TY - JOUR T1 - Single-Cell Imaging of Intracellular Ca<sup>2+</sup> and Phospholipase C Activity Reveals That RGS 2, 3, and 4 Differentially Regulate Signaling via the Gα<sub>q/11</sub>-Linked Muscarinic M<sub>3</sub> Receptor JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1453 LP - 1464 DO - 10.1124/mol.104.005827 VL - 66 IS - 6 AU - Stephen C. Tovey AU - Gary B. Willars Y1 - 2004/12/01 UR - http://molpharm.aspetjournals.org/content/66/6/1453.abstract N2 - 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-mediated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], diacylglycerol, and Ca2+ signaling. In human embryonic kidney (HEK) 293 cells expressing recombinant Gαq/11-coupled muscarinic M3 receptors, transient coexpression of RGS proteins with fluorescently-tagged biosensors for either Ins(1,4,5)P3 or diacylglycerol demonstrated 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 inhibitory activity on the order RGS3 ≥ RGS2 &gt; RGS4. In HEK293 cells, stable expression of myc-tagged RGS2, -3, or -4 at equivalent levels also inhibited phosphoinositide and Ca2+ signaling by endogenously expressed muscarinic M3 receptors in the order RGS3 ≥ RGS2 &gt; 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 submaximal levels that were consistent with those of RGS2 and -3. ER -