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Molecular Pharmacology

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Research ArticleArticle

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

Stephen C. Tovey and Gary B. Willars
Molecular Pharmacology December 2004, 66 (6) 1453-1464; DOI: https://doi.org/10.1124/mol.104.005827
Stephen C. Tovey
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Gary B. Willars
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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 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 > 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.

  • Received August 5, 2004.
  • Accepted September 17, 2004.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 66 (6)
Molecular Pharmacology
Vol. 66, Issue 6
1 Dec 2004
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Research ArticleArticle

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

Stephen C. Tovey and Gary B. Willars
Molecular Pharmacology December 1, 2004, 66 (6) 1453-1464; DOI: https://doi.org/10.1124/mol.104.005827

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Research ArticleArticle

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

Stephen C. Tovey and Gary B. Willars
Molecular Pharmacology December 1, 2004, 66 (6) 1453-1464; DOI: https://doi.org/10.1124/mol.104.005827
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