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

Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom

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 G_q/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 > 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 > 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.

Address correspondence to: Dr. Gary B. Willars, Department of Cell Physiology and Pharmacology, Medical Sciences Building, University of Leicester, University Road, LE1 9HN, United Kingdom. E-mail: gbw2{at}le.ac.uk

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