Received for publication August 5, 2004.
Revised September 17, 2004.
Accepted for publication September 17, 2004.

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

Abstract

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 Ins(1,4,5)P₃, diacylglycerol and Ca²⁺ signaling. In HEK293 cells expressing recombinant G_q/11-coupled muscarinic M₃ receptors, transient co-expression 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 in 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, either 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 whilst 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 behaviour of Ca²⁺ signaling at sub-maximal levels that were consistent with those of RGS2 and 3.

Key words: Gq/11 family, Phospholipase C's, IP3/DAG, Calcium (G Protein Coupled Signals), G protein regulation, RGS proteins, Ca imaging, Fluorescence techniques

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