βγ Subunits of Pertussis Toxin-sensitive G Proteins Mediate A1 Adenosine Receptor Agonist-induced Activation of Phospholipase C in Collaboration with Thyrotropin
1997; Elsevier BV; Volume: 272; Issue: 37 Linguagem: Inglês
10.1074/jbc.272.37.23130
ISSN1083-351X
AutoresHideaki Tomura, Hiroshi Itoh, Kimie Sho, Kōichi Sato, Motoshi Nagao, Michio Ui, Yoichi Kondo, Fumikazu Okajima,
Tópico(s)Cellular transport and secretion
ResumoCOS-7 cells were transiently transfected with human thyrotropin receptor and dog A1 adenosine receptor cDNAs. An A1 agonist,N 6-(l-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced the thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition. These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of Gi2α or Gi3α, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the βγ subunits of the G proteins enhanced the TSH-induced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of Gi2α, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation but hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects on the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G11α mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the βγ subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A1 adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; α subunits of the PTX-sensitive G proteins mediate the inhibitory action on adenylyl cyclase, and the βγ subunits mediate the stimulatory action on PLC. In the case of the latter stimulatory action on PLC, the βγ subunits may not directly activate PLC. The possible mechanism by which βγ subunits enhance the TSH-induced PLC activation is discussed. COS-7 cells were transiently transfected with human thyrotropin receptor and dog A1 adenosine receptor cDNAs. An A1 agonist,N 6-(l-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced the thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition. These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of Gi2α or Gi3α, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the βγ subunits of the G proteins enhanced the TSH-induced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of Gi2α, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation but hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects on the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G11α mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the βγ subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A1 adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; α subunits of the PTX-sensitive G proteins mediate the inhibitory action on adenylyl cyclase, and the βγ subunits mediate the stimulatory action on PLC. In the case of the latter stimulatory action on PLC, the βγ subunits may not directly activate PLC. The possible mechanism by which βγ subunits enhance the TSH-induced PLC activation is discussed. The activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) 1The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding protein; PTX, pertussis toxin; TSH, thyrotropin; TSHR, TSH receptor; A1R, A1 adenosine receptor; PLC, phospholipase C; AC, adenylyl cyclase; Gα, Gβ, and Gγ, G protein α, β, and γ subunits, respectively; IP, inositol phosphate; PIA,N 6-(l-2-phenylisopropyl) adenosine. 1The abbreviations used are: G protein, heterotrimeric guanine nucleotide-binding protein; PTX, pertussis toxin; TSH, thyrotropin; TSHR, TSH receptor; A1R, A1 adenosine receptor; PLC, phospholipase C; AC, adenylyl cyclase; Gα, Gβ, and Gγ, G protein α, β, and γ subunits, respectively; IP, inositol phosphate; PIA,N 6-(l-2-phenylisopropyl) adenosine. is involved in the stimulation of a variety of signaling pathways by hormone, neurotransmitter, and sensory receptors with seven transmembrane domains (1Kaziro Y. Itoh H. Kozasa T. Nakafuku M. Satoh T. Annu. Rev. Biochem. 