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• W1968536472 abstract "Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Giand Gq class α 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 α 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. Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Giand Gq class α 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 α 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. Heterotrimeric G proteins of the Gq class transduce Ca2+ signaling by coupling heptahelical transmembrane receptors to the β isoforms of phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase Cβ; RGS proteins, regulators of G protein signaling; GAP, GTPase-activating proteins; WT, wild type; CCK, cholecystokinin; SLO, streptolysin O; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GDPβS, guanyl-5′-yl thiophosphate; IP3, inositol 1,4,5-trisphosphate. (1Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (925) Google Scholar). Many cells express multiple receptors that each activates the Gqsignaling pathway (2Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6175) Google Scholar). For example, pancreatic acinar cells respond to acetylcholine, bombesin and cholecystokinin (CCK) by intense activation of PLC to generate IP3 and mobilize Ca2+ from internal stores. In a recent study we showed that, although these three agonists activate the same Gq-regulated signaling pathway to mobilize the same Ca2+ pool, each agonist evokes a distinct pattern of Ca2+ wave propagation (3Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Ca2+ signaling in pancreatic acini triggers the exocytotic release of digestive enzymes from granules adjacent to the secretory membrane (4Muallem S. Lee M.L. Cell Calcium. 1997; 22: 1-4Crossref PubMed Scopus (21) Google Scholar). An intriguing question in cell signaling is how different agonists can stimulate the same Gq-coupled pathway to generate distinct spatial and/or temporal patterns of Ca2+ response within the same cell. Although signaling in pancreatic acinar cells is dependent on agonist binding to Gq-coupled receptors, several experiments suggested that Ca2+ release was also regulated by a novel mechanism that controlled G protein signaling. First, Ca2+ release evoked by carbachol, bombesin, or CCK was equally inhibited by titration with proteins that sequestered Gβγ and with antisera that specifically recognized Gqclass α subunits (3Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Second, these receptors promiscuously coupled to members of the Gq class α subunits (6Xu X. Croy J.T. Zeng W. Zhao L.-P. Davignon I. Popov S. Yu K. Jiang H. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 27275-27279Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Third, maximal stimulation with each of these agonists generated roughly equal levels of IP3. These results suggest that all three agonists stimulate their receptors to activate the same amount of Gαq/11. Nevertheless, we found that GTPγS differentially activated, and GDPβS differentially inhibited, signaling by the various agonists acting in these cells (3Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Xu X. Zeng W. Muallem S. J. Biol. Chem. 1996; 271: 11737-11744Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These effects of guanine nucleotides suggested that G protein activity was differentially regulated by an unknown intracellular protein(s) that acted in a receptor-dependent manner. G protein activity is regulated both by receptor-catalyzed GTP binding to the α subunit and by the rate of GTP hydrolysis. Regulators of G protein signaling (RGS) proteins are a recently identified family of intracellular GTPase-activating proteins (GAPs) (8Dolman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 9Berman D.M. Gilman A.G. J Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar) that accelerate GTP hydrolysis by Gα subunits (10Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 11Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 12Faurobert E. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2945-2950Crossref PubMed Scopus (94) Google Scholar, 13Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (310) Google Scholar, 14Popov S. Yu K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (149) Google Scholar, 15Heximer S.P. Cristillo A.D. Forsdyke D.R. DNA Cell Biol. 1997; 16: 589-598Crossref PubMed Scopus (59) Google Scholar), thus limiting the duration of G protein activation. RGS proteins may regulate signaling by uncoupling the cycle of GTP binding and hydrolysis from effector protein activation by the Gα subunit, even in the presence of persistent agonist stimulation. RGS proteins were first identified by genetic techniques to be inhibitors of G protein signaling (8Dolman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 16Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (477) Google Scholar, 17Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). Recent work has identified over 20 RGS proteins expressed in mammals (12Faurobert E. