Abstract
Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active Gα subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be Gq-dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical, functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.
Footnotes
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This work was supported by the Association pour la Recherche contre le Cancer [Grant ARC 3756]; the Fonds National de la Recherche Scientifique, Belgium; and by Fondation pour la Recherche Medicale.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; RGS, regulator of G protein signaling; GAP, GTPase-activating protein; CCK, cholecystokinin; CCK2R, cholecystokinin-2 receptor; CCK9s, sulfated [Thr28,Nle31]-CCK25-33; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; IP, inositol phosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; PAGE, polyacrylamide gel electrophoresis; GST, glutathione transferase.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received August 29, 2008.
- Accepted December 5, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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