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• W2064507764 abstract "A Ser to Asn mutation at position 54 of the α subunit of Gs (designated N54-αs) was characterized after transient expression of it with various components of the receptor-adenylyl cyclase pathway in COS-1, COS-7, and HEK 293 cells. Previous studies of the N54-αs mutant revealed that it has a conditional dominant negative phenotype that prevents hormone-stimulated increases in cAMP without interfering with the regulation of basal cAMP levels (Cleator, J. H., Mehta, N. D., Kurtz, D. K., Hildebrandt, J. D. (1999) FEBS Lett. 243, 205–208). Experiments reported here were conducted to localize the mechanism of the dominant negative effect of the mutant. Competition studies conducted with activated αs* (Q212L) showed that the N54 mutant did not work down-stream by blocking the interaction of endogenous αs with adenylyl cyclase. The co-expression of wild type or N54-αs along with the thyroid-stimulating hormone (TSH) receptor and adenylyl cyclase isotypes differing with respect to βγ stimulation (AC II or AC III) revealed that the phenotype of the mutant is not dependent upon the presence of adenylyl cyclase isoforms regulated by βγ. These studies ruled out a downstream site of action of the mutant. To investigate an upstream site of action, N54-αs was co-expressed with either the TSH receptor that activates both αs and αq or with the α1B-adrenergic receptor that activates only αq. N54-αs failed to inhibit α1B-adrenergic receptor stimulation of inositol 1,4,5-trisphosphate production but did inhibit TSH stimulation of inositol 1,4,5-trisphosphate. These results show that Gs and Gq compete for activation by the TSH receptor. They also indicate that the N54 protein has a dominant negative phenotype by blocking upstream receptor interactions with normal G proteins. This phenotype is different from that seen in analogous mutants of other G protein α subunits and suggests that either regulation or protein-protein interactions differ among G protein α subunits. A Ser to Asn mutation at position 54 of the α subunit of Gs (designated N54-αs) was characterized after transient expression of it with various components of the receptor-adenylyl cyclase pathway in COS-1, COS-7, and HEK 293 cells. Previous studies of the N54-αs mutant revealed that it has a conditional dominant negative phenotype that prevents hormone-stimulated increases in cAMP without interfering with the regulation of basal cAMP levels (Cleator, J. H., Mehta, N. D., Kurtz, D. K., Hildebrandt, J. D. (1999) FEBS Lett. 243, 205–208). Experiments reported here were conducted to localize the mechanism of the dominant negative effect of the mutant. Competition studies conducted with activated αs* (Q212L) showed that the N54 mutant did not work down-stream by blocking the interaction of endogenous αs with adenylyl cyclase. The co-expression of wild type or N54-αs along with the thyroid-stimulating hormone (TSH) receptor and adenylyl cyclase isotypes differing with respect to βγ stimulation (AC II or AC III) revealed that the phenotype of the mutant is not dependent upon the presence of adenylyl cyclase isoforms regulated by βγ. These studies ruled out a downstream site of action of the mutant. To investigate an upstream site of action, N54-αs was co-expressed with either the TSH receptor that activates both αs and αq or with the α1B-adrenergic receptor that activates only αq. N54-αs failed to inhibit α1B-adrenergic receptor stimulation of inositol 1,4,5-trisphosphate production but did inhibit TSH stimulation of inositol 1,4,5-trisphosphate. These results show that Gs and Gq compete for activation by the TSH receptor. They also indicate that the N54 protein has a dominant negative phenotype by blocking upstream receptor interactions with normal G proteins. This phenotype is different from that seen in analogous mutants of other G protein α subunits and suggests that either regulation or protein-protein interactions differ among G protein α subunits. Heterotrimeric GTP-binding proteins or G proteins transmit signals from receptors on the surface of the cell to various intracellular effector enzymes. G proteins have traditionally been defined by their α subunits, which consist of four families: αs; αi/o; αq/11; and α15/16. One of the best-characterized α subunits, αs, stimulates adenylyl cyclase in response to receptor-mediated hormone stimulation (1Neer E.J. Cell. 1995; 80: 249-258Abstract Full Text PDF PubMed Scopus (1287) Google Scholar, 2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4714) Google Scholar, 3Spiegel A.M. Med. Res. Rev. 1992; 12: 55-71Crossref PubMed Scopus (13) Google Scholar, 4Birnbaumer L. Annu. Rev. Pharmacol. Toxicol. 