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

Geneviève Laroche, Patrick M. Giguère, Bryan L. Roth, Jo Ann Trejo, David P. Siderovski

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

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 Gα 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.

Original languageEnglish
Pages (from-to)C654-C664
JournalAmerican Journal of Physiology - Cell Physiology
Volume299
Issue number3
DOIs
StatePublished - Sep 2010

Keywords

  • Calcium flux
  • Muscarinic acetylcholine receptor
  • Protease-activated receptor-1
  • Regulators of G protein signaling
  • Short interfering RNA duplexes

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