TY - JOUR
T1 - RNA interference screen for RGS protein specificity at muscarinic and protease-activated receptors reveals bidirectional modulation of signaling
AU - Laroche, Geneviève
AU - Giguère, Patrick M.
AU - Roth, Bryan L.
AU - Trejo, Jo Ann
AU - Siderovski, David P.
PY - 2010/9
Y1 - 2010/9
N2 - 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.
AB - 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.
KW - Calcium flux
KW - Muscarinic acetylcholine receptor
KW - Protease-activated receptor-1
KW - Regulators of G protein signaling
KW - Short interfering RNA duplexes
UR - http://www.scopus.com/inward/record.url?scp=77956609549&partnerID=8YFLogxK
U2 - 10.1152/ajpcell.00441.2009
DO - 10.1152/ajpcell.00441.2009
M3 - Article
C2 - 20573995
AN - SCOPUS:77956609549
VL - 299
SP - C654-C664
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
SN - 0363-6143
IS - 3
ER -