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• W1986248919 abstract "The stimulatory GTP-binding protein of adenylyl cyclase (AC) regulates hormone-stimulated production of cAMP. Here, we demonstrate that Cu2+ and Zn2+ inhibit the steady-state GTPase activity of the α subunit of GTP-binding protein (Gαs) but do not alter its intrinsic GTPase activity. Cu2+ and Zn2+ decrease steady-state GTPase activity by inhibiting the binding of GTP to Gαs. Moreover, Cu2+ and Zn2+ increase GDP dissociation from Gαs and render the G protein in a nucleotide-free state. However, these cations do not alter the dissociation of the guanosine 5′-3-O-(thio)triphosphate (GTPγS) that is already bound to the Gαs. Because of their ability to inhibit GTPγS binding, preincubation of Cu2+ or Zn2+ with Gαs does not permit GTPγS to activate Gαs and stimulate AC activity. However, preincubation of Gαs with GTPγS followed by addition of Cu2+ or Zn2+ did not alter the ability of Gαs to stimulate AC activity. Interestingly, AlF4− partially restored the ability of Gαs, which had been preincubated with Cu2+ or Zn2+, to stimulate AC; AlF4− does not permit the re-association of unbound GDP with Gαs. Thus, the interaction of AlF4− with the nucleotide-free Gαs is sufficient to activate AC. Using antibodies to the N and C termini of Gαs, we show that the Cu2+ interaction site on the G protein is in the C terminus. We conclude that Cu2+ and Zn2+ generate a nucleotide-free state of Gαs and that, in the absence of any nucleotide, the γ-phosphate mimic of GTP, AlF4−, alters Gαs structure sufficiently to permit stimulation of AC activity. Moreover, our finding that isoproterenol-stimulated AC activity was more sensitive to inhibition by Cu2+ and Zn2+ as compared with forskolin-stimulated activity is consistent with Gαs being a primary target of these cations in regulating the signaling from receptor to AC. The stimulatory GTP-binding protein of adenylyl cyclase (AC) regulates hormone-stimulated production of cAMP. Here, we demonstrate that Cu2+ and Zn2+ inhibit the steady-state GTPase activity of the α subunit of GTP-binding protein (Gαs) but do not alter its intrinsic GTPase activity. Cu2+ and Zn2+ decrease steady-state GTPase activity by inhibiting the binding of GTP to Gαs. Moreover, Cu2+ and Zn2+ increase GDP dissociation from Gαs and render the G protein in a nucleotide-free state. However, these cations do not alter the dissociation of the guanosine 5′-3-O-(thio)triphosphate (GTPγS) that is already bound to the Gαs. Because of their ability to inhibit GTPγS binding, preincubation of Cu2+ or Zn2+ with Gαs does not permit GTPγS to activate Gαs and stimulate AC activity. However, preincubation of Gαs with GTPγS followed by addition of Cu2+ or Zn2+ did not alter the ability of Gαs to stimulate AC activity. Interestingly, AlF4− partially restored the ability of Gαs, which had been preincubated with Cu2+ or Zn2+, to stimulate AC; AlF4− does not permit the re-association of unbound GDP with Gαs. Thus, the interaction of AlF4− with the nucleotide-free Gαs is sufficient to activate AC. Using antibodies to the N and C termini of Gαs, we show that the Cu2+ interaction site on the G protein is in the C terminus. We conclude that Cu2+ and Zn2+ generate a nucleotide-free state of Gαs and that, in the absence of any nucleotide, the γ-phosphate mimic of GTP, AlF4−, alters Gαs structure sufficiently to permit stimulation of AC activity. Moreover, our finding that isoproterenol-stimulated AC activity was more sensitive to inhibition by Cu2+ and Zn2+ as compared with forskolin-stimulated activity is consistent with Gαs being a primary target of these cations in regulating the signaling from receptor to AC. The heterotrimeric stimulatory GTP-binding protein of adenylyl cyclase (Gs) 1The abbreviations used are: Gs, stimulatory GTP-binding protein of adenylyl cyclase; Gαs, and Gαi1, α subunits of the stimulatory and inhibitory GTP-binding proteins of adenylyl cyclase, respectively; AC, adenylyl cyclase; DTT, dithiothreitol; GTPγS, guanosine-5′-O-(3-thiotriphosphate). mediates the hormonal of neurotransmitter-elicited increase in cyclic AMP (cAMP). Its activation by heptahelical transmembrane receptors for hormones, neurotransmitters, and chemokines, results in the exchange of GDP for GTP on the α subunit of Gs (see Refs. 1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar, 2Neer E. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1289) Google Scholar, 3Bourne H.R. Curr. Opin. Cell Biol. 1997; 9: 134-142Crossref PubMed Scopus (530) Google Scholar, 4Neves S.R. Ram P.T. Iyengar R. Science. 2002; 296: 1636-1639Crossref PubMed Scopus (999) Google Scholar for reviews). The binding of GTP to the α subunit of Gs (Gαs) results in its dissociation from Gβγ subunits (reviewed in Refs. 1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar and 2Neer E. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1289) Google Scholar). The active, GTP-bound, Gαs subunit subsequently stimulates the downstream effectors such as adenylyl cyclase (AC) with a resultant increase in cAMP production (5Gilman A.G. Adv. Second Messenger Phosphoprotein Res. 1990; 24: 51-57PubMed Google Scholar, 6Patel T.B. Du Z. Pierre S. Cartin L. Scholich K. Gene (Amst.). 2001; 269: 13-25Crossref PubMed Scopus (143) Google Scholar, 7Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (272) Google Scholar). cAMP is an important second messenger that regulates various cellular functions such as cell growth, cell differentiation, and gene expression (8Gilman A.G. Harvey Lect. 1989; 85: 153-172PubMed Google Scholar, 9Chin K.V. Yang W.L. Ravatn R. Kita T. Reitman E. Vettori D. Cvijic M.E. Shin M. Iacono L. Ann. N. Y. Acad. Sci. 2002; 968: 49-64Crossref PubMed Scopus (102) Google Scholar). The divalent metal cations such as Cu2+ or Zn2+ are catalytic or structural components for a variety of enzymes such as cytochrome c oxidase (10Gennis R.B. Science. 1998; 280: 1712-1713Crossref PubMed Scopus (43) Google Scholar), Cu/Zn-superoxide dismutase (11Koningsberger J.C. van Asbeck B.S. van Faassen E. Wiegman L.J. van Hattum J. van Berge Henegouwen G.P. Marx J.J. Clin. Chim. Acta. 1994; 230: 51-61Crossref PubMed Scopus (24) Google Scholar), and dopamine-β-hydroxylase (12Abudu N. Banjaw M.Y. Ljones T. Eur. J. Biochem. 1998; 257: 622-629Crossref PubMed Scopus (8) Google Scholar). As such, these metal cations are involved in the regulation of a wide array of important biological processes that range from gene expression, neurotransmission, hormonal storage and release, to memory (13Beyersmann D. Haase H. Biometals. 2001; 14: 331-341Crossref PubMed Scopus (514) Google Scholar, 14Bush A.I. Curr. Opin. Chem. Biol. 2000; 4: 184-191Crossref PubMed Scopus (702) Google Scholar, 15Frederickson C.J. Suh S.W. Silva D. Thompson R.B. J. Nutr. 2000; 130: 1471S-1483SCrossref PubMed Google Scholar). Excess of Cu2+ or Zn2+ are toxic and are the cause of severe symptoms such as those observed in Wilson's disease. This latter disease is caused by an inherited defect in copper excretion into the bile by the liver (16Rathbun J.K. Int. J. Neurosci. 1996; 85: 221-229Crossref PubMed Scopus (91) Google Scholar, 17Thomas G.R. Forbes J.R. Roberts E.A. Walshe J.M. Cox D.W. Nat. Genet. 1995; 9: 210-217Crossref PubMed Scopus (493) Google Scholar). The accumulation of copper results in liver and neurological disease (17Thomas G.R. Forbes J.R. Roberts E.A. Walshe J.M. Cox D.W. Nat. Genet. 1995; 9: 210-217Crossref PubMed Scopus (493) Google Scholar, 18Waggoner D.J. Bartnikas T.B. Gitlin J.D. Neurobiol. Dis. 1999; 6: 221-230Crossref PubMed Scopus (771) Google Scholar). Excess Cu2+ and Zn2+ are also reported to be associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases (18Waggoner D.J. Bartnikas T.B. Gitlin J.D. Neurobiol. Dis. 1999; 6: 221-230Crossref PubMed Scopus (771) Google Scholar, 19Moir R.D. Atwood C.S. Huang X. Tanzi R.E. Bush A.I. Eur. J. Clin. Invest. 1999; 29: 569-570Crossref PubMed Scopus (21) Google Scholar, 20Kocaturk P.A. Akbostanci M.C. Tan F. Kavas G.O. Pathophysiology. 2000; 7: 63-67Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The cellular concentrations of Cu2+ or Zn2+ have been reported to be 10–200 μm (21Tholey G. Ledig M. Mandel P. Sargentini L. Frivold A.H. Leroy M. Grippo A.A. Wedler F.C. Neurochem. Res. 1988; 13: 45-50Crossref PubMed Scopus (49) Google Scholar), and in neuronal cells the concentration of Zn2+ can reach even higher values in the millimolar range (15Frederickson C.J. Suh S.W. Silva D. Thompson R.B. J. Nutr. 2000; 130: 1471S-1483SCrossref PubMed Google Scholar). Because cAMP plays a crucial role in the regulation of a number of cellular processes, one study has investigated the actions of Zn2+ on ACs and shown that Zn2+ inhibits the activity of type I, V, and VI ACs (22Klein C. Sunahara R.K. Hudson T.Y. Heyduk T. Howlett A.C. J. Biol. Chem. 2002; 277: 11859-11865Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The hormonal stimulation of cAMP was also attenuated by Zn2+ (22Klein C. Sunahara R.K. Hudson T.Y. Heyduk T. Howlett A.C. J. Biol. Chem. 2002; 277: 11859-11865Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). However, it is not clear whether and how Cu2+ and Zn2+ would influence the function of trimeric GTP-binding proteins that play an important role in regulating the activity of AC and synthesis of second messenger cAMP. Here we demonstrate that, at concentrations as low as 10 μm, Cu2+ and Zn2+ severely impair the function of Gαs. Both Cu2+ and Zn2+ cause the release of bound GDP from Gαs and inhibit the further binding of GTP, resulting in the formation of a nucleotide-free state of Gαs that is incapable in stimulating its effector, AC. Interestingly, the addition of aluminum fluoride, which mimics the γ phosphate of GTP on the GDP-bound form of G protein α subunits (23Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (757) Google Scholar, 24Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (538) Google Scholar), permits the nucleotide-free Gαs to activate AC. Our results provide a possible prime target for Cu2+ and Zn2+ in their toxicity in vivo. Our findings also imply that Cu2+ or Zn2+ by altering the function of G proteins can interfere with hormone-mediated changes in AC activity. Expression and Purification of Recombinant Gαs—N-terminal 6× His-tagged recombinant bovine Gαs and myristoylated rat Gαi1 were expressed and purified as described previously (25Graziano M.P. Freissmuth M. Gilman A.G. Methods Enzymol. 1991; 195: 192-202Crossref PubMed Scopus (21) Google Scholar, 26Wittpoth C. Scholich K. Bilyeu J.D. Patel T.B. J. Biol. Chem. 2000; 275: 25915-25919Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The proteins were stored in buffer containing 50 mm Tris-HCl, pH 8.0, and 20%(v/w) glycerol. The Gαs used in our experiments was the short form (380 amino acids long). Wild type and cyc– S49 cell membranes that contain predominantly type VI and VII ACs (27Premont R.T. Jacobowitz O. Iyengar R. Endocrinology. 1992; 131: 2774-2784Crossref PubMed Google Scholar) were isolated as described previously (26Wittpoth C. Scholich K. Bilyeu J.D. Patel T.B. J. Biol. Chem. 2000; 275: 25915-25919Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). The N terminus of 6× His-tagged Gαs contains an anti-Xpress antibody epitope. Thus, the protein was detected on Western blots with either the anti-Xpress antibody purchased from Invitrogen or a C-terminal epitope recognizing antibody obtained from Upstate Biotechnology (Lake Placid, NY). As monitored by GTPγS binding, the G protein preparations derived by these methods were 10–20% active. Steady-state GTPase Activity—Steady-state GTPase activity was measured in 100 μl of buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 1 μm [γ-32P]GTP (∼5000 dpm/pmol), and the indicated concentration of Gαs in the presence and absence of Cu2+ or Zn2+ at room temperature for 10–20 min. The reactions were terminated with ice-cold charcoal slurry (5% (w/v) in 50 mm NaH2PO4), and after centrifugation (20,000 × g for 10 min) aliquots of the supernatant were counted for 32Pi release. Single Cycle GTPase Assays—The single cycle GTPase activity was measured as described by Berman et al. (28Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar) with the following modification. Gαs (100 μg/ml) was first incubated at room temperature for 10 min in 50 mm Tris-HCl, pH 8.0, 1.0 mm EDTA, 1 μm [γ-32P]GTP (60 dpm/fmol). The temperature was lowered to 4 °C, and incubation continued for another 10 min. For time zero, 10-μl aliquots (1 μg of Gαs) were withdrawn and quenched with 0.99 ml of charcoal suspension in 50 mm NaH2PO4 described above. Within 10 s of withdrawing the zero time point, 100 μl of the reaction mixture was diluted to 1 ml with buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 200 μm CuSO4 or ZnSO4, and 1 mm GTP. At the indicated time points, 100-μl aliquots were withdrawn and mixed with 0.9 ml of charcoal suspension. 32Pi release was monitored as described for steady-state GTPase activity assay. GDP or GTPγS Release and GTPγS Binding—GDP release in absence or presence of AlF4− was measured by incubating Gαs (100 μg/ml) with trace amount of [α-32P]GTP in buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm MgSO4 without or with 30 μm AlCl3 and 2 mm NaF for 1 h at room temperature. The reaction mixture was then diluted 10 times in a buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm MgSO4, 1 mm GTP, and without or with AlF4− (30 μm AlCl3 and 2 mm NaF), and 100 μm CuSO4 or ZnSO4. Aliquots (100 μl) were withdrawn at the indicated times and filtered through PROTRAN BA85 filters (Schleicher and Schuell, Keene, NH) as described previously (29Higashijima T. Ferguson K.M. Sternweis P.C. Smigel M.D. Gilman A.G. J. Biol. Chem. 1987; 262: 762-766Abstract Full Text PDF PubMed Google Scholar). GTPγS release was determined by first incubating Gαs (100 μg/ml) with 1 μm [35S]GTPγS in buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm MgSO4 for 1 h at room temperature, and subsequently diluted 10 times in a buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm MgSO4, 1 mm GTP, and the 100 μm CuSO4 or ZnSO4. Aliquots (100 μl) were withdrawn at the indicated times and filtered through PROTRAN BA85 filters as described above. GTPγS binding was measured by incubating Gαs (1 μg of protein) at room temperature in 100 μl of buffer containing 50 mm Tris-HCl, pH 8.0, 5 mm MgSO4, 1 μm [35S]GTPγS (∼3000 dpm/pmol), and indicated concentrations of CuSO4 or ZnSO4. After 1-h incubation, 90-μl aliquots were withdrawn and filtered as described above. Adenylyl Cyclase Activity Assays—AC activity was measured as described previously (30Nair B.G. Parikh B. Milligan G. Patel T.B. J. Biol. Chem. 1990; 265: 21317-21322Abstract Full Text PDF PubMed Google Scholar) with 10 μg of S49 membranes in 100 μl of reaction mix containing 50 mm Tris-HCl, pH 7.4, 5 mm MgCl2, 10 μm GTP or GTPγS, 1 mm 3-isobutyl-1-methylxanthine, 0.1 mm ATP, 1 mm cAMP, ∼15,000 dpm [3H]cAMP, and ∼400 dpm/pmol [α-32P]ATP, and an ATP-regenerating system at room temperature for 20 min. Reactions were initiated by adding the assay mixture and were stopped with 50 μl of ice-cold, 3× stopping buffer (150 mm Tris-HCl, pH 7.4, 6% SDS, 3 mm cAMP, and 3 mm ATP). cAMP was separated by chromatography on two sequential columns of Dowex AG50 WX-4 and alumina, and the amount of 32P label in this fraction was determined by liquid scintillation counting. Cleavage of Gαs by Cu2+ in the Presence of Ascorbate and H2O2—Gαs (4 μg) was incubated with 10 μm CuSO4 in 20 μl of buffer containing 50 mm Tris-HCl, pH 8.0, and 5 mm MgSO4 for 10 min at room temperature. Cleavage of Gαs was initiated by adding 1 mm H2O2 and 10 mm ascorbate. Reactions were stopped after 10 min with 10 mm EDTA and 10 mm DTT. The cleaved Gαs was mixed with Laemmli sample buffer and separated on 10% SDS-polyacrylamide gels (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Proteins were transferred onto nitrocellulose, and the membranes were blocked by incubation in 10% newborn calf serum in phosphate-buffered saline. Duplicate sets of membranes were incubated either with anti-Xpress antibody (1:5000 dilution) or anti-Gαs C-terminal antibody (1:1000 dilution) for 1 h, respectively, followed by horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 h at room temperature. Proteins were detected using an enhanced chemiluminescence kit from Pierce. Cu2+ and Zn2+ Inhibit the Steady-state GTPase Activities of Gαs—To determine whether Cu2+ and Zn2+ alter Gαs function, we first examined the effects of these metal cations on the steady-state GTPase activity. The divalent metal cations, Cu2+ and Zn2+, were added directly in the reaction mixture without preincubation with Gαs. As shown in Fig. 1A, the steady-state GTPase activity of Gαs was completely inhibited by Cu2+ at concentrations higher than 10 μm with an IC50 for Cu2+ of ∼3 μm. Copper at this concentration has been reported to accumulate inside cells (21Tholey G. Ledig M. Mandel P. Sargentini L. Frivold A.H. Leroy M. Grippo A.A. Wedler F.C. Neurochem. Res. 1988; 13: 45-50Crossref PubMed Scopus (49) Google Scholar). Moreover, the onset of inhibition of the steady-state GTPase activity of Gαs by Cu2+ was very fast (Fig. 1A, inset). Within minutes of the addition of 20 μm CuSO4, the steady-state GTPase activity of Gαs was completely inhibited. Because the experiments in Fig. 1A were performed with 6× His-tagged Gαs, we investigated whether Cu2+ or Zn2+ also inhibited untagged Gαs. Essentially, both metal cations inhibited untagged Gαs in a manner similar to the N-terminally tagged Gαs (data not shown). Thus, Cu2+ and Zn2+ do not inhibit the steady-state GTPase activity by interacting with the N-terminal 6× His tag on Gαs. The inhibition of steady-state GTPase activity by Cu2+ and Zn2+ is not specific for Gαs only. Hence, at concentrations similar to those shown for Gαs in Fig. 1A, the metal cations also inhibit the steady-state GTPase activity of the inhibitory GTP-binding protein of AC, Gαi1 (Fig. 1B). That the inhibition of Gαs steady-state GTPase activity by Cu2+ or Zn2+ is specific is shown by the findings that other divalent cations such as Ni2+ or Fe2+ did not inhibit the steady-state GTPase activity of Gαs (Fig. 1C). If anything, Ni2+ modestly stimulated the steady-state GTPase activity of Gαs. Nevertheless, the data with Ni2+ (Fig. 1C) further emphasize the fact that the N-terminal tag on Gαs is not responsible for the inhibition that is observed with Cu2+ or Zn2+. It is possible that the inhibition of Gαs function may be due to the oxidation of sulfhydryl groups caused by Cu2+ and Zn2+. However, this seems not to be the case, because preincubation of Gαs with Cu2+ or Zn2+ for 10 min followed by incubation with 1 mm DTT did not result in any significant recovery of steady-state GTPase activity (Fig. 2A). On the other hand, preincubation of Cu2+ or Zn2+ with 1 mm DTT prior to the addition of Gαs completely neutralized their ability to inhibit steady-state GTPase activity (not shown). To determine if EDTA by chelating the Cu2+ or Zn2+ could reverse the inhibition of steady state GTPase activity of Gαs, the experiments shown in Fig. 2B were performed. Essentially, after incubation of Gαs with 20 μm each of Cu2+ or Zn2+, 1 mm EDTA was added to the incubations. The calculated free Cu2+ and Zn2+ concentrations in the presence of EDTA would be 4 × 10–15 m and 2.6 × 10–12 m, respectively. As shown in Fig. 2B, under these conditions the inhibition of steady-state GTPase activity was not reversed. These data suggest that either the metal cations are bound to Gαs with a very high affinity as demonstrated for interactions between protein phosphatase 1 and cobalt (32Chu Y. Lee E.Y. Schlender K.K. J. Biol. Chem. 1996; 271: 2574-2577Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) or that the Cu2+ and Zn2+ somehow modify the Gαs. Whatever the mechanism, the inhibition of steady-state GTPase activity of Gαs is not reversed by either EDTA or DTT. Cu2+ or Zn2+ Do Not Affect Catalysis but Inhibit the GTPγS Binding—The steady-state GTPase activity of Gαs can be decreased either by inhibiting the rate of GTP hydrolysis or by inhibiting the rate of GTP-GDP exchange (33Higashijima T. Ferguson K.M. Smigel M.D. Gilman A.G. J. Biol. Chem. 1987; 262: 757-761Abstract Full Text PDF PubMed Google Scholar, 34Landis C.A. Masters S.B. Spada A. Pace A.M. Bourne H.R. Vallar L. Nature. 