Single-Cell Imaging of Intracellular Ca2 and Phospholipase C Activity Reveals That RGS 2, 3, and 4 Differentially Regulate Signaling via the Gq/11-Linked Muscarinic M3 Receptor

Using single cell, real-time imaging, this study compared the impact of members of the B/R4 subfamily of the regulators of G-protein signaling (RGS) (RGS2,-3, and-4) on receptor-me-diated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], diacylglycerol, and Ca2 signaling. In human embryonic kidney (HEK) 293 cells expressing recombinant Gq/11-coupled muscarinic M3 recep-tors, transient coexpression of RGS proteins with fluorescently-tagged biosensors for either Ins(1,4,5)P3 or diacylglycerol dem-onstrated that RGS2 and 3 inhibited receptor-mediated events. Although gross indices of signaling were unaffected by RGS4, it slowed the rate of increase in Ins(1,4,5)P3 levels. At equivalent levels of expression, myc-tagged RGS proteins showed inhib-itory activity on the order RGS3 RGS2 RGS4. In HEK293 cells, stable expression of myc-tagged RGS2,-3, or-4 at equivalent levels also inhibited phosphoinositide and Ca2 sig-naling by endogenously expressed muscarinic M3 receptors in the order RGS3 RGS2 RGS4. In these cells, RGS2 or-3 reduced receptor-mediated inositol phosphate generation in cell populations and reduced both the magnitude and kinetics (rise-time) of single cell Ca2 signals. Furthermore, at low levels of receptor activation, oscillatory Ca2 signals were dampened or abolished, whereas at higher levels, RGS2 and-3 promoted the conversion of more stable Ca2 elevations into oscillatory signals. Despite little or no effect on responses to maximal receptor activation, RGS4 produced effects on the magnitude, kinetics, and oscillatory behavior of Ca2 signaling at submaxi-mal levels that were consistent with those of RGS2 and-3. The family of regulators of G-protein signaling (RGS) neg-atively regulate signaling by G-protein-coupled receptors (GPCRs) by binding to activated G-subunits and acting as either GTPase-activating proteins (GAPs) or effector antag