1991; 60: 349-400Crossref PubMed Scopus (547) Google Scholar). Agonist-bound receptors activate G proteins by stimulating the exchange of GDP for GTP on α subunits in a trimeric form of the proteins, which, in turn, accelerates dissociation of the βγ subunits from the α subunits (2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4669) Google Scholar). In early studies, only the α subunits were studied as transducers, but now both the α and βγ subunits are recognized to be involved in the regulation of various effector systems such as adenylyl cyclase (AC), phospholipase C (PLC), or ion channels (3Birnbaumer L. Cell. 1992; 71: 1069-1072Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 4Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (384) Google Scholar). At least 16, 5, and 6 species of the α, β, and γ subunits, respectively, have been identified in molecular cloning studies (5Watson A.J. Katz A. Simon M.I. J. Biol. Chem. 1994; 269: 22150-22156Abstract Full Text PDF PubMed Google Scholar, 6Watson S. Arkinstall S. The G-protein Linked Receptor FactsBook. Academic Press, Inc., San Diego, CA1994: 296-355Google Scholar). Furthermore, several isoforms of each subunit may be expressed within the same cell type (7Clapham D.E. Neer E.J. Nature. 1993; 365: 403-406Crossref PubMed Scopus (587) Google Scholar, 8Strathmann M. Wilkie T.M. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7407-7409Crossref PubMed Scopus (120) Google Scholar, 9Strathmann M. Simon M.I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9113-9117Crossref PubMed Scopus (385) Google Scholar). Thus, it would be reasonable to assume that a variety of G-protein-dependent actions are executed by each different molecular species of the G proteins or their subunits. In this context, identification of molecular species that participate in certain signaling systems is necessary for understanding its mechanism. In thyroid cells, thyrotropin (TSH) activates PLC through the Gq/G11 protein as well as AC through the Gs protein, resulting in mobilization of Ca2+and accumulation of cAMP in the cells (10Allgeier A. Offermanns S. Van Sande J. Spicher K. Schultz G. Dumont J.E. J. Biol. Chem. 1994; 269: 13733-13735Abstract Full Text PDF PubMed Google Scholar). We have shown that in rat FRTL-5 thyroid cells, adenosine and its derivatives such as phenylisopropyl adenosine (PIA) inhibited TSH-induced AC activation and, in contrast, enhanced TSH-induced PLC activation and subsequent Ca2+ mobilization through the A1 type receptor (A1R) and PTX-sensitive G protein (11Sho K. Okajima F. Majid M.A. Kondo Y. J. Biol. Chem. 1991; 266: 12180-12184Abstract Full Text PDF PubMed Google Scholar). This PTX-sensitive G protein-mediated PLC activation is not restricted to the cross-talk between the TSH and adenosine signaling mechanisms. In FRTL-5 thyroid cells, PIA, through A1R, also enhanced PLC activation induced by α1-adrenergic receptor agonists (12Okajima F. Sato K. Sho K. Kondo Y. FEBS Lett. 1989; 248: 145-149Crossref PubMed Scopus (51) Google Scholar) and P2-purinergic receptor agonists (13Okajima F. Sato K. Nazarea M. Sho K. Kondo Y. J. Biol. Chem. 1989; 264: 13029-13037Abstract Full Text PDF PubMed Google Scholar, 14Nazarea M. Okajima F. Kondo Y. Eur. J. Pharmacol. 1991; 206: 47-52Crossref PubMed Scopus (22) Google Scholar) as well as TSH. Likewise, A1R agonists enhance PLC activation induced by IgE in RBL2H3 cells and those induced by ATP and bradykinin in the smooth muscle cell line (15Ali H. Cunha-Melo J.R. Saul W.F. Beaven M.A. J. Biol. Chem. 1990; 265: 745-753Abstract Full Text PDF PubMed Google Scholar, 16Gerwins P. Fredholm B.