1989; 340: 692-696Crossref PubMed Scopus (1237) Google Scholar). Therefore, additional experiments were performed to determine which of the two aforementioned possibilities contributes to the inhibition of steady-state Gαs-GTPase activity by Cu2+ and Zn2+. First, we examined the effects of Cu2+ and Zn2+ on the single cycle GTP hydrolysis by Gαs. This assay measures the ability of the G protein α subunits to hydrolyze prebound GTP and, therefore, is independent of the rate of GTP-GDP exchange (28Berman D.M. Kozasa T. Gilman A.G. J. Biol. Chem. 1996; 271: 27209-27212Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Essentially, Gαs was first loaded with γ-32P-labeled GTP in the presence of EDTA followed by dilution into buffer containing Mg2+ and excess of GTP in the absence or presence of 200 μm each of Cu2+ or Zn2+. As shown in Fig. 3A, neither Cu2+ nor Zn2+ altered the single cycle GTP hydrolysis by Gαs. Under the conditions of the experiment in Fig. 3A, the calculated free Cu2+ and Zn2+ concentrations were 2.6 and 27 μm, respectively. Notably, at these concentrations, Cu2+ and Zn2+ inhibit the steady-state GTPase activity by ∼50% and ≥90%, respectively (Fig. 1). Therefore, the data in Fig. 3A demonstrate that alterations in the intrinsic GTPase activity of Gαs do not contribute to Cu2+- and Zn2+-mediated inhibition of its steady-state GTPase activity. The crystal structure of Gαs shows that Mg2+ is coordinated with the β- and γ-phosphate groups of GTP and directly participates in the catalysis (7Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (272) Google Scholar). In this respect, the inability of Cu2+ or Zn2+ to inhibit the single cycle GTP hydrolysis by Gαs suggests that the inhibition of the steady-state GTPase activity of Gαs by Cu2+ or Zn2+ is not due to competition with Mg2+ for binding at the catalytic site on Gαs. Moreover, these results (Fig. 3A) suggest that Cu2+ or Zn2+ interact at one or more sites distinct from the Mg2+ binding site in the catalytic pocket of Gαs. Next, we examined if Cu2+ or Zn2+ altered the binding of GTPγS to Gαs. As demonstrated in Fig. 3B, Cu2+ and Zn2+ abrogated GTPγS binding to Gαs at concentrations >5 μm. The IC50 ∼ 2.5 μm for Cu2+-mediated inhibition of GTPγS binding is similar to that required for inhibition of steady-state GTPase activity. Overall, the findings in Fig. 3 demonstrate that the divalent metal cations, Cu2+ and Zn2+, inhibit steady-state GTPase activity of Gαs by decreasing the binding of GTP and not the intrinsic GTPase activity of the G protein. Cu2+ and Zn2+ Induce an Empty State of Gαs by Promoting the Release of GDP and Blocking Further Binding of Guanine Nucleotides—The purified Gαs has a very tightly bound GDP at its nucleotide binding site (35Ferguson K.M. Higashijima T. Smigel M.D. Gilman A.G. J. Biol. Chem. 1986; 261: 7393-7399Abstract Full Text PDF PubMed Google Scholar). Therefore, the binding of GTPγS has frequently been used as a reflection of GDP release (35Ferguson K.M. Higashijima T. Smigel M.D. Gilman A.G. J. Biol. Chem. 1986; 261: 7393-7399Abstract Full Text PDF PubMed Google Scholar). The inhibition of GTPγS binding by Cu2+ or Zn2+ (Fig. 3B) would suggest that Cu2+ or Zn2+ may decrease the release of GDP. To directly test this possibility, we preincubated Gαs with a trace amount of α-32P-labeled GTP in the presence of Mg2+ to ensure the complete hydrolysis of the bound GTP on Gαs, and subsequently diluted the radioactivity with excess GTP in the absence or presence of 100 μm each of Cu2+ or Zn2+. Surprisingly, instead of inhibiting the rate of GDP release, Cu2+ and Zn2+ dramatically accelerated the release of GDP (Fig. 4A). The t½ for GDP release in the absence of Cu2+ or Zn2+ was about 5 min. However, within that time all GDP was released when Cu2+ or Zn2+ was present (Fig. 4A). These results combined with the inhibition of GTPγS binding (Fig. 3B) suggest that Cu2+ or Zn2+ cause the release of GDP from Gαs and prevent further binding of nucleotide and imply that the resulting Gαs is in a nucleotide-free state. It is not clear whether this empty state has any resemblance to the assumed transitional empty state that occurs when Gαs is activated by ligand-bound receptor (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar). Both biochemical and structural evidence has shown that there are significant conformational changes among GDP-, GDP plus AlF4−-, and GTPγS-bound states of α subunits of heterotrimeric G proteins (23Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (757) Google Scholar, 24Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (538) Google Scholar, 36Raw A.S. Coleman D.E. Gilman A.G. Sprang S.R. Biochemistry. 