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2945-2950Crossref PubMed Scopus (94) Google Scholar, 16Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (477) Google Scholar, 18Yu J.H. Wieser J. Adams T.H. EMBO J. 1996; 15: 5184-5190Crossref PubMed Scopus (267) Google Scholar, 19Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (407) Google Scholar, 20De Vries L. Mousli M. Wurmser A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11916-11920Crossref PubMed Scopus (266) Google Scholar, 21Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry W.L.R. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar, 22Chen C.K. Wieland T. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12885-12889Crossref PubMed Scopus (124) Google Scholar).In vitro studies suggest that several mammalian RGS proteins, including RGS1, RGS4, and RGS16, have GAP activity toward different Gi and/or Gq class α subunits but not Gαs or Gα12 (10Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 11Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 15Heximer S.P. Cristillo A.D. Forsdyke D.R. DNA Cell Biol. 1997; 16: 589-598Crossref PubMed Scopus (59) Google Scholar, 23Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11184-11189Crossref PubMed Scopus (202) Google Scholar, 24Chen C. Zheng B. Han J. Lin S.C. J. Biol. Chem. 1997; 272: 8679-8685Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 25Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). RGS4 inhibited Gq-dependent PLCβ activation inXenopus oocytes and transfected COS and HEK293 cells (24Chen C. Zheng B. Han J. Lin S.C. J. Biol. Chem. 1997; 272: 8679-8685Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 25Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 26Huang C. Hepler J.R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6159-6163Crossref PubMed Scopus (151) Google Scholar, 27Doupnik C. Davidson N. Laster H.A. Kofugi P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (297) Google Scholar). Furthermore, addition of recombinant RGS4 protein to NG-108 cell membranes inhibited Gq-dependent PLCβ activity (28Saugstad J.A. Marino J.J. Folk J.A. Hepler J.R. Conn P.J. J. Neurosci. 1998; 18: 905-913Crossref PubMed Google Scholar, 29Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (336) Google Scholar). RGS2 was reported as a specific GAP for Gαq in an in vitro assay (30Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (314) Google Scholar), although it inhibited Gi-dependent signaling when expressed in transfected cells (31Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snow B.E. Barnes C.A. Lanahan A.A. Siderovski D.P. Ross E.M. Gilman A.G. Worley P.F. J. Neurosci. 1998; 18: 7178-7188Crossref PubMed Google Scholar). Because RGS proteins with similar GAP activities are co-expressed in cells within a single tissue (16Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (477) Google Scholar, 18Yu J.H. Wieser J. Adams T.H. EMBO J. 1996; 15: 5184-5190Crossref PubMed Scopus (267) Google Scholar,23Huang L.J. Durick K. Weiner J.A. Chun J. Taylor S.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11184-11189Crossref PubMed Scopus (202) Google Scholar, 29Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (336) Google Scholar, 30Heximer S.P. Watson N. Linder M.E. Blumer K.J. Hepler J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14389-14393Crossref PubMed Scopus (314) Google Scholar, 31Ingi T. Krumins A.M. Chidiac P. Brothers G.M. Chung S. Snow B.E. Barnes C.A. Lanahan A.A. Siderovski D.P. Ross E.M. Gilman A.G. Worley P.F. J. Neurosci. 1998; 18: 7178-7188Crossref PubMed Google Scholar), 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 α 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. In the present study, we examined the role of RGS4 and other RGS proteins in regulating Ca2+ signaling in pancreatic acinar cells. RGS4 inhibited Ca2+ signaling assayed either by measuring agonist-dependent Ca2+ mobilization in streptolysin O (SLO)-permeabilized cells or Ca2+-activated Cl− current in intact cells. The potency of RGS4 was exceedingly high in intact cells, and GTPγS reversed the inhibitory action of RGS4. This suggests that catalysis of GTPase activity is the dominant mechanism by which RGS4 regulates Ca2+ signaling. Furthermore, we provide the first evidence for receptor selectivity in RGS4 inhibition of PLCβ and Ca2+ signaling. Even more pronounced receptor selectivity was measured with RGS1 and RGS16. On the other hand, RGS2 showed similar potency in inhibiting m3- and CCK-dependent Ca2+ signaling. The potential role of the α subunits in determining differential sensitivity to RGS4 was analyzed using knockout mice. Deletion of individual Gq class α subunit genes or combinations thereof had no effect on the receptor-specific action of RGS4. Thus, specificity of RGS protein actions depends on their interaction with the receptor complex rather than their interaction with a specific Gq class α subunit. Recombinant RGS1, RGS2, RGS4, and RGS16 proteins were tagged at the N terminus with the sequence MH6MG using the pET19b and a modified pQE60 expression vector, respectively (Qiagen), expressed inEscherichia coli and purified as described (10Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar, 14Popov S. Yu K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (149) Google Scholar, 15Heximer S.P. Cristillo A.D. Forsdyke D.R. DNA Cell Biol. 1997; 16: 589-598Crossref PubMed Scopus (59) Google Scholar). Briefly, a 10-ml overnight culture grown at 37 °C in T7 medium supplemented with 100 μg/ml ampicillin and 1% glucose was used to inoculate 2 liter of T7/100 μg/ml ampicillin medium at 30 °C. Isopropyl-1-thio-β-d-galactopyranoside (0.5 mm) induction was performed at OD600 of 0.6–0.8, and cell cultures were shaken 4 h prior to harvest. Cells were pelleted and resuspended in TBP (50 mm Tris-HCl, pH 8.0, 20 mm β-mercaptoethanol, and 0.1 mmphenylmethylsulfonyl fluoride). Lysozyme (0.2 mg/ml) and DNase I (5 μg/ml) were added, and cells were inoculated on ice to complete lysis and DNA digestion. The total lysate was centrifuged (12,000 ×g for 30 min) at 4 °C, and the supernatant was loaded onto a 2-ml nickel-NTA column pre-equilibrated with TBP buffer. The column was washed with 20 ml of TBP and 0.2 m NaCl. The final wash was performed using 10 ml of TBP with 5 mmimidazole (pH 8.0). The protein was eluted twice with 2 ml elution buffer (TBP containing 200 mm imidazole, pH 8.0), dialyzed overnight against 1 liter of 50 mm HEPES, 2 mmdithiothreitol buffer (pH 8.0) at 4 °C, and further concentrated with a Centricon 10 device (Amicon). Mice deficient in Gα11 were produced as described (33Offermanns S. Zhao L.-P. Gohla A. Sarosi I. Simon M.I. Wilkie T.M. EMBO J. 1998; 17: 4304-4312Crossref PubMed Scopus (204) Google Scholar). Briefly, the murine Gα11 gene was disrupted by homologous recombination in mouse embryonic stem cells. Integration of the Gα11targeting vector replaced exons 3, 4, and a portion of 5 with several translation termination codons present in the reverse orientation of the PGK::Neo transgene. Gα11−/− mice are viable and fertile. Gα14 knockout mice were produced by disrupting the Gα14 gene in ES cells by homologous integration of a PGK::Neo expression cassette into the exon that encodes amino acid residues Ser-121 to Lys-154. Gα14−/− mice are viable and fertile with no apparent behavioral or morphologic defects. Gα11−/−;Gα14−/− double homozygous null mice were obtained from intercrossing the offspring of the single knockout mice. To produce Gα15−/− knockout mice, integration of the Gα15 targeting vector replaced exons 3, 4, and a portion of 5 with PGK::Neo in the inverse orientation. Gα15−/− mice are viable and fertile with no apparent behavioral or morphologic defects. Production of Gαq−/− mice was described (34Offermanns S. Hashimoto K. Watanabe M. Sun W. Kurihara H. Thompson R.F. Inoue Y. Kano M. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14089-14094Crossref PubMed Scopus (232) Google Scholar). Gαq−/−;Gα15−/− double homozygous null mice were obtained by intercrossing the offspring of the single knockout mice. All WT and mutant mice were of 129/SvEv × C57BL/6 genetic background. Two strains of double homozygous null mice were unavailable for this study. Gαq−/−;Gα11−/− mice could not be obtained as they die during embryogenesis (33Offermanns S. Zhao L.-P. Gohla A. Sarosi I. Simon M.I. Wilkie T.M. EMBO J. 1998; 17: 4304-4312Crossref PubMed Scopus (204) Google Scholar), and obtaining Gαq−/−;Gα14−/− mice from intercrossing the single knockouts is impractical because these genes are tandemly duplicated on mouse chromosome 19 (35Wilkie T.M. Gilbert D.J. Olsen A.S. Chen X.-N. Amatruda T.T. Korenberg J.R. Trask B.J. de Jong P. Reed R.R. Simon M.I. Jenkins N.A. Copeland N.G. Nat. Genet. 1992; 1: 85-91Crossref PubMed Scopus (213) Google Scholar) and are thus too close together to expect recombination to place both null mutations on the same chromosome. Expression of Gαq, Gα11, Gα14, and Gα15 in WT and knockout mice was assayed by Western blot as described (6Xu X. Croy J.T. Zeng W. Zhao L.-P. Davignon I. Popov S. Yu K. Jiang H. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 27275-27279Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 36Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar). Analysis of membrane proteins from pancreatic acini showed that the mutated α subunits were not expressed in the respective single and double knockout mice and the remaining Gq class α subunits were expressed at similar levels in WT and mutant mice (see also Refs. 6Xu X. Croy J.T. Zeng W. Zhao L.-P. Davignon I. Popov S. Yu K. Jiang H. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 27275-27279Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 33Offermanns S. Zhao L.-P. Gohla A. Sarosi I. Simon M.I. Wilkie T.M. EMBO J. 1998; 17: 4304-4312Crossref PubMed Scopus (204) Google Scholar, and 34Offermanns S. Hashimoto K. Watanabe M. Sun W. Kurihara H. Thompson R.F. Inoue Y. Kano M. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14089-14094Crossref PubMed Scopus (232) Google Scholar). Pancreatic acini and single pancreatic acinar cells from the rat and mouse pancreas were prepared by standard collagenase and trypsin digestion procedures as described (3Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 7Xu X. Zeng W. Muallem S. J. Biol. Chem. 1996; 271: 11737-11744Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The cells were suspended in solution A containing (in mm): NaCl 140, KCl 5, Hepes 10 (pH 7.4), MgCl2 1, CaCl2 1, and glucose 10. The cells were kept on ice until use. The procedures used to measure agonist- and IP3-evoked Ca2+ release in SLO-permeabilized pancreatic acini are essentially as described (3Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Xu X. Zeng W. Muallem S. J. Biol. Chem. 1996; 271: 11737-11744Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In brief, the acini were washed and suspended in a KCl-based permeabilization medium containing ATP regeneration system and SLO. The cells were allowed to reduce medium [Ca2+] to 50–100 nm before stimulation. The same protocol was used to measure IP3 production except that the volume of the reaction medium was increased from 400 to 500 μl. At designated times monitoring of [Ca2+] was interrupted to remove 25–50 μl of duplicate samples to 25–50 μl of an ice-cold 15% perchloric acid. At the end of the incubation periods with all agonists and at least 10 min after the last stimulation, precipitated proteins were removed by centrifugation and IP3 content of the supernatant were evaluated by a radioligand assay (3Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The whole cell configuration of the patch clamp technique (37Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15149) Google Scholar) was used to measure the effect of agonists on Cl−current. The pipette solution and recording conditions were set to optimize detection of the Ca2+-activated Cl−current as a reporter of changes in [Ca2+]i (for details, see Ref. 5Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). All experiments were performed at room temperature. The patch clamp output was filtered at 20 Hz. Recording was performed with pClamp 6 and a digi-Data 1200 interface (Axon Instruments). In all experiments, the Cl− and cation equilibrium potentials were about 0 mV. Cl− current was recorded at a holding potential of −40 mV. Current amplitude of stimulated cells was within 15% in a given cell preparation but varied between 50 and 150 pA/pF between cell preparations. Therefore, each experiment included at least one control and the inhibition of signaling by RGS proteins was compared with a control performed with a cell from the same preparation. Previous studies showed that RGS4 added to isolated membranes (28Saugstad J.A. Marino J.J. Folk J.A. Hepler J.R. Conn P.J. J. Neurosci. 1998; 18: 905-913Crossref PubMed Google Scholar, 29Hepler J.R. Berman D.M. Gilman A.G. Kozasa T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 428-432Crossref PubMed Scopus (336) Google Scholar) or overexpressed in cell lines (24Chen C. Zheng B. Han J. Lin S.C. J. Biol. Chem. 1997; 272: 8679-8685Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 25Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 26Huang C. Hepler J.R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6159-6163Crossref PubMed Scopus (151) Google Scholar, 27Doupnik C. Davidson N. Laster H.A. Kofugi P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (297) Google Scholar) inhibited Gq-dependent PLCβ activation. Fig.1 shows that addition of RGS4 to permeabilized rat pancreatic acinar cells effectively blocked agonist-dependent, Gq-coupled Ca2+release from internal stores. The permeabilized cells reduced [Ca2+] in the incubation medium to about 50 nm and responded to maximal stimulation with carbachol by releasing about 75% of the Ca2+ that is accessible to IP3 (Fig. 1 a). Addition of 50 nmRGS4 to the permeabilization medium inhibited about 85% of Ca2+ release triggered by carbachol (Fig. 1 b). Inhibition by RGS4 was fully reversible by addition of GTPγS (2.5 μm), a non-hydrolyzable