1990; 30: 675-705Crossref PubMed Google Scholar). Inactive Gs consists of a GDP-bound α subunit and a βγ dimer. Upon receptor activation, the αs·GTP complex dissociates from the receptor and its βγ dimer and then stimulates adenylyl cyclase to increase cAMP levels in the cell. Certain isotypes of adenylyl cyclase (e.g. AC II and AC IV) are also stimulated by βγ in the presence of activated αs (5Iyengar R. FASEB J. 1993; 7: 768-775Crossref PubMed Scopus (266) Google Scholar, 6Taussig R. Tang W.J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar), whereas other isotypes of adenylyl cyclase are inhibited by βγ (e.g. AC I). Deactivation occurs when the α subunit hydrolyzes its bound GTP to GDP, allowing it to reassociate with βγ. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; TSH, thyroid-stimulating hormone; PI, phosphatidylinositol; TSH-R, TSH receptor; αs*, activated αs Q212L mutant; VIP, vasoactive intestinal polypeptide; VIP-R, vasoactive intestinal polypeptide receptor; HEK, human embryonic kidney; GMP-P(NH)P, guanyl-5′-yl imido-diphosphate; AC I and AC II, adenylyl cyclases I and II; AR, adrenergic receptor; IP, inositol phosphate. activate G proteins by catalyzing GTP exchange on the α subunit. Although some GPCRs activate only one G protein isotype (e.g. β-adrenergic receptor activation of αs), others are described as promiscuous and can activate multiple G protein isotypes (e.g. the receptor for TSH). The TSH receptor has been shown to couple to adenylyl cyclase and phospholipase C (7Van Sande J. Raspe E. Perret J. Lejeune C. Maenhaut C. Vassart G. Dumont J.E. Mol. Cell. Endocrinol. 1990; 74: R1-R6Crossref PubMed Scopus (182) Google Scholar, 8Kosugi S. Okajima F. Ban T. Hidaka A. Shenker A. Kohn L.D. Mol. Endocrinol. 1993; 7: 1009-1018Crossref PubMed Scopus (82) Google Scholar, 9Kosugi S. Okajima F. Ban T. Hidaka A. Shenker A. Kohn L.D. J. Cell Biol. 1992; 267: 24153-24156Google Scholar), in part because the receptor can couple to both Gs and Gq/11 (10Allgeier A. Offermanns S. Van Sande J. Spicher K. Schultz G. Dumont J. J. Cell Biol. 1994; 269: 13733-13735Google Scholar). It has not been determined whether one population of TSH receptors exists that possesses the ability to couple both Gs and Gq/11 or whether multiple populations of receptor exist (e.g. arising from either splice variants, post-translational modifications, or compartmentalization) that could account for the ability of the TSH-R to activate both Gs and Gq/11. The superfamily of GTP-binding proteins, which include the α subunits of heterotrimeric G proteins and the monomeric GTP-binding proteins, share four conserved amino acid sequences that form the core of the GTP regulatory mechanism of these proteins (11Kaziro Y. Itoh H. Kozasa T. Nakafuku M. Satoh T. Annu. Rev. Biochem. 1991; 60: 349-400Crossref PubMed Scopus (549) Google Scholar, 12Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2690) Google Scholar, 13Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1585) Google Scholar). The consensus sequence of the first conserved region of the nucleotide binding site is GX4GK(S/T), the residues of which include Ser17 in Ras and the equivalent Ser54 in Gαs. These residues are involved in binding Mg2+ ion when it is complexed with nucleotide (14Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (966) Google Scholar). Ras with an Asn mutation at this site (N17) has an increased preference for GDP over GTP and a dominant negative phenotype (15Feig L.A. Cooper G.M. Mol. Cell. Biol. 1988; 8: 3235-3243Crossref PubMed Scopus (679) Google Scholar). The dominant negative phenotype of N17-Ras can be explained by its ability to sequester its own guanine nucleotide exchange factor (16Stacey D.W. Feig L.A. Gibbs