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7330-7334Crossref PubMed Scopus (81) Google Scholar, 17Gerwins P. Fredholm B.B. J. Biol. Chem. 1992; 267: 16081-16087Abstract Full Text PDF PubMed Google Scholar). All of these A1R-mediated actions were abolished by PTX treatment. In addition, this cross-talk is not specific to the A1R agonist; in NG108–15 cells, enkephalin, somatostatin, α2-adrenergic agonist, and carbachol have also been shown to enhance P2 receptor agonist- or bradykinin-induced PLC activation and Ca2+mobilization, in a PTX-sensitive manner (18Okajima F. Kondo Y. FEBS Lett. 1992; 301: 223-226Crossref PubMed Scopus (23) Google Scholar, 19Tomura H. Okajima F. Kondo Y. Neurosci. Lett. 1992; 148: 93-96Crossref PubMed Scopus (20) Google Scholar, 20Okajima F. Tomura H. Kondo Y. Biochem. J. 1993; 290: 241-247Crossref PubMed Scopus (69) Google Scholar). In a previous study, we also showed that the PTX-sensitive G protein-mediated modulation of the TSH and muscarinic acetylcholine actions by adenosine are reconstituted by expressing both the TSH receptor (TSHR) and A1R in COS-7 cells (21Okajima F. Tomura H. Sho K. Akbar M. Majid M.A. Kondo Y. Biochem. J. 1995; 306: 709-715Crossref PubMed Scopus (9) Google Scholar) and both the m3 muscarinergic acetylcholine receptor and A1R in CHO cells (22Akbar M. Okajima F. Tomura H. Shimegi S. Kondo Y. Mol. Pharmacol. 1994; 45: 1036-1042PubMed Google Scholar), respectively. Thus, our findings in concert with those of others have suggested the presence of a universal cross-talk mechanism mediated by a PTX-sensitive G protein(s) between AC-inhibitory and PLC-stimulatory signaling mechanisms resulting in the enhancement of PLC activation. Two receptors involved in this cross-talk regulation of PLC are characterized as one type of receptor that couples to PTX-sensitive Gi/Go proteins whose stimulation inhibits AC but exhibits only a small or undetectable effect on PLC, and the other type of receptor, the so-called Ca2+-mobilizing receptor, couples to Gq/G11 proteins whose stimulation leads to activation of PLC. Thus, AC-inhibiting receptor agonists, through Gi/Go proteins, permissively or synergistically enhance Ca2+-mobilizing receptor agonist-induced PLC activation. The molecular mechanism by which the respective receptor agonists induce stimulation of each signaling pathway leading to AC inhibition and PLC activation through Gi/Go and Gq/G11, respectively, has been well characterized (6Watson S. Arkinstall S. The G-protein Linked Receptor FactsBook. Academic Press, Inc., San Diego, CA1994: 296-355Google Scholar). However, it has not been well elucidated how a single type of AC-inhibiting receptor simultaneously links to two signaling pathways with the aid of the PTX-sensitive G protein(s). In this study, we aimed to further define the role of PTX-sensitive G proteins in the cross-talk phenomena in COS-7 cells where TSHR and A1R were expressed. The specific objectives were to determine (a) whether a single type of PTX-sensitive G protein mediates the modulation of the two signaling pathways and (b) how the G protein participates in the two signaling pathways. In the reconstituted cross-talk systems constructed by recombinant receptors and manipulated G-protein pools in the cells, we found that at least a single molecular species of Gi2 or Gi3 can mediate the modulation of two signaling pathways; enhancement of PLC is mediated by βγ subunits, and inhibition of AC is mediated by a single species of the Giα subunit. Furthermore, our results suggest that the primary target of the βγ subunits may not be PLC itself but may instead be located upstream of the enzyme in the signaling pathway, possibly at the level of the α subunits of the Gq/G11 proteins. PIA was purchased from Sigma; staurosporin was from Kyowa Medex Co. (Tokyo, Japan); and myo-[2-3H]inositol (23.0 Ci/mmol) was from NEN Life Science Products. Human TSHR cDNA in the pSVL expression vector (23Libert F. Lefort A. Gerard C. Parmentier M. Perret J. Ludgate M. Dumont J.E. Vassart G. Biochem. Biophys. Res. Commun. 