RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors

Shmuel Muallem,Serguei Popov,Thomas M. Wilkie,Weizhong Zeng,Stefan Offermanns,David Yowe,Kan Yu,Xin Xu,David M. Berman,Isabelle Davignon

doi:10.1074/jbc.274.6.3549

Shmuel Muallem, Serguei Popov + Show 8 more

Open Access

https://doi.org/10.1074/jbc.274.6.3549

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Abstract

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Gi and Gq class alpha subunits. In the present study, we used knockouts of Gq class genes in mice to evaluate the potency and selectivity of RGS4 in modulating Ca2+ signaling transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+ signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+ signaling by RGS4 was observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol was about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent inhibitors of Gq-dependent Ca2+ signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 was observed in acinar cells from wild type and several single and double Gq class knockout mice. Thus, these receptors appear to couple Gq class alpha subunit isotypes equally. Difference in receptor selectivity of RGS proteins action indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes.

Highlights

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by G␣ subunits, thereby attenuating signaling
We examined the role of RGS4 and other RGS proteins in regulating Ca2ϩ signaling in pancreatic acinar cells
Inhibition of Ca2ϩ Signaling by RGS4 in Permeable Cells—Previous studies showed that RGS4 added to isolated membranes [28, 29] or overexpressed in cell lines (24 –27) inhibited Gq-dependent phospholipase C (PLC)␤ activation

Summary

RGS Proteins and Signaling Specificity

RGS4 protein to NG-108 cell membranes inhibited Gq-dependent PLC␤ activity [28, 29]. RGS2 was reported as a specific GAP for G␣q in an in vitro assay [30], it inhibited Gi-dependent signaling when expressed in transfected cells [31]. Because RGS proteins with similar GAP activities are co-expressed in cells within a single tissue (16, 18, 23, 29 –31), the mechanisms that may provide more precise regulatory specificity have been enigmatic. To date there is no information on the potency or selectivity with which mammalian RGS proteins modulate the same G protein ␣ subunit during its response to different receptors. This highlights the need to analyze RGS proteins in intact cells under controlled conditions to assess their potency and specificity of action. The potency of RGS4 was exceedingly high in intact cells, and GTP␥S reversed the inhibitory action of RGS4 This suggests that catalysis of GTPase activity is the dominant mechanism by which RGS4 regulates Ca2ϩ signaling. Specificity of RGS protein actions depends on their interaction with the receptor complex rather than their interaction with a specific Gq class ␣ subunit

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION