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• W2100629398 abstract "Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gqclass heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cβ either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and β-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gsand Gi. This inference was based on the inhibitory action of antibodies specific for Gαs, Gαi, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of β-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Gα15 −/− knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors. Many Gs-coupled receptors can activate both cAMP and Ca2+ signaling pathways. Three mechanisms for dual activation have been proposed. One is receptor coupling to both Gs and G15 (a Gqclass heterotrimeric G protein) to initiate independent signaling cascades that elevate intracellular levels of cAMP and Ca+2, respectively. The other two mechanisms involve cAMP-dependent protein kinase-mediated activation of phospholipase Cβ either directly or by switching receptor coupling from Gs to Gi. These mechanisms were primarily inferred from studies with transfected cell lines. In native cells we found that two Gs-coupled receptors (the vasoactive intestinal peptide and β-adrenergic receptors) in pancreatic acinar and submandibular gland duct cells, respectively, evoke a Ca2+ signal by a mechanism involving both Gsand Gi. This inference was based on the inhibitory action of antibodies specific for Gαs, Gαi, and phosphatidylinositol 4,5-bisphosphate, pertussis toxin, RGS4, a fragment of β-adrenergic receptor kinase and inhibitors of cAMP-dependent protein kinase. By contrast, Ca2+ signaling evoked by Gs-coupled receptor agonists was not blocked by Gq class-specific antibodies and was unaffected in Gα15 −/− knockout mice. We conclude that sequential activation of Gs and Gi, mediated by cAMP-dependent protein kinase, may represent a general mechanism in native cells for dual stimulation of signaling pathways by Gs-coupled receptors. A family of heterotrimeric guanine nucleotide-binding proteins (G proteins) transduces a variety of signals across the plasma membrane by sequential interactions with receptor and effector proteins (e.g. second messenger-generating enzymes and ion channels). These interactions result from guanine nucleotide-driven conformational changes in G protein α subunits (1Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (922) Google Scholar). Agonist-bound receptors catalyze the exchange of GDP for GTP on the α subunits of their cognate G proteins to promote dissociation of α from a high affinity complex of βγ subunits. Dissociated subunits are competent to modulate the activity of effectors. GTP hydrolysis ultimately returns Gα to the GDP-bound state, thus allowing reformation of inactive heterotrimer. Sixteen distinct genes encode G protein α subunits in mammals. The family is commonly divided into four classes based on amino acid sequence identity and function: Gs, Gi, Gq, and G12. Members of a newly identified family of regulators of G protein signaling (RGS proteins) 1The abbreviations used are: RGS, regulator of G protein signaling; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol trisphosphate; PLCβ, phospholipase Cβ; PKA, cAMP-dependent protein kinase; VIP, vasoactive intestinal peptide; Iso, isoprenaline; βARK1, β-adrenergic receptor kinase 1; PTX, pertussis toxin; SMG, submandibular gland; SLO, streptolysin O toxin; Rs, Gs-coupled receptor; Rq, Gq-coupled receptor; Rp-8-CPT-cAMP-S, 8-(4-chlorophenylthio)adenosine cyclic 3′,5′-phosphorothioate; [Ca2+]i, intracellular Ca2+. 1The abbreviations used are: RGS, regulator of G protein signaling; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol trisphosphate; PLCβ, phospholipase Cβ; PKA, cAMP-dependent protein kinase; VIP, vasoactive intestinal peptide; Iso, isoprenaline; βARK1, β-adrenergic receptor kinase 1; PTX, pertussis toxin; SMG, submandibular gland; SLO, streptolysin O toxin; Rs, Gs-coupled receptor; Rq, Gq-coupled receptor; Rp-8-CPT-cAMP-S, 8-(4-chlorophenylthio)adenosine cyclic 3′,5′-phosphorothioate; [Ca2+]i, intracellular Ca2+. have been shown to stimulate the GTPase activity of Gi and Gqclass α subunits, thus attenuating signaling (2Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). One of the more thoroughly characterized examples of G protein-mediated signal transduction is carried out by the hormone-sensitive adenylyl