1989; 165: 1250-1255Crossref PubMed Scopus (390) Google Scholar) and dog A1R (24Libert F. Schiffmann S.N. Lefort A. Parmentier M. Gerard C. Dumont J.E. Vanderhaeghen J.J. Vassart G. EMBO J. 1991; 10: 1677-1682Crossref PubMed Scopus (171) Google Scholar) cDNA were generously provided by Dr. G. Vassart (Université Libre de Bruxelles, Belgium), bovine Gβ1 cDNA by Dr. M. I. Simon (California Institute of Technology), bovine Gγ2 cDNA by Dr. T. Nukada (Tokyo Institute of Psychiatry, Tokyo, Japan), and pCDL-SRα 296 vector (25Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar) by Dr. Y. Takebe (National Institute of Health, Tokyo, Japan). Rabbit antiserum specific to Gi2α was generously provided by Dr. Y. Kanaho (Tokyo Institute of Technology, Yokohama, Japan), and the β subunit of the G protein was supplied by Dr. T. Katada (Tokyo University, Tokyo, Japan). The radioimmunoassay of cAMP used a Yamasa cAMP assay kit, which was a gift from Yamasa Shoyu Co. (Choshi, Chiba, Japan). The sources of all other reagents were the same as described previously (11Sho K. Okajima F. Majid M.A. Kondo Y. J. Biol. Chem. 1991; 266: 12180-12184Abstract Full Text PDF PubMed Google Scholar, 14Nazarea M. Okajima F. Kondo Y. Eur. J. Pharmacol. 1991; 206: 47-52Crossref PubMed Scopus (22) Google Scholar, 26Okajima F. Kondo Y. J. Biol. Chem. 1990; 265: 21741-21748Abstract Full Text PDF PubMed Google Scholar). The PTX-insensitive mutant Gi2α(C352G) expression plasmid was obtained by polymerase chain reaction mutagenesis using wild type rat Gi2α cDNA (27Itoh H. Kozasa T. Nagata S. Nakamura S. Katada T. Ui M. Iwai S. Ohtsuka E. Kawasaki H. Suzuki K. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3776-3780Crossref PubMed Scopus (259) Google Scholar) as a template. The 5′-primer, 5′-gggaattcCCACCATGGGCTGCACCGTGAG-3′, contains the EcoRI site, Kozak sequence (CCACC) (28Kozak M. Cell. 1986; 44: 283-292Abstract Full Text PDF PubMed Scopus (3556) Google Scholar), and the first six amino acids of the Gi2α. The 3′-primer, 5′-gggcggccgcTCAGAAGAGGCCGCCGTCCTTCAGCGTTGTTCTTGATGATGACGT-3′, contains the NotI site, a stop codon, and 14 amino acids in the C-terminal region of Gi2α, except for GCC (underlined), which encodes glycine instead of cysteine at position 352 from the N terminus. The PTX-insensitive mutant Gi3α(C351G) expression plasmid was obtained by polymerase chain reaction mutagenesis using wild type rat Gi3α cDNA (29Itoh H. Toyama R. Kozasa T. Tsukamoto T. Matsuoka M. Kaziro Y. J. Biol. Chem. 1988; 263: 6656-6664Abstract Full Text PDF PubMed Google Scholar) as a template. The 5′-primer, 5′-gggaattcCCACCATGGGCTGCACGTTGAGCGCCGAGGAC-3′, contains theEcoRI site, Kozak sequence (CCACC), and the first nine amino acids of Gi3α. The 3′-primer, 5′-gggcggccgcTCAGTAAAGCCCGCCTTCCTTTAAGTTGTT-3′, contains the NotI site, a stop codon, and nine amino acids in the C-terminal region of Gi3α, except for GCC (underlined), which encodes glycine instead of cysteine at position 351 from the N terminus. The amplified Gi2α(C352G) or Gi3α(C351G) was digested with EcoRI andNotI and then inserted into theEcoRI/NotI site of the pcDNA/AMP expression plasmids (Invitrogen, CA). The constitutively active mutant G11α(Q209L) expression plasmid was obtained by polymerase chain reaction mutagenesis using wild type mouse G11α cDNA from mouse S49 lymphoma cells as a template. The 5′-primer, 5′-tagcaagcttcatATGACTCTGGAGTCCATGATGGC-3′, contains the HindIII site and the first eight amino acids of G11α. The 3′-primer, 5′-caatggatccACTTCCTGCGCTCTGACCTCAGGCCTCCC3′, contains theBamHI site and 14 amino acids including the leucine codon (underlined) instead of the glutamine codon at position 209 from the N terminus. The amplified fragment was digested with HindIII and BamHI and then inserted into the corresponding region of the wild G11α cDNA. The cDNA was subcloned into the HindIII site of the pCMV5 expression plasmid (30Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar) in the right orientation. The constitutively active mutant Gi2α(Q205L) was obtained by replacing the glutamine codon at position 205 from the N terminus to the leucine codon, using a uracil-containing single-stranded DNA of the rat Gi2α as a template. DNA sequences of the mutants were confirmed by dideoxynucleotide sequencing (31Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52251) Google Scholar). The Gβ1 and Gγ2 cDNAs were subcloned into pCMV5 as described previously (32Yamauchi J. Kaziro Y. Itoh H. Bioche. Biophys. Res. Commun. 