1997; 36: 15660-15669Crossref PubMed Scopus (60) Google Scholar). Because Cu2+ and Zn2+ cause a rapid release of GDP from Gαs, we tested whether these metal cations would have different effects on the release of GDP in the presence of AlF4−. As shown in Fig. 4B, the presence of AlF4− markedly reduced the rate of release of GDP from Gαs (Fig. 4, compare A and B). However, the ability of Cu2+ and Zn2+ to accelerate the release of GDP from Gαs persisted even in the presence of AlF4− (Fig. 4B). The rate constants for release of GDP in Fig. 4B are: none (AlF4− alone): 0.072 min–1, AlF4− plus Zn2+: 0.17 min–1, and AlF4− plus Cu2+: 0.206 min–1. Although, to the best of our knowledge, the interactions between AlF4− and Cu2+ or Zn2+ have not been studied, it is possible that the difference in the GDP off rates in Fig. 4B could be attributed to some type of an interaction between the divalent metal cations and AlF4−. In contrast to the effects on GDP off rates, Cu2+ and Zn2+ did not significantly affect the release of GTPγS from Gαs (Fig. 4C). Analysis of the data in Fig. 4C demonstrated that the best fit is obtained by a one-binding site model and the average rate constants for GTPγS off were as follows: control (none in Fig. 4C): 0.109 ± 0.05 min–1; plus Cu2+: 0.104 ± 0.03 min–1; and plus Zn2+: 0.076 ± 0.009 min–1. None of these rate constants were significantly different from the others. Thus, Cu2+ and Zn2+ alter the rates of GDP off but do not affect the GTPγS off rates. These results (Fig. 4) are consistent with previous reports (23Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (757) Google Scholar, 24Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (538) Google Scholar, 36Raw A.S. Coleman D.E. Gilman A.G. Sprang S.R. Biochemistry. 1997; 36: 15660-15669Crossref PubMed Scopus (60) Google Scholar) that the structural and biochemical properties of GDP-, GDP plus AlF4−-, and GTPγS-bound forms of Gαs are different. Cu2+ and Zn2+ Modulate the Ability of Gαs to Stimulate AC—Because Cu2+ and Zn2+ alter the binding of GTPγS to Gαs and the release of GDP from this G protein, we tested the ability of the metal cations to alter the ability of Gαs to stimulate AC activity. For this purpose membranes of cells (S49 cyc–) that do not express endogenous Gαs (37Bourne H.R. Coffino P. Tomkins G.M. Science. 1975; 187: 750-752Crossref PubMed Scopus (145) Google Scholar) were used. In this system, the stimulation of AC by Gαs in the presence of GTP is modest but can be substantially enhanced by forskolin. Therefore, these experiments were performed in the presence of forskolin to improve the sensitivity of the assay that monitors Gαs activity. Moreover, to preclude any effects of Cu2+ or Zn2+ on AC activity per se, after incubation of the Gαs in the presence or absence of the metal cations, DTT (1 mm) was added. It has been shown that in the presence of DTT, Zn2+ does not alter AC activity (22Klein C. Sunahara R.K. Hudson T.Y. Heyduk T. Howlett A.C. J. Biol. Chem. 2002; 277: 11859-11865Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Indeed, in our experiments also this treatment did not alter control activity of AC (Fig. 5, A and B, cf. none with Cu2+ or Zn2+). As shown in Fig. 5, preincubation of Gαs with GTP or GTPγS or AlF4− resulted in stimulation of AC activity. However, when Gαs was preincubated first with Cu2+ or Zn2+ and then incubated with GTP or GTPγS for 1 h, the G protein did not stimulate AC activity (Fig. 5, A and B). This is consistent with our findings (Fig. 3B) that GTPγS binding to the G protein is inhibited by Cu2+ and Zn2+ and, therefore, the G protein is in an inactive state. On the other hand, if the Gαs was first preincubated with GTPγS and then incubation with Cu2+ or Zn2+, the ability of the G protein to stimulate AC activity was retained. This latter finding is also predictable from the observation (Fig. 4C) that once GTPγS is bound to the G protein neither Cu2+ nor Zn2+ alter its release and the Gαs is maintained in an active state. However, preincubation with GTP followed by incubation with the metal cations did not retain the ability of Gαs to stimulate AC (Fig. 5, A and B). This would be expected, because the intrinsic GTPase activity rapidly converts GTP to GDP that is then rapidly released in the presence of Cu2+ or Zn2+. Surprisingly, after preincubation of Gαs with Cu2+ or Zn2+, AlF4− partially restored the ability of Gαs to stimulate AC (Fig. 5C). These findings demonstrate that Cu2+ and Zn2+ do not irreversibly inactivate the G protein by some nonspecific action or denature the Gαs. The ability of AlF4− to partially restore the ability of Gαs that was preincubated with Cu2+ or Zn2+ to stimulate AC activity is similar to the ability of AlF4− to partially restore the ability of nucleotide-free transducin to activate cGMP phosphodiesterase (38Bigay J. Deterre P. Pfister C. Chabre M. FEBS Lett. 1985; 191: 181-185Crossref PubMed Scopus (324) Google Scholar). Thus, AlF4−, by mimicking the γ-phosphate of GTP (23Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (757) Google Scholar, 24Sondek J. Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 372: 276-279Crossref PubMed Scopus (538) Google Scholar), may be sufficient to alter the conformation or the guanine nucleotide-free Gαs so that the G protein can stimulate AC activity. However, it is also possible that AlF4− increases the affinity for GDP such that, after the preincubation with Cu2+ or Zn2+, in the presence of AlF4−, GDP that is released binds back to the Gαs. To distinguish between the last two possibilities mentioned above, the experiment depicted in Fig. 6 was performed. Essentially, after loading Gαs with trace amounts of α-32P-labeled GTP and Mg2+, the G protein was passed over a Sephadex column to remove any unbound guanine nucleotide. Because of the high intrinsic GTPase activity of Gαs, under these conditions, the 32P label associated with the protein is in the GDP form. Thereafter, the Gαs was incubated with Cu2+ or Zn2+ or AlF4− followed by another incubation in the presence or absence of one of these reagents, and the amount of 32P-labeled GDP associated with the G protein was determined. As shown in Fig. 6 and consistent with the findings in Fig. 4A, incubation with Cu2+ or Zn2+ decreased the amount of 32P-labeled GDP associated with Gαs. Moreover, as shown in Fig. 4B, when AlF4− was added prior to Cu2+ or Zn2+, the dissociation of GDP from Gαs was decreased. However, when AlF4− was added after Cu2+ or Zn2+, the GDP associated with Gαs was minimal and the same as that in the presence of Cu2+ or Zn2+ alone (Fig. 6). These findings (Fig. 6) demonstrate that the AlF4− does not facilitate the re-association of the GDP that has been released from Gαs by Cu2+ or Zn2+. Thus, the data in Fig. 6 support the notion that the ability of AlF4− to stimulate AC after treatment of the Gαs with Cu2+ or Zn2+ (Fig. 5) is independent of GDP reassociation to the G protein and most likely due to the ability of AlF4− to mimic the γ-phosphate of GTP and alter the conformation of the nucleotide-free Gαs sufficiently to activate AC. An identical scenario has previously been presented for the nucleotide-free transducin and its partial activation by AlF4− to stimulate cGMP phosphodiesterase (38Bigay J. Deterre P. Pfister C. Chabre M. FEBS Lett. 1985; 191: 181-185Crossref PubMed Scopus (324) Google Scholar). In this context, it should also be noted that studies from the Iyengar laboratory (39Chen Y. Yoo B. Lee J. Weng G. Iyengar R. J. Biol. Chem. 2001; 276: 45751-45754Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) have shown that small peptides corresponding to the switch regions of Gαs can stimulate AC. Therefore, small and subtle changes in the switch regions of Gαs manifested by AlF4− binding in the absence of GDP may be sufficient to activate AC. Interestingly, the guanine nucleotide-free state of Gαs induced by Cu2+ or Zn2+ appears to be different from the guanine nucleotide-free form of Gαi that is induced by activated receptors or guanine nucleotide exchange factors such as Ric-8 (40Tall G.G. Krumins A.M. Gilman A.G. J. Biol. Chem. 2002; 278: 8356-8362Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). In the latter case, the G protein can bind GTPγS (40Tall G.G. Krumins A.M. Gilman A.G. J. Biol. Chem. 2002; 278: 8356-8362Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar), whereas in the case of Cu2+ or Zn2+ pretreatment, the G protein loses the ability to bind GTPγS. Nevertheless, Cu2+ and Zn2+ provide a means by which a guanine nucleotide-free state of Gαs can be achieved to study the structure of this form of the G protein. Location of Cu2+ Binding Region on Gαs—To locate the possible Cu2+ binding site(s) on Gαs, we took the advantage of the fact that Cu2+ can catalyze the Fenton reaction in the presence of H2O2 and ascorbate and hydrolyze the peptide backbone at the Cu2+ binding site(s) (41Marx G. Chevion M. Biochem. J. 1986; 236: 397-400Crossref PubMed Scopus (206) Google Scholar). Moreover, to determine the fragment sizes and whether the N or C terminus of Gαs was cleaved, we utilized one antibody that recognizes the anti-Xpress tag on the N terminus of the recombinant Gαs and another antibody against the last 10 amino acids of the G protein. As shown in Fig. 7 (A and B), incubation of Gαs with CuSO4, ascorbate, and H2O2 for 10 min resulted in the cleavage of Gαs, and as determined with the N-terminal antibody, the appearance of a new band that migrated as a 39- to 40-kDa protein. In control reactions, no cleavage was observed in the absence of ascorbate (lane 2, Fig. 7A). Interestingly, Western analysis of the cleaved Gαs with the C-terminal anti-Gαs antibody did not detect the smaller band that migrated as a 39- to 40-kDa protein (Fig. 7B). These data (Fig. 7, A and B) show that the cleavage site is at the C terminus of Gαs. Moreover, the data in Fig. 7B show that Cu2+ by itself does not alter the migration of Gαs and that longer (1 