RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling.
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.
Laroche
Geneviève
G
Department of Pharmacology, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Giguère
Patrick M
PM
Roth
Bryan L
BL
Trejo
Joann
J
Siderovski
David P
DP
D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D006801:Humans; D018721:Muscarinic Agonists / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction
D018721:Muscarinic Agonists; D009842:Oligopeptides; D020710:RGS Proteins; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1; D011976:Receptors, Muscarinic; C483910:TFLLRNPNDK-NH2; D002217:Carbachol; D002118:Calcium