1995; 214: 694-700Crossref PubMed Scopus (14) Google Scholar). COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (JRH Biosciences, KS) in a 5% CO2 atmosphere at 37 °C. For the transfection experiments, the cells were harvested with 0.05% trypsin and 0.53 mm EDTA (Life Technologies, Inc.), washed once with Mg2+- and Ca2+-free phosphate-buffered saline, and suspended in the same phosphate-buffered saline solution. The cell suspensions (about 107 cells in 0.8 ml) were transfected by electroporation (0.3 kV, 500 microfarads, Gene Pulser II, Bio-Rad) with dog A1R cDNA in pCDL-SRα 296 vector (20 μg) and human TSHR cDNA in pSVL (20 μg) expression plasmids in combination with cDNAs encoding bovine Gβ1 (40 μg), bovine Gγ2 (40 μg), Gi2α(C352G) (50 μg), Gi3α(C351G) (50 μg), and G11α(Q209L) (1, 2, or 4 μg) in pCMV5 (β1, γ2, and G11α(Q209L)) or pcDNA/AMP (Gi2α(C352G) and Gi3α(C351G)) expression plasmids, unless otherwise specified. The total amount of DNA for transfection was adjusted by the empty pCMV5 and pcDNA/AMP expression plasmids. After transfection, the cells (about 2 × 105) were cultured for 2 days on 12-well plates (Costar) in inositol-free Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum and myo-[2-3H]inositol (2 μCi/ml), unless otherwise specified. The [3H]inositol-labeled cells were washed twice with Hepes-buffered medium, which consisted of 20 mm Hepes (pH 7.5), 134 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 1.2 mmMgSO4, 2 mm CaCl2, 2.5 mm NaHCO3, 5 mm glucose, and 0.1% (w/v) bovine serum albumin (fraction V). The cells were then incubated for 30 min at 37 °C with the same medium containing 10 mm LiCl and 0.5 units/ml adenosine deaminase, 0.1 mm Ro20–1724, and 1 μg/ml staurosporin, and the agents were tested in a final volume of 0.6 ml. The reaction was terminated by the addition of 0.1 ml of 1 n HCl. The contents of cAMP and3H-labeled inositol phosphate were measured in the same HCl extract. Cyclic AMP was measured by radioimmunoassay. [3H]Inositol mono-, di-, and trisphosphates were separated from inositol and glycerophosphoinositol on Dowex 1-X8 (formate form, Bio-Rad) columns as described previously (11Sho K. Okajima F. Majid M.A. Kondo Y. J. Biol. Chem. 1991; 266: 12180-12184Abstract Full Text PDF PubMed Google Scholar). The radioactivity of the trichloroacetic acid (5%)-insoluble fraction was measured as the total radioactivity incorporated into the cellular inositol lipids. Where indicated, the results were normalized to 105 cpm of the total radioactivity. Crude plasma membranes and their cholate extracts were prepared as described previously (33Okajima F. Katada T. Ui M. J. Biol. Chem. 1985; 260: 6761-6768Abstract Full Text PDF PubMed Google Scholar, 34Okajima F. Tokumitsu Y. Kondo Y. Ui M. J. Biol. Chem. 1987; 262: 13483-13490Abstract Full Text PDF PubMed Google Scholar). The cholate extract (25 μg of protein) was resolved on SDS-12.5% polyacrylamide slab gel electrophoresis and then electrophoretically transferred to a Millipore Immobilon sheet (35Sato K. Okajima F. Katada T. Kondo Y. Arch. Biochem. Biophys. 1990; 281: 298-304Crossref PubMed Scopus (4) Google Scholar). The expressions of Gi2α(Q205L) and the β1 subunits in addition to endogenous Gi2α and the β subunits were visualized by incubating the sheet with each specific rabbit antiserum, with alkaline phosphate-conjugated goat antibody against rabbit IgG and finally with 5-bromo-4-chloro-3-indoylphosphate and nitro blue tetrazolium as described previously (35Sato K. Okajima F. Katada T. Kondo Y. Arch. Biochem. Biophys. 