J.B. Mol. Cell. Biol. 1991; 11: 4053-4064Crossref PubMed Scopus (137) Google Scholar, 17Schweighoffer F. Cai H. Chevallier-Multon M.C. Fath I. Cooper G. Tocque B.T. Mol. Cell. Biol. 1993; 13: 39-43Crossref PubMed Scopus (49) Google Scholar), which is functionally analogous to a GPCR. An extension of the N17-Ras mutation to other members of the Ras superfamily, such as Ran (18Kornbluth S. Dasso M.J.N. J. Cell Biol. 1994; 125: 705-719Crossref PubMed Scopus (107) Google Scholar), Rab (19Nuoffer C. Davidson H.W. Matteson J. Meinkoth J. Balch W.E. J. Cell Biol. 1994; 125: 225-237Crossref PubMed Scopus (193) Google Scholar), Ral (20Jiang H. Luo J. Urano T. Frankel P. Lu Z. Foster D.A. Feig L.A. Nature. 1995; 378: 409-412Crossref PubMed Scopus (247) Google Scholar), Rho (21Qiu R. Chen J. McMormick F. Symons M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11781-11785Crossref PubMed Scopus (488) Google Scholar), and Rac (22Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3076) Google Scholar), also results in a dominant negative phenotype. Gαs with a Ser to Asn mutation at this site (N54) also has an increased preference for GDP over GTP (23Hildebrandt J.D. Day R. Farnsworth C.L. Feig L.A. Mol. Cell. Biol. 1991; 11: 4830-4838Crossref PubMed Google Scholar) and a conditional dominant negative phenotype (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar). Its phenotype is conditional, because it prevents hormone-stimulated increases in cAMP, even though it itself activates adenylyl cyclase down-stream and increases basal cAMP levels (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar). The site analogous to Ser54 of αs in αi and αo is Ser47. Cys mutants of this site also have a dominant negative phenotype (25Slepak V.Z. Katz A. Simon M.I. J. Biol. Chem. 1995; 270: 4037-4041Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Slepak V.Z. Quick M.W. Aragay A.M. Davidson N. Lester H.A. Simon M.I. J. Biol. Chem. 1993; 268: 21889-21894Abstract Full Text PDF PubMed Google Scholar). However, the mechanism in this case appears to be different from that of the N17-Ras mutant. In particular, others have suggested that C47-αo and C47-αi interfere with hormone activation of G proteins by tightly binding βγ dimers (25Slepak V.Z. Katz A. Simon M.I. J. Biol. Chem. 1995; 270: 4037-4041Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Slepak V.Z. Quick M.W. Aragay A.M. Davidson N. Lester H.A. Simon M.I. J. Biol. Chem. 1993; 268: 21889-21894Abstract Full Text PDF PubMed Google Scholar). These mutants are presumably not active themselves, because G protein subunit dissociation is required for activation (2Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4714) Google Scholar). Further, their dominant negative phenotype is explained by their ability to sequester βγ dimers away from other G protein α subunits and by the fact that intact G protein heterotrimers facilitate greatly receptor interactions. Because Ras mutants and αi/o mutants of this essential Ser residue involved in complexing Mg and nucleotide seem to have different mechanisms of action, we sought to determine the analogous mechanism for the N54-αs mutant. Surprisingly, the N54-αs mutant has a mechanism of action more similar to that of Ras than its more closely related αi/o counterparts. These results suggest differences either in the mechanism of regulation of αs and αi proteins by magnesium and nucleotides or in the protein-protein interactions of these different G proteins. Materials—[3H]adenine, [3H]inositol, donkey anti-rabbit horseradish peroxidase-linked antibody, and ECL reagents were purchased from Amersham Biosciences. Bovine TSH (Lot-AFP-5555B) was obtained from the National Hormone and Pituitary Program, NIDDK, National Institutes of Health. All of the other materials were of the highest quality grade available. Construction of Vectors—cDNAs encoding the long form of Gαs and the N54 mutation of Gαs in pMV7 (23Hildebrandt J.D. Day R. Farnsworth C.L. Feig L.A. Mol. Cell. Biol. 1991; 11: 4830-4838Crossref PubMed Google Scholar) were subcloned into the HindIII site of the mammalian expression vector pcDNA3 (Invitrogen) driven by the cytomegalovirus promoter. The activated (Q212L) αs mutant (αs*) in the mammalian expression vector pCr-cytomegalovirus was kindly provided by R. Iyengar. Types II and III adenylyl cyclase cDNAs (27Bakalyar H.A. Reed R.R. Science. 