RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling

• Published in: American Journal of Physiology: Cell Physiology 2010-09, Vol.299 (3), p.C654-C664
• Main Authors: Laroche, Geneviève, Giguère, Patrick M, Roth, Bryan L, Trejo, Joann, Siderovski, David P
• Format: Article
• Language: English
• Subjects: Calcium - metabolism; Carbachol - pharmacology; Cells, Cultured; Cytokines; Gene expression; Gene Knockdown Techniques; Glycoproteins; Humans; Muscarinic Agonists - pharmacology; Oligopeptides - pharmacology; Peptides; Receptor, PAR-1 - agonists; Receptor, PAR-1 - metabolism; Receptors and Signal Transduction; Receptors, Muscarinic - metabolism; RGS Proteins - genetics; RGS Proteins - metabolism; Ribonucleic acid; RNA; RNA Interference; RNA, Small Interfering - genetics; Signal Transduction; T cell receptors
• ISSN: 0363-6143; 1522-1563; 1522-1563
• DOI: 10.1152/ajpcell.00441.2009

Regulator of G protein signaling (RGS) proteins are considered key modulators of G protein-coupled receptor (GPCR)-mediated signal transduction. These proteins act directly on Galpha subunits in vitro to increase their intrinsic rate of GTP hydrolysis; this activity is central to the prevailing view of RGS proteins as negative regulators of agonist-initiated GPCR signaling. However, the specificities of action of particular RGS proteins toward specific GPCRs in an integrated cellular context remain unclear. Here, we developed a medium-throughput assay to address this question in a wholly endogenous context using RNA interference. We performed medium-throughput calcium mobilization assays of agonist-stimulated muscarinic acetylcholine and protease-activated receptors in human embryonic kidney 293 (HEK293) cells transfected with individual members of a "pooled duplex" short interfering RNA library targeting all conventional human RGS transcripts. Only knockdown of RGS11 increased both carbachol-mediated calcium mobilization and inositol phosphate accumulation. Surprisingly, we found that knockdown of RGS8 and RGS9, but not other conventional RGS proteins, significantly decreased carbachol-mediated calcium mobilization, whereas only RGS8 knockdown decreased protease-activated receptor-1 (PAR-1)-mediated calcium mobilization. Loss of responsiveness toward carbachol and PAR-1 agonist peptide upon RGS8 knockdown appears due, at least in part, to a loss in respective receptor cell surface expression, although this is not the case for RGS9 knockdown. Our data suggest a cellular role for RGS8 in the stable surface expression of M3 muscarinic acetylcholine receptor and PAR-1, as well as a specific and opposing set of functions for RGS9 and RGS11 in modulating carbachol responsiveness similar to that seen in Caenorhabditis elegans.