cyclase system. Relevant receptors communicate with homologous G proteins, one of which (Gs) activates adenylyl cyclase while others (Gi) inhibit the enzyme (1Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (922) Google Scholar). The second messenger (cAMP) mediates diverse cellular responses, primarily by activating cAMP-dependent protein kinase (PKA). In the case of Ca2+-mobilizing agonists, G protein activation is followed by stimulation of phospholipase Cβ (PLCβ) to generate IP3 in the cytosol, which initiates the [Ca2+]i signal by release of Ca2+from internal stores (1Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (922) Google Scholar, 3Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6165) Google Scholar). PLCβ can be activated by each of the four Gq class α subunits or by Gβγ subunits released from Gi class proteins (4Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (349) Google Scholar). Only Gi-mediated PLCβ activation is inhibited by pertussis toxin (4Singer W.D. Brown H.A. Sternweis P.C. Annu. Rev. Biochem. 1997; 66: 475-509Crossref PubMed Scopus (349) Google Scholar). In this study we sought to learn the mechanism by which Gs-coupled receptors evoke Ca2+ signaling. Several Gs-coupled receptors can activate dual signaling cascades. For example, increases in both cAMP and [Ca2+]i have been observed by histamine acting on H2 receptors in parietal cells (5Chew C.S. Am. J. Physiol. 1986; 250: G814-G823PubMed Google Scholar), parathyroid hormone acting on osteoblasts (6Yamaguchi D.T. Hahn T.J. Iida-Klein A. Kleeman C.R. Muallem S. J. Biol Chem. 1987; 262: 7711-7718Abstract Full Text PDF PubMed Google Scholar), and isoprenaline acting on cardiac myocytes (7Xiao R.P. Ji X. Lakatta E.G. Mol. Pharmacol. 1995; 47: 322-329Crossref PubMed Scopus (199) Google Scholar) or salivary gland cells (8Dehaye J.P. Valdez I.H. Turner R.J. Am. J. Physiol. 1993; 265: C1356-C1362Crossref PubMed Google Scholar, 9Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar). In contrast to the simple paradigm that each receptor molecule can activate a single class of G protein (10Gudermann T. Schoneberg T. Schultz G. Annu. Rev. Neurosci. 1997; 20: 399-427Crossref PubMed Scopus (252) Google Scholar), activation of more than one signaling cascade could be due to coupling of one receptor type to two classes of G proteins. This model is supported by experiments in heterologous expression systems. Overexpression of histaminergic H2 (11Kuhn B. Schmid A. Harteneck C. Gudermann T. Schultz G. Mol. Endocrinol. 1996; 10: 1697-1707Crossref PubMed Scopus (74) Google Scholar), parathyroid hormone (12Offermanns S. Iida-Klein A. Segre G.V. Simon M.I. Mol. Endocrinol. 1996; 10: 566-574Crossref PubMed Google Scholar), luteinizing hormone (13Herrlich A. Kuhn B. Grosse R. Schmid A. Schultz G. Gudermann T. J. Biol. Chem. 1996; 271: 16764-16772Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), P2Y11 (14Communi D. Govaerts C. Parmentier M. Boeynaems J.M. J. Biol. Chem. 1997; 272: 31969-31973Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar), vasopressin V2, dopamine D1A, and adenosine A2A (15Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar) receptors resulted in stimulation of adenylyl cyclase and PLCβ. The β-adrenergic receptor (which is considered to be a classical Gs-coupled receptor) and the vasopressin V2, dopamine D1A, and adenosine A2A can functionally interact with the Gq family member, G15, when both proteins are overexpressed in COS cells (15Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 16Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16011Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). An alternate mechanism for stimulation of Ca2+ signaling by Gs-coupled receptors is activation of PLCβ by PKA. In several cell types, increasing cellular cAMP with forskolin (5Chew C.S. Am. J. Physiol. 1986; 250: G814-G823PubMed Google Scholar, 8Dehaye J.P. Valdez I.H. Turner R.J. Am. J. Physiol. 1993; 265: C1356-C1362Crossref PubMed Google Scholar, 9Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar) or membrane permeable cAMP analogues (5Chew C.S. Am. J. Physiol. 1986; 250: G814-G823PubMed Google Scholar) increased [Ca2+]i similar to stimulation of Gs-coupled receptors. In a recent study we showed that stimulation of submandibular gland (SMG) duct cells with forskolin results in PLCβ-mediated and IP3-dependent Ca2+ release from internal stores (9Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar). These findings suggest that, at least in some cell types, stimulation of PKA can activate PLCβ to generate a Ca2+ signal. Phosphorylation-dependent switching of receptor specificity for G proteins is another mechanism by which a single receptor could activate more than one G protein (17Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1070) Google Scholar). As outlined recently by Lefkowitz (18Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar), receptor-dependent activation of Gs stimulates adenylyl cyclase, generates cAMP, and activates PKA. Phosphorylation of the receptor by PKA is proposed to switch its coupling specificity from Gs to Gi. Receptor-dependent activation of Gi could thus release sufficient Gβγ to activate PLCβ. Activation of PLCβ generates IP3 (which releases Ca2+ from internal stores) and diacylglycerol to activate protein kinase C. Hence, PKA-dependent switching of receptor coupling to different classes of G proteins (the Gs/Giswitching model) is a potential mechanism for activation of multiple signal transduction cascades by the same receptor. In the work presented here we sought to determine if any of the above models applied to classical Gs-coupled receptors that evoke Ca2+ signals in cells freshly isolated from native tissues. We used vasoactive intestinal peptide (VIP) stimulation of pancreatic acinar cells and isoprenaline (Iso) stimulation of SMG duct cells to show that switching or augmentation of receptor coupling to Gi could account for activation of cAMP and Ca2+ signaling systems in vivo. Affinity purified B087, C260, and C267 polyclonal antibodies specific for Gαi1 and Gαi2(Gαi1,2), Gαi3, and Gαo (Gαi3,0) and Gαs, respectively (19Linder M.E. Middleton P. Hepler J.R. Taussig R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3675-3679Crossref PubMed Scopus (297) Google Scholar), and anti-Gαq IgG (20Gutowski S. Smrcka A. Nowak L. Wu D.G. Simon M. Sternweis P.C. J. Biol. Chem. 1991; 266: 20519-20524Abstract Full Text PDF PubMed Google Scholar, 21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) were prepared as described. Monoclonal antibody against PIP2 was purchased from Preseptive Diagnostics. Pertussis toxin (PTX) (from List Biological Laboratories) was reconstituted into distilled H2O and diluted into a pipette solution containing 0.5 mm dithiothreitol. A glutathione-tagged fragment of β-adrenergic receptor kinase (βARK1) was kindly provided by Dr. Robert Lefkowitz (Duke University, Durham, NC). His-tagged RGS4 was expressed in Escherichia coli and purified as described (22Xin X. Croy J.T. Zeng W. Zhao L. 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). Stock solutions of all antibodies, the βARK1 fragment, and RGS4 were dialyzed against a solution containing 100 mm KCl and 10 mm HEPES (pH 7.2 with NaOH) and stored at −20 °C until dilution into the pipette solution. H89 and Rp-8-CPT-cAMP-S were obtained from Biomole and BioLog, respectively. The pipette solution contained (in mm): 150 KCl, 10 HEPES (pH 7.2 with NaOH), 2 MgCl2, 1 ATP, and 0.1 EGTA. The standard bath solution A contained (in mm): 150 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH 7.2 with NaOH), and 10 glucose. When this solution was supplemented with 10 mm pyruvate, 1 mg/ml bovine serum albumin, and 0.02% soybean trypsin inhibitor, it was abbreviated PSA. Production of Gα15(−/−)-mutant mice was described (22Xin X. Croy J.T. Zeng W. Zhao L. 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, 23Xu X. Zeng W. Popov S. Berman D.B. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Single pancreatic acinar and submandibular gland (SMG) duct cells from wild type (WT) and Gα15 (−/−)-mice were prepared by standard collagenase and trypsin digestion procedures (24Xu X. Zeng W. Diaz J. Muallem S. J. Biol. Chem. 1996; 271: 24684-24690Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 25Zeng W. Lee M.G. Muallem S. J. Biol. Chem. 1997; 272: 32956-32965Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In brief, mice were sacrificed by exposure to a methoxyflurane-saturated atmosphere. The pancreas and SMG were removed and cleaned by injection of PSA. Minced tissues were incubated in a PSA solution containing 0.1 mg/ml collagenase (type CLSP, Worthington) before a short treatment with a trypsin/EDTA solution to release single cells. The cells were washed with PSA and kept on ice until use. The Ca2+-activated Cl− current of pancreatic acinar and SMG duct cells was recorded as detailed (21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), using the whole cell configuration of the patch clamp technique (26Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15138) Google Scholar). The cells were dialyzed with the pipette solution for 8–10 min before the first stimulation to allow equilibration of proteins and antibodies when included in the pipette solution. Membrane potential was held at −40 mV to record the inward current. The output signal recorded with a pClamp 6 and DigiData 1200 interface was filtered at 20 Hz. Due to significant variations in the current magnitude between preparations, results are given primarily as the number of responding cells. For each protocol similar results were obtained with cells from at least three mice. Fig. 1 summarizes the signaling pathways by which Gs-coupled receptors (Rs) may trigger a Ca2+ signal. Stimulation of a Gq-coupled cholinergic receptor with carbachol (Rq) was used as a positive control. Three mechanisms were tested: (a) activation of Gα15 by Rs, (b) direct activation of PLCβ by PKA, and (c) switching or augmentation of coupling specificity of Rs from Gs to Gi. We tested these mechanisms using two Gs-coupled receptors which evoke different types of Ca2+ signals: pancreatic acinar cells stimulated with VIP and SMG duct cells stimulated with Iso. Ca2+ signaling was followed by measuring the activity of the Ca2+-activated Cl− current in each cell type. Previous work showed that pancreatic acinar and SMG cells express the Ca2+-activated Cl− channel (21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 25Zeng W. Lee M.G. Muallem S. J. Biol. Chem. 1997; 272: 32956-32965Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 27Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (366) Google Scholar) and this current faithfully reflects changes in [Ca2+]i (21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 27Petersen O.H. J. Physiol. 1992; 448: 1-51Crossref PubMed Scopus (366) Google Scholar). Fig. 2 a shows that stimulation of pancreatic acinar cells with a saturating concentration of VIP-induced [Ca2+]i oscillations which lasted for the duration of cell stimulation, as previously reported (28Kase H. Wakui M. Petersen O.H. Pflugers Arch. 1991; 419: 668-670Crossref PubMed Scopus (22) Google Scholar). Maximal stimulation of the Gq-coupled muscarinic m3 receptor with 1 mm carbachol in the same cells resulted in a typical biphasic response of a spike and a plateau. This response was highly reproducible in mouse pancreatic acinar cells; similar responses were observed in 15/15 cells from 13 mice. Fig. 2 b shows that stimulation of SMG duct cells with the β-adrenergic agonist Iso caused a sustained increase in the Ca2+-activated Cl− current with no apparent oscillations. Following removal of Iso, stimulation with carbachol caused a large biphasic response. The Cl− current responses are similar in shape and time course to the previously reported changes in [Ca2+]i caused by these agonists in SMG cells (8Dehaye J.P. Valdez I.H. Turner R.J. Am. J. Physiol. 1993; 265: C1356-C1362Crossref PubMed Google Scholar,9Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar). Among cells which responded to carbachol, prior stimulation with Iso elicited a response similar to that in Fig. 2 b in 19/25 SMG duct cells from 17 mice. β-Adrenergic, vasopressin V2, dopamine D1A, and adenosine A2A receptors overexpressed in COS cells can couple to Gα15, but not other members of the Gq class, and stimulate PLCβ activity (15Offermanns S. Simon M.I. J. Biol. Chem. 1995; 270: 15175-15180Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 16Wu D. Kuang Y. Wu Y. Jiang H. J. Biol. Chem. 1995; 270: 16008-16011Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This would suggest that Gα15 has the unique ability to couple to receptors which are usually coupled to Gs. Currently, there are no good biochemical tools to specifically evaluate Gα15 function in native cells. Genetics provide an alternative approach. We measured the effect of VIP and Iso on Ca2+ signaling in cells prepared from mutant Gα15 (−/−)-mice to rule out the possibility that Gα15 contributes to Ca2+ signaling by Gs-coupled receptors in SMG and pancreatic acinar cells. Fig. 2 c shows that VIP- and carbachol-induced Ca2+ signaling was completely normal in pancreatic acini for Gα15 (−/−)-mice. The same results were obtained in six out of six experiments with acini from six mice. Fig. 2 dshows that Iso- and carbachol-induced Ca2+ signaling was normal in SMG duct cells from Gα15 (−/−)-mice. Similar results were obtained in four out of six experiments with SMG ducts prepared from the six mice that were used to study the response of pancreatic acinar cells. These findings exclude coupling to Gα15 as obligatory for activation of Ca2+signaling by the Gs-coupled receptors. Coupling of Rs to other members of the Gq class is also excluded by experiments with antibodies described below. Experiments with RGS4 supplied our first evidence that activation of Ca2+ signaling by VIP and Iso involves more than activation of Gs. RGS4 accelerates GTP hydrolysis by Gqand Gi class α subunits but not Gαs (29Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar,30Popov S. Yu K. Kozasa T. Wilkie T.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7216-7220Crossref PubMed Scopus (148) Google Scholar). In Fig. 3, infusion of 100 pm RGS4 through a patch pipette into pancreatic acinar (Fig. 3 a) or SMG duct (Fig. 3 b) cells completely inhibited the Ca2+ response to VIP and Iso, respectively. The control shows that the response to subsequent stimulation with carbachol was markedly reduced, as we reported recently (23Xu X. Zeng W. Popov S. Berman D.B. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Measurement of cAMP production in streptolysinO-permeabilized cells showed that inhibition of Ca2+ signaling by RGS4 was not due to inhibition of cAMP production by the Gs-coupled receptors (not shown). The results with RGS4 exclude model b of Fig. 1 as the mechanism by which Rs evokes a Ca2+ signal. In the next set of experiments we systematically tested the model for PKA-dependent Gs/Gi switching (or augmentation) of receptor specificity shown in Fig. 1 c (17Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1070) Google Scholar,18Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). We first tested if stimulation of Gs is obligatory for launching a Ca2+ signal by the VIP and Iso receptors. This was achieved by introducing antibodies specific for Gαsinto the cells through a patch pipette. Antibodies to the carboxyl terminus of Gαs were used because they have been reported to block receptor-mediated activation of adenylyl cyclase (31Simonds W.F. Golgsmith P.K. Woodard C.J. Unson C.G. Spiegel A.M. FEBS Lett. 1989; 249: 189-194Crossref PubMed Scopus (147) Google Scholar). Fig.4 shows that the antibodies specific for Gαs inhibited Ca2+ oscillations induced by VIP stimulation of pancreatic acinar cells and the Ca2+signal stimulated by Iso acting on SMG duct cells without affecting the oscillations or the biphasic response evoked by stimulation of the Gq-coupled m3 receptor with carbachol. Similar findings were observed in 4 additional acinar and 3 additional duct cells. As discussed below, infusion of Gαq specific antibodies did not effect VIP- or Iso-evoked Ca2+ signaling. Therefore, Gs stimulation was essential for launching a Ca2+ signal by the two classical Gs-coupled receptors. If PKA-dependent phosphorylation were involved, then inhibition of PKA activity should block Gs- but not Gq-dependent signaling (Fig. 1 c). The Rs in both cell types met this criterion as shown in Fig. 5. In control experiments, Ca2+ oscillations were initiated by stimulation of pancreatic acinar cells with VIP. After termination of VIP stimulation by removing the agonist, very similar oscillations were initiated by stimulating the same cells with low concentrations of carbachol, which acts through the Gq-coupled muscarinic receptor. Finally, the cell was stimulated with a supermaximal concentration of carbachol (Fig. 5). Similar results were obtained in 14 cells. In four separate experiments, the VIP response was completely abolished when pancreatic acinar cells were treated with 10 μm H89, a selective and potent inhibitor of PKA (32Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar), whereas the ability of a low concentration of carbachol to induce oscillations or of a supermaximal concentration to induce a biphasic response was unaltered (Fig.5 b). Similarly, treatment of SMG duct cells with 10 μm H89 abolished Iso-dependent [Ca2+]i increase, without affecting the carbachol-dependent response (Fig. 5 d). Inhibition of the response to Iso was observed in all 6 SMG duct cells treated with H89. The requirement for PKA stimulation was further verified by testing the effect of the potent and