1990; 250: 1403-1405Crossref PubMed Scopus (525) Google Scholar, 28Feinstein P.G. Schrader K.A. Bakalyar H.A. Tang W.J. Krupinski J. Gilman A.G. Reed R.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10173-10177Crossref PubMed Scopus (224) Google Scholar), kindly provided by Dr. R. Reed, were subcloned from Bluescript KS between the HindIII and BamHI sites of pcDNA1 and into the EcoRI site of pcDNA3, respectively. The rat TSH-R cDNA inserted in the simian virus 40 promoter-driven expression vector, pSG5 (29Akamizu T. Ikuyama S. Saji M. Kosugi S. Kozak C. McBride O.W. Kohn L.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 55677-55681Crossref Scopus (241) Google Scholar), was kindly provided by Dr. L. Kohn. The rat vasoactive intestinal polypeptide receptor (VIP-R) cDNA (30Ishihara T. Shigemoto R. Mori K. Takahashi K. Nagata S. Neuron. 1992; 8: 811-819Abstract Full Text PDF PubMed Scopus (730) Google Scholar) in the expression vector CDM8 under control of the cytomegalovirus promoter was a kind gift from Dr. S. Nagata. The α1B-adrenergic receptor in the expression vector pMT2 under control of the SV40 major late promoter was kindly provided by Dr. D. Perez. Transient Transfections and cAMP Determination—COS-1 and HEK 293 cells grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C were transfected with LipofectAMINE as previously described (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar). Cyclic AMP was measured by use of the [3H]adenine uptake assay as described previously (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar). Immunoblotting—Samples were prepared by washing cells in phosphate-buffered saline followed by addition of Laemmli sample buffer containing 10% β-mercaptoethanol (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). Proteins were separated by SDS-polyacrylamide gel electrophoresis on 11% polyacrylamide gels by the procedure of Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar) and transferred to nitrocellulose using a Bio-Rad semi-dry transfer apparatus. Proteins were incubated with anti-αs antibody, ASC, produced according to the protocol of Spiegel and co-workers (32McKenzie F.R. Mullaney I. Unson C.G. Spiegel A.M. Milligan G. Biochem. Soc. Trans. 1988; 16: 434-437Crossref PubMed Scopus (11) Google Scholar). Bands were visualized by ECL. Inositol Phosphate Determination—Inositol phosphate production was measured by use of the [3H]inositol uptake assay (33De vivo M. Methods Enzymol. 1994; 238: 131-141Crossref PubMed Scopus (11) Google Scholar, 34Clarke S. Curr. Opin. Cell Biol. 1993; 5: 977-983Crossref PubMed Scopus (200) Google Scholar, 35Hildebrandt J.D. Biochem. Pharmacol. 1997; 54: 325-339Crossref PubMed Scopus (127) Google Scholar, 36Conklin B.R. Chabre O. Wong Y.H. Federman A. Bourne H.R. J. Biol. Chem. 1992; 267: 31-34Abstract Full Text PDF PubMed Google Scholar). Cells were labeled with 2 μCi/ml myo-[3H]inositol ∼24 h prior to treatment with hormone. Cells were washed once with Dulbecco's modified Eagle's medium followed by incubation in Dulbecco's modified Eagle's medium/20 mm NaHepes (or Hanks' balanced salt solution where indicated) containing 10 mm LiCl for 10 min prior to hormone treatment. Cells were incubated for 40 min at 35 °C in the presence or absence of the hormone. The reaction was terminated by the addition of 0.75 ml of ice-cold 10 mm formic acid to the cells. 0.75 ml of each reaction (1 well of a 24-well dish) was diluted in 3 ml of 20 mm ammonium hydroxide (pH of final solution, 8.0–9.0) and applied to regenerated Dowex anion-exchange columns (AG1-X8 resin, Bio-Rad 140-1444) followed by the addition of 1 ml of H20. The combined sample and water wash was collected as the [3H]inositol fraction. The columns then were washed with 4 ml of 40 mm ammonium formate in 0.1 m formic acid. The [3H]inositol phosphates were collected by eluting the columns with 5 ml of 2 m ammonium formate in 0.1 m formic acid. Data are presented as the percent of total [3H]inositol recovered as [3H]inositol phosphates where total [3H]inositol equals [3H]inositol phosphates plus [3H]inositol. Previous characterization of the N54 mutant revealed that it possessed a conditional dominant negative phenotype with respect to the TSH-R and the β-adrenergic receptor (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar). The nature of this phenotype is shown in Fig. 1A where the rat VIP-R (30Ishihara T. Shigemoto R. Mori K. Takahashi K. Nagata S. Neuron. 