1990; 281: 298-304Crossref PubMed Scopus (4) Google Scholar). All experiments were performed in triplicate or quadruplicate with more than three different batches of cells. The results were presented as means ± S.E. of three or four values in a representative experiment, unless otherwise stated. In accordance with our previous study (21Okajima F. Tomura H. Sho K. Akbar M. Majid M.A. Kondo Y. Biochem. J. 1995; 306: 709-715Crossref PubMed Scopus (9) Google Scholar), when both TSHR and A1R were expressed in COS-7 cells, although PIA, an A1 agonist, alone hardly affected the basal activities of PLC and AC, this A1agonist enhanced TSH-stimulated PLC but inhibited the TSH-stimulated AC in a way similar to those in FRTL-5 thyroid cells (11Sho K. Okajima F. Majid M.A. Kondo Y. J. Biol. Chem. 1991; 266: 12180-12184Abstract Full Text PDF PubMed Google Scholar) (Fig. 1, A and C). These PIA actions were completely abolished by a PTX treatment without an appreciable effect on intrinsic TSH actions (Figs. 1, B andD). In COS cells, several types of PTX-sensitive G proteins are expressed (36Yasuda K. Rens-Domiano S. Breder C.D. Law S.F. Saper C.B. Reisine T. Bell G.I. J. Biol. Chem. 1992; 267: 20422-20428Abstract Full Text PDF PubMed Google Scholar, 37Rens Domiano S. Law S.F. Yamada Y. Seino S. Bell G.I. Reisine T. Mol. Pharmacol. 1992; 42: 28-34PubMed Google Scholar, 38Wu D. LaRosa G.J. Simon M.I. Science. 1993; 261: 101-103Crossref PubMed Scopus (332) Google Scholar). To ascertain the role of a particular PTX-sensitive G protein subtype in the bidirectional A1R agonist action, we planned to use the cells where only a species of PTX-insensitive and active Giα mutant is expressed under the conditions where native Gi/Go is inactivated by the PTX treatment. We constructed a mutant cDNA, Gi2α(C352G), in which a glycine residue substitutes for a cysteine residue at position 352 of Gi2α. COS-7 cells were transfected with this PTX-insensitive Gi2α cDNA together with cDNAs of the β1 and γ2 subunits of the G protein, expecting the expression of the PTX-insensitive Gi2(C352G) (Fig. 2). The transfection of these subunit-cDNAs did not appreciably affect the TSH- and PIA-induced IP responses (compare panels A of Figs. 1 and 2) and cAMP responses (compare panels C of Figs. 1 and 2) in the control cells. However, when the cells were treated with PTX, a clear difference with respect to PIA actions was observed; PIA was still effective at enhancing the TSH-induced PLC activation (comparepanels B of Figs. 1 and 2) and inhibiting the TSH-induced AC activation (compare panels D of Figs. 1 and 2) in the cells transfected with these subunit cDNAs. Thus, Gi2 can couple to A1R and mediate the reduction of the TSH-induced AC activation as well as the enhancement of the TSH-dependent PLC activation. Another PTX-insensitive α subunit of Gi3, Gi3α(C351G), which was expressed in COS-7 cells, also coupled to A1R and modulated the TSH-induced actions even in the PTX-treated cells; the TSH (100 nm)-induced levels were 857 ± 83 or 1162 ± 45 dpm for IP production and 2.42 ± 0.05 or 1.75 ± 0.06 nmol/mg for cAMP accumulation in the absence or presence of PIA, respectively. In all of the cases using or not using either the Gi2 or Gi3 mutant, PIA alone exerted no significant effect on either the AC or PLC activities. It has been reported that the C-terminal region of α subunits of G proteins is important for their receptor recognition (39West Jr., R.E. Moss J. Vaughan M. Liu T. Liu T.-Y. J. Biol. Chem. 1985; 260: 14428-14430Abstract Full Text PDF PubMed Google Scholar). Therefore, we examined whether the cysteine to glycine substitution affects the ability of PTX-sensitive G proteins to enhance the TSH-induced PLC activation. As shown in Fig. 3, no significant difference was detected between mock- and Gi2α(C352G)-transfected cells in terms of their response to any PIA dose. This result indicates that Gi2α(C352G) still retains the ability to associate with A1R in COS-7 cells in a way similar to endogenous PTX-sensitive G proteins. The response of Gi2α(C352G) transfected cells to higher than 100 nm PIA was slightly stronger than those of the mock-transfected cells. However, this feature of the mutant transfected cells is not specific for the PIA effect on the TSH-induced PLC activation, because in the mutant G protein-transfected cells, PIA alone