selective inhibitor of PKA, Rp-8-CPT-cAMP-S. Infusing the cells with 10 μmRp-8-CPT-cAMP-S through the pipette abolished the response to VIP (n = 7) and Iso (n = 5) in all cells tested (Fig. 5, c and e). Again, control experiments in the same cells showed that all forms of Gq-dependent responses were unaffected by inhibition of PKA with Rp-8-CPT-cAMP-S. These inhibitory effects of the two PKA inhibitors argue against the possibilities that unregulated VIP or β-adrenergic receptors are coupled directly to Gi (33Asano T. Katada T. Gilman A.G. Ross E.M. J Biol. Chem. 1984; 259: 9351-9354Abstract Full Text PDF PubMed Google Scholar) or that Gαs directly modulates Ca2+ channels (34Mattera R. Graziano M.P. Yatani A. Zhou Z. Graf R. Codina J. Birnbaumer L. Gilman A.G. Brown A.M. Science. 1989; 243: 804-807Crossref PubMed Scopus (148) Google Scholar) in these systems. To directly address a role for Gi in Ca2+signaling by VIP and Iso we measured the effect of infusing the cells with PTX or antibodies specific for certain members of the Gi subclass of α subunits. Preliminary studies showed that concentrations of PTX below 20 ng/ml in the pipette solution did not consistently inhibit VIP-induced signaling. At concentrations above 50 ng/ml, PTX rapidly caused a large, time-dependent, nonselective increase in membrane conductance, as if PTX caused cell permeabilization. We therefore limited our testing to the effect of 20 ng/ml PTX on Ca2+ signaling in pancreatic acinar cells. Fig. 6 shows that treatment with PTX inhibited VIP but not carbachol-dependent Ca2+signaling. Similar results were obtained in four experiments. In 13 additional experiments, PTX-treated acinar cells lysed before the experimental protocol could be completed. We were unable to find a concentration of PTX that inhibited the Iso response in SMG duct cell without causing cell lysis. Antibodies generated against peptides representing the carboxyl termini of Gαi and Gαq subunits inhibit receptor-initiated activation of these G proteins (20Gutowski S. Smrcka A. Nowak L. Wu D.G. Simon M. Sternweis P.C. J. Biol. Chem. 1991; 266: 20519-20524Abstract Full Text PDF PubMed Google Scholar, 21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 35Simonds W.F. Golgsmith P.K. Codina J. Unson C.G. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7809-7813Crossref PubMed Scopus (309) Google Scholar). The results obtained by infusing antibodies into pancreatic acinar cells are illustrated in Fig. 7. Two types of polyclonal antibodies against Gi were used, one recognizing Gαi3 and Gαo or one specific for Gαi1 and Gαi2 (19Linder M.E. Middleton P. Hepler J.R. Taussig R. Gilman A.G. Mumby S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3675-3679Crossref PubMed Scopus (297) Google Scholar). Fig. 7 ashows that infusing 17.5 μg/ml antibodies specific for Gαi3 and Gαo had no effect on Ca2+ signaling induced by Gs- or Gq-coupled receptors. Similar results were obtained in four cells. However, these antibodies were not without effect, as seen for SMG cells (described below). Fig. 7 b shows that infusing pancreatic acinar cells with 9 μg/ml Gαi1,i2-specific antibodies completely inhibited the response to VIP without affecting the response to carbachol. Similar results were observed in six cells. An important control is shown in Fig. 7 c. In contrast to the effect of Gi-specific antibodies, infusing the cells with Gαq,11 antibodies (at sufficient concentration to abolish the oscillation and largely inhibit the sustained response to carbachol) had no effect on the ability of VIP to induce oscillations. In seven experiments with cells infused with 80 μg/ml anti-Gαq IgG the response to VIP remained normal, while the response to the low concentration of carbachol was abolished and the response to supermaximal concentration of carbachol was inhibited by 83 ± 7%. Activation of Gi by Iso is further suggested by the results for SMG duct cells shown in Fig. 8. In six cells infused with Gαq,11 reactive IgG, the response to supermaximal concentrations of carbachol was reduced by 91 ± 6% while the response to Iso was not affected (Fig. 8 b). Unlike the findings in pancreatic acinar cells stimulated with VIP, both Gi antibody preparations effectively inhibited the response to Iso in SMG duct cells. Gαi1,i2-specific antibodies, at a concentration of 9 μg/ml, completely inhibited the Ca2+ response to Iso (Fig. 8 c). Infusion of only 3.5 μg/ml Gαi3,o antibodies completely inhibited the response to Iso in two cells and partially (63 ± 14%) in three cells (Fig. 8 d). At a concentration of 7 μg/ml the anti-Gαi3,o completely inhibited the response to Iso in five cells (Fig. 8 e). The findings in Figs. 7 and 8 provide strong evidence that activation of Ca2+ signaling by Gs-coupled receptors is independent of members of the Gq class. The inhibitory Gq antibodies used in the present work recognizes the predominant Gq class α subunits expressed in these cells, Gαq, Gα11, and Gα14 (22Xin X. Croy J.T. Zeng W. Zhao L. 