1992; 8: 811-819Abstract Full Text PDF PubMed Scopus (730) Google Scholar) was co-expressed in COS-1 cells with either wild type or N54-αs. The expression of αs resulted in elevated basal cAMP levels with the mutant being slightly more effective than wild type. Co-expression with wild type αs was substantially more effective at increasing VIP-stimulated cAMP levels than co-expression with the N54-αs mutant. When basal cAMP levels were subtracted from those in the presence of VIP (Fig. 1A, right graph), N54-αs was seen to actually decrease VIP-stimulated cAMP levels. These results were similar to what is seen with the TSH receptor and the β-adrenergic receptors (Fig. 1B). Interestingly, whereas the expression of wild type αs increased hormone-stimulated levels, N54-αs decreased both TSH and VIP-stimulated cAMP levels by ∼50%. Based on dose-response curves for the expression of wild type and mutant αs (data not shown and Ref, 24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar), this appears to be a maximal effect of the mutant. The mutant also decreased stimulation of cAMP levels through the endogenous β-adrenergic receptor but to a lesser degree than for the transfected receptors. The lower inhibition seen in this case probably reflects the relative transfection efficiency, and stimulation through this receptor is probably blunted by ∼50% as well. Fig. 1 and our previous results (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar) indicate that N54-αs interferes with receptor stimulation of adenylyl cyclase. The N17-Ras homolog of N54-αs interferes with Ras signaling by binding unproductively to its upstream regulator (16Stacey D.W. Feig L.A. Gibbs J.B. Mol. Cell. Biol. 1991; 11: 4053-4064Crossref PubMed Scopus (137) Google Scholar, 17Schweighoffer F. Cai H. Chevallier-Multon M.C. Fath I. Cooper G. Tocque B.T. Mol. Cell. Biol. 1993; 13: 39-43Crossref PubMed Scopus (49) Google Scholar, 37Farnsworth C.L. Feig L.A. Mol. Cell. Biol. 1991; 11: 4822-4829Crossref PubMed Scopus (184) Google Scholar). Signaling by G proteins is more complex than for the monomeric G proteins, and hence, there are more possible sites of action for N54-αs. In fact, the mutants of the cognate site in αo and αi (S47C) appear to have a dominant negative phenotype because they bind tightly to βγ (25Slepak V.Z. Katz A. Simon M.I. J. Biol. Chem. 1995; 270: 4037-4041Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Slepak V.Z. Quick M.W. Aragay A.M. Davidson N. Lester H.A. Simon M.I. J. Biol. Chem. 1993; 268: 21889-21894Abstract Full Text PDF PubMed Google Scholar). Therefore, we sought to determine whether N54-αs had a mechanism analogous to its related αi/o proteins or one similar to the more distantly related Ras protein. Co-expression of Wild Type or N54-αs with Activated αs in COS-7 Cells—One possible mechanism of action of a dominant negative effect of N54-αs would be for it to bind to adenylyl cyclase, thus blocking stimulation by wild type αs. Although at first thought this mechanism might seem inconsistent with the fact that N54-αs has inherent basal activity (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar), this is not necessarily true. For example, N54-αs could have a lower intrinsic efficacy for activating adenylyl cyclase than does wild type αs. Thus, its binding to adenylyl cyclase could activate the enzyme to a low level but preclude its interaction with an activated wild type αs with greater efficacy and generated by receptor activation of Gs. In fact, our previous data could be consistent with the idea that N54-αs does have less efficacy than does a persistently activated αs protein (24Cleator J.H. Mehta N.D. Kurtz D.T. Hildebrandt J.D. FEBS Lett. 1999; 243: 205-208Crossref Scopus (18) Google Scholar). To test whether this idea is probable, we determined whether N54-αs could effectively compete with an activated αs (Q212L