slightly stimulates PLC in the absence of TSH stimulation (Fig. 3). We next examined which subunits, i.e. the α subunit or βγ subunits of the PTX-sensitive G protein, mediate each A1R-mediated signaling. To clarify this point, we transfected mutant DNAs encoding GTPase-deficient Gi2α(Q205L), Gβ1, Gγ2, or a combination of them. Gi2α(Q205L) is mutated by substituting a leucine residue for a glutamine residue at position 205. This mutant is lacking in GTPase activity and thereby constitutively stimulates an effector enzyme (40Wong Y.H. Federman A. Pace A.M. Zachary I. Evans T. Pouyssegur J. Bourne H.R. Nature. 1991; 351: 63-65Crossref PubMed Scopus (184) Google Scholar). Expression of the mutated α and β subunits was verified by immunoblotting using specific antisera against the Gi2α (Fig. 4 A) or β (Fig. 4 B) subunit. Consistent with previous results (41Simonds W.F. Butrynski J.E. Gautam N. Unson C.G. Spiegel A.M. J. Biol. Chem. 1991; 266: 5363-5366Abstract Full Text PDF PubMed Google Scholar), no significant increase in the expression of the β subunit was detected when this subunit cDNA was transfected alone. A β subunit might be unstable in an intracellular environment unless it forms a complex with a γ subunit. We measured cAMP accumulation and IP production in the absence or presence of TSH in the cells overexpressing these α or βγ subunits. As shown in Fig. 4 D, TSH-induced cAMP accumulation was significantly inhibited by the expression of Gi2α(Q205L), whereas the transfection of β and/or γ subunit cDNAs hardly affected the cAMP response. Conversely, the overexpression of βγ subunits stimulated the TSH-induced PLC activation, whereas the expression of Gi2α(Q205L) did not appreciably influence it (Fig. 4 C). These results suggest the potential roles of the α and βγ subunits for the inhibition of the TSH-induced AC activation and the enhancement of the TSH-induced PLC activation, respectively. To determine whether these G protein subunits actually mediate the A1R agonist-induced modulation of TSH-induced actions, we examined the effects of PIA on the dose-dependent TSH activation of AC and PLC in the cells overexpressing Gi2α(Q205L) or βγ subunits (Fig. 5). As previously shown (Ref. 21Okajima F. Tomura H. Sho K. Akbar M. Majid M.A. Kondo Y. Biochem. J. 1995; 306: 709-715Crossref PubMed Scopus (9) Google Scholar and Figs. 1 and 2), PIA enhanced the dose-dependent TSH activation of IP production (Fig. 5 A) and conversely inhibited the TSH stimulation of cAMP accumulation (Fig. 5 D). When the βγ subunits were overexpressed, similar to the results shown in Fig. 4, the level of TSH-induced PLC (Fig. 5 B) but not cAMP accumulation (Fig. 5 E) was increased from that of the control cells at any TSH dose. Under these conditions, the addition of PIA together with TSH did not change the PLC level obtained by TSH alone but reduced the cAMP accumulation induced by the hormone. On the other hand, when the constitutively active mutant of Gi2α was expressed in the cells, the level of the TSH-induced accumulation of cAMP, but not of PLC activation, was lower than that in the control cells. In this case, the further addition of PIA did not change the level of TSH-induced cAMP accumulation but induced a further increase in the PLC level from that obtained by TSH alone. Thus, the overexpressed βγ subunit complex mimicked the stimulatory action of PIA on the TSH-induced PLC, while the constitutively active α subunit mimicked the inhibitory action of PIA on the TSH-induced AC. The direct inhibitory action of an α subunit of the Gi protein on activated AC has been reported by several groups (40Wong Y.H. Federman A. Pace A.M. Zachary I. Evans T. Pouyssegur J. Bourne H.R. Nature. 1991; 351: 63-65Crossref PubMed Scopus (184) Google Scholar, 42Taussig R. Iniguez Lluhi J.A. Gilman A.G. Science. 1993; 261: 218-221Crossref PubMed Scopus (321) Google Scholar, 43Chen J. Iyengar R. J. Biol. Chem. 1993; 268
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