Single-cell imaging of intracellular Ca2+ and phospholipase C activity reveals that RGS 2, 3, and 4 differentially regulate signaling via the Galphaq/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-mediated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], diacylglycerol, and Ca2+ signaling. In human embryonic kidney (HEK) 293 cells expressing recombinant Galpha(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 > or = 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 > or = 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.
Tovey
Stephen C
SC
Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.
Willars
Gary B
GB
D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D016259:Genes, myc; D006706:Homeostasis; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007668:Kidney; D016210:Methacholine Chloride / Q000494:pharmacology; D018613:Microscopy, Confocal; D020710:RGS Proteins / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D020710:RGS Proteins; C087182:RGS2 protein, human; C485898:RGS3 protein, human; D043587:Receptor, Muscarinic M3; D011994:Recombinant Proteins; D016210:Methacholine Chloride; C098799:RGS4 protein; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D002118:Calcium