 and arrestin-3 play the primary role in regulating ERK activation.

Address correspondence to: Dr. Jeffrey L. Benovic, Department of Biochemistry and Molecular Biology, Thomas Jefferson University, BLSB 350, Philadelphia, PA 19107. E-mail: benovic{at}mail.jci.tju.edu

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