Abstract
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 Gαq binding (GRK2-D110A, GRK2-R106A, and GRK2-R106A/K220R) had no effect on calcium signaling, suggesting that GRK2 primarily regulates Gq 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-1α (CK1α) and found that knockdown of CK1α 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.
Footnotes
-
This work was supported by National Institutes of Health grants GM44944 and GM47417 (to J.L.B). J.M.B. is supported by a predoctoral fellowship from the American Heart Association.
-
J.L. and J.M.B. contributed equally to this work.
-
ABBREVIATIONS: GPCR, G protein-coupled receptor; Bis I, bisindolymaleimide I; CK1α, casein kinase 1-α; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; GRK, G protein-coupled receptor kinase; IP3, inositol trisphosphate; M3 mAChR, muscarinic acetylcholine receptor subtype 3; PKC, protein kinase C; PLC-β, phospholipase C-β; HEK, human embryonic kidney; siRNA, small interfering RNA; AM, acetoxymethyl ester; McN-A-343, 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis; MEK, mitogen-activated protein kinase kinase.
- Received December 31, 2007.
- Accepted April 2, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