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). Furthermore, these antibodies were shown to inhibit Ca2+signaling evoked by several Gq-coupled receptors in pancreatic (21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and other cell types (36Wilk-Blaszczak M.A. Gutowski S. Sternweis P.C. Belardetti F. Neuron. 1994; 12: 109-116Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 37Stehno-Bittel L. Krapivinsky G. Krapivinsky L. Perez-Terzic C. Clapham D.E. J. Biol. Chem. 1995; 270: 30068-30074Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). At a concentration inhibiting the oscillatory and the biphasic response to cholinergic stimulation, the antibodies had no apparent effect on the response to either VIP or Iso. This data supports the conclusion that inhibition of VIP- and Iso-induced Ca2+ signaling by RGS4 was due to acceleration of GTPase activity of a Gi class α subunit(s). The use of PTX and Gαi antibodies indicates that receptor-mediated activation of Gi was required for activation of Ca2+ signaling by VIP or Iso. It is notable that both Gi antibody preparations inhibited Iso-stimulated Ca2+ signaling in SMG duct cells whereas only the Gαi1,i2-specific preparation was effective for inhibiting VIP-stimulated signaling in the pancreatic acinar cells. This minor difference between the two systems may be attributed to cell type-specific expression patterns of Gαi isoforms or the degree of Gαi selectivity exhibited by putative PKA-phosphorylated VIP and β-adrenergic receptors. It is puzzling that the β-adrenergic Ca2+ response is inhibited completely by either Gi antibody preparation. If the β-adrenergic receptor couples to all members of the Giclass, then each antibody preparation would be expected to only partially inhibit and a mixture of the antibodies to completely inhibit signaling by these receptors. The complete inhibition of signaling by either antibody preparation suggests that partial inhibition of IP3 production by stimulation of the β-adrenergic receptor had reduced IP3 below a threshold level needed to trigger Ca2+ release. This interpretation is supported by previous work showing that Iso released Ca2+ from the IP3 mobilizable Ca2+ pool (9Xu X. Diaz J. Zhao H. Muallem S. J. Physiol. 1996; 491: 647-662Crossref PubMed Scopus (58) Google Scholar) without causing a detectable increase in global IP3 concentration (8Dehaye J.P. Valdez I.H. Turner R.J. Am. J. Physiol. 1993; 265: C1356-C1362Crossref PubMed Google Scholar). In the Gs/Gi switching model, activated receptor, phosphorylated by PKA, couples to Gi (18Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar). This predicts that Gβγ released from Gi could activate PLCβ. Thus, inhibition of Gβγ or PLCβ activity is expected to inhibit the effect of the Gs-coupled receptors on [Ca2+]i. To test these predictions, we measured the effect of the Gβγ scavenging protein βARK1 (21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 38Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar) and of the inhibitory PIP2 antibody (39Matuoka K. Fukami K. Nakanishi O. Kawai S. Takenawa T. Science. 1988; 239: 640-643Crossref PubMed Scopus (102) Google Scholar, 40Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (455) Google Scholar) on VIP-dependent Ca2+ signaling. Fig.9 a shows that infusing 5 μm βARK1 into pancreatic acinar cells completely inhibited the response to VIP. As we (21Zeng W. Xu X. Muallem S. J. Biol. Chem. 1996; 271: 18520-18526Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and others (37Stehno-Bittel L. Krapivinsky G. Krapivinsky L. Perez-Terzic C. Clapham D.E. J. Biol. Chem. 1995; 270: 30068-30074Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) reported earlier, βARK1 also inhibited the response to stimulation of the Gq-coupled muscarinic receptor. Inhibition by βARK1 was upstream of the Ca2+ increase because elevation of [Ca2+]i with A23187 strongly activated the