GTP analog that activates Gα subunits, suggesting that inhibition of signaling reflects the GAP activity of RGS4 under these conditions. Consistent with this interpretation, RGS4 at concentrations up to 1.87 μm had no effect on Ca2+ release evoked by IP3 (Fig.1 e), which excludes the possibility that RGS4 inhibited the IP3-activated Ca2+ channel. An important finding shown in Fig. 1 is that RGS4 inhibited Ca2+ release stimulated by three different Gq-coupled receptors with markedly different potencies. Fig. 1 (b, d, ande) shows that, whereas 0.05 μm RGS4 inhibited 85% of Ca2+ release evoked by carbachol, 0.65 μm RGS4 only partially inhibited Ca2+ release and 1.87 μm RGS4 inhibited 85% of Ca2+release by CCK. The dependence of inhibition of Ca2+release on RGS4 concentration is illustrated in Fig.2. Half-maximal inhibition of Ca2+ mobilization induced by carbachol, bombesin, and CCK occurred at [RGS4] of approximately 35, 110, and 380 nm, respectively. Hence, cholinergic receptors were 3- and 10-fold more sensitive to RGS4 than were bombesin and CCK receptors. Addition of higher concentrations of any agonist did not alter the IC50of RGS4 (data not shown). The differential sensitivity of the Gq-coupled receptors to RGS4 inhibition was also reflected in activation of PLC and IP3 production in SLO-permeabilized cells. Fig.3 shows that the three agonists stimulated PLC activity to the same extent, indicating similar activation of Gq class α subunits by each agonist. Consistent with the concentration dependence of Ca2+signaling inhibition shown above, 50 nm and 0.2 μm RGS4 inhibited carbachol-stimulated IP3production by 60 ± 4% and 91 ± 7%, respectively. By contrast, 0.1 μm and 0.5 μm RGS4 was needed to inhibit bombesin-stimulated IP3 production by 41 ± 6% and 94 ± 7%, respectively. Finally, 0.4 μm and 2 μm RGS4 inhibited the effect of CCK on IP3production by 43 ± 7% and 77 ± 8%, respectively. Thus, RGS4 inhibition of both PLC activity and Ca2+ signaling were receptor-selective. The receptor-selective action of RGS4 might reflect preferential coupling of the receptors to different Gq class α subunits that respond differentially to RGS4. Alternatively, selectivity might be determined by receptor-specific interactions. We used mice genetically deficient in one or more of the Gq class α subunits that are expressed in pancreatic acinar cells (Gαq, Gα11, and Gα14) (Fig. 4 a; Ref. 6Xu X. Croy J.T. Zeng W. Zhao L.-P. Davignon I. Popov S. Yu K. Jiang H. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 27275-27279Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) to distinguish these possibilities. When Ca2+signaling was measured in intact acinar cells of homozygous knockout mice (Gαq−/−, Gα11−/−, and the double knockout lines Gα11−/−;Gα14−/− and Gαq−/−;Gα15−/−), maximal Ca2+ responses were identical in wild type and all mutant mice tested (6Xu X. Croy J.T. Zeng W. Zhao L.-P. Davignon I. Popov S. Yu K. Jiang H. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 27275-27279Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To evaluate the importance of the α subunit identity in conferring selectivity to RGS4 action, we compared the inhibition of Ca2+ signaling by RGS4 in permeabilized pancreatic acini from WT and Gq class knockout mice. Representative experiments are illustrated in Figs. 4 and5, and the results of all experiments are summarized in Table I. Fig. 4(b–e) shows the concentration of RGS4 needed to inhibit 50–60% of Ca2+ signaling evoked by each agonist in acini from WT mice. Fig. 5 (a–d, e–h, andi–l) show similar experiments for 50–60% inhibition of Ca2+ signaling by comparable concentrations of RGS4 in acini from the Gαq−/−;Gα15−/−, Gα11−/−, and Gα11−/−;Gα14−/− mice, respectively. The relative inhibitory potency of RGS4 was unaltered for each of the three agonists used here (Table I). The differential sensitivity to RGS4 among the different receptors was somewhat higher in mouse than in rat pancreas. In cells from WT mice, the response to carbachol was about 4.5- and 33-fold more sensitive to RGS4 than that of bombesin and CCK, respectively. Similar results were obtained in cells from Gα11−/−, Gα11−/−;Gα14−/−, and Gαq−/−;Gα15−/− mice. The findings in Table I and Figs. 4 and 5 suggest that interactions with receptor, rather than the identity of the G protein α subunit, dictate differential sensitivity to RGS4.Table IReceptor selectivity of RGS4 is unaltered in mice deficient in Gq class genesAgonistRelative inhibitory potency of RGS4 (agonist:carbachol ratio)aFold increase in RGS4 concentration in the incubation medium that is n" @default.
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• W1968536472 title "RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors" @default.
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