h) incubation of the G protein in the presence of ascorbate and H2O2 results in near complete degradation. Notably, we did not observe any protective effect of GTPγS against the Cu2+-mediated cleavage in the Fenton reaction (not shown) demonstrating that, although Cu2+ does not alter the ability of the GTPγS-bound form of Gαs to stimulate AC activity (Fig. 5), the divalent cation can still bind to the GTPγS-bound form of Gαs. Based on the approximate molecular weight of the cleaved band (from its migration on SDS-PAGE gels) we propose that the potential cleavage site is located in the region encompassed by amino acid residues 310–330 (numbering according to the long form of Gαs) (Fig. 7C). This region is on the opposite side of switches 1, 2, and 3 and AC binding site on Gαs (7Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (272) Google Scholar). This region (amino acids 310–330) has also been shown to be the dimerization site in the Gαs crystal structure (7Sunahara R.K. Tesmer J.J. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (272) Google Scholar). However, whether Gαs forms dimers in vivo and whether this dimerization is of functional significance are presently not clear. Receptor-mediated Activation of AC Is More Sensitive to Inhibition by Zn2+ Than Forskolin-stimulated AC—Zn2+ has been demonstrated to inhibit certain isoforms of AC, including ACVI (22Klein C. Sunahara R.K. Hudson T.Y. Heyduk T. Howlett A.C. J. Biol. Chem. 2002; 277: 11859-11865Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Because our studies demonstrate that Zn2+ also inhibits Gαs activation, we determined the relative sensitivity of receptor-mediated stimulation of AC versus direct inhibition of the enzyme by Zn2+. For this purpose, membranes of S49 cells that express ACVI and ACVII (27Premont R.T. Jacobowitz O. Iyengar R. Endocrinology. 1992; 131: 2774-2784Crossref PubMed Google Scholar) were used. Essentially, the ability of different concentrations of Zn2+ and Cu2+ to inhibit AC activity that was stimulated by either isoproterenol or forskolin was monitored. The data in Fig. 8A demonstrate that, as shown previously by Klein et al. (22Klein C. Sunahara R.K. Hudson T.Y. Heyduk T. Howlett A.C. J. Biol. Chem. 2002; 277: 11859-11865Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), Zn2+ inhibited forskolin-stimulated AC activity with an IC50 value of ∼5.5 μm. Likewise, Cu2+ also inhibited forskolin-stimulated AC activity with an IC50 value of ∼6 μm (Fig. 8B). However, isoproterenol-stimulated AC activity was more sensitive to inhibition by Zn2+ and Cu2+ as compared with forskolin-stimulated AC activity (Fig. 8, A and B). Thus, although Zn2+ and Cu2+ can inhibit AC activity directly, the additional inhibition at the level of the Gαs activation also plays a role in inhibiting AC activity in response to neurotransmitters or hormones. In fact, the inhibition of Gαs apparently occurs at much lower concentrations of the metal cations and, therefore, the more sensitive response of receptor-activated AC as compared with forskolin-stimulated AC activity. Notably, the IC50 value for Zn2+- and Cu2+-mediated inhibition of isoproterenol-stimulated AC activity was ∼1.5 μm (Fig. 8) a value similar (∼2 μm) to that observed for inhibition of GTPγS binding to Gαs (Fig. 3B). Overall, these data (Fig. 8) are consistent with the notion that inhibition of Gαs function is one of the primary sites of Cu2+ and Zn2+ action in regulating receptor-mediated stimulation of AC. In conclusion, our data show that Cu2+ and Zn2+ dissociate the GDP that is bound to Gαs and that these divalent metal cations inhibit the binding of GTPγS to the G protein thus generating a nucleotide-free form of the G protein. Both of these effects render the G protein inactive as a stimulator of AC when Cu2+ and Zn2+ are added before GTP or GTPγS. However, once bound, GTPγS is not readily dissociated from the G protein by either Cu2+ or Zn2+, and, therefore, the G protein is capable of activating AC. Interestingly, AlF4− can partially restore the ability of Gαs to activate AC even after treatment of the G protein with the divalent metal cations. This effect of AlF4− is not dependent on the re-association of GDP with the G protein. Thus, AlF4− by itself is sufficient to alter the structure of the guanine nucleotide-free form of the Gαs induced by Cu2+ and Zn2+ to permit activation of AC. In this respect, this is the first evidence to show the presence of a nucleotide-free form of Gαs and that the γ-phosphate of GTP may be sufficient to alter the structure of Gαs sufficiently to stimulate AC. We also show that the Cu2+ interaction site on Gαs is in the C terminus of the G protein and is most likely located between amino acid residues 310–330. Finally, by modulating the activity of Gαs, Cu2+ and Zn2+ can interfere with the regulation of AC activity by hormones and neurotransmitters that mediate their actions via GTP-binding protein-coupled receptors." @default.
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