Cl− current. Results similar to those in Fig.9 a, including the positive control with A23187, were obtained in five experiments. Fig. 9 b shows that cytoplasmic PIP2 antibodies completely inhibited the response to VIP and reduced the response to carbachol by 88 ± 11% (n = 7). These experiments indicate that both VIP and carbachol stimulate PLCβ to cause the hydrolysis of PIP2. In summary, our examination of the [Ca2+]iincrease triggered by Gs-coupled receptors supports a model for switching or augmentation of receptor coupling to extend to Gi in native cells freshly isolated from tissue. We conclude that the pathway involves activation of Gs and PKA, receptor stimulation of Gi, and activation of PLCβ by Gβγ (derived from Gi). We acknowledge that the PKA substrate(s) responsible for activation of Gi are not known but, as suggested by the switching model (17Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1070) Google Scholar, 18Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (905) Google Scholar), they could be the same receptors that were initially coupled only to Gs. We use caution, however, in referring to the Ca2+ pathway (Fig. 1 c), as a receptor switching model. PKA-dependent phosphorylation of the VIP or β-adrenergic receptors could allow Gi to replace Gs but the data are also consistent with broadening of receptor coupling to Gs plus Gi. One mode for augmentation of receptor coupling can be envisioned if it is assumed that most β-adrenergic or VIP receptors are productively coupled to Gs but a smaller subpopulation are poised to couple to Gi. Effective Gi coupling would occur only when the receptors are phosphorylated by PKA. Because expression of a mutant (phosphorylation negative) β-adrenergic receptor prevented PKA-dependent activation of Gi in HEK 293 cells (17Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1070) Google Scholar), it is unlikely that phosphorylation of proteins downstream of the VIP or β-adrenergic receptors are responsible for activation of Gi in pancreatic acinar or submandibular gland cells. An alternative to the assumption that mutant receptor is unable to couple to Gi (17Daaka Y. Luttrell L.M. Lefkowitz R.J. Nature. 1997; 390: 88-91Crossref PubMed Scopus (1070) Google Scholar) is that the mutant cannot regulate its interaction with an RGS protein that may ordinarily suppress Gi activation stimulated by the β-adrenergic receptor. A role for regulation of RGS protein function by receptor phosphorylation is attractive, not only because RGS proteins exhibit selectivity among receptor signaling complexes (23Xu X. Zeng W. Popov S. Berman D.B. Davignon I. Yu K. Yowe D. Offermanns S. Muallem S. Wilkie T.M. J. Biol. Chem. 1999; 274: 3549-3556Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 41Zeng W. Xu X. Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K. Fisher S. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 42Zhang Y. Neo S.Y. Han J. Yaw L.P. Lin S.C. J. Biol. Chem. 1999; 274: 2851-2857Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), but also because phosphorylation is not necessary for purified β-adrenergic receptors to activate Gi in vitro (33Asano T. Katada T. Gilman A.G. Ross E.M. J Biol. Chem. 1984; 259: 9351-9354Abstract Full Text PDF PubMed Google Scholar). Additional experimental tools are needed to distinguish between these and other potential mechanisms. Independent of the mode of coupling it is clear that in pancreatic acinar and submandibular cells Gs-coupled receptors activate Ca2+ signaling by coupling to Gi and this coupling requires activation of Gs. An equally important conclusion is that VIP and β-adrenergic receptor regulation of Ca2+ release is completely independent of Gq class proteins. The observation that PKA-dependent switching/augmentation in receptor/G protein coupling occurs in two different native cell types via two different receptors (that generate different types of Ca2+ signals) suggests a generalization of the mechanism by which Gs-coupled receptors generate a second signal to activate a distinct signaling cascade. We thank Dr. Paul C. Sternweis for providing the Gq antibodies and Dr. Robert Lefkowitz for the βARK fragment and our colleagues for helpful discussions." @default.
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• W2100629398 title "Alternate Coupling of Receptors to Gs and Gi in Pancreatic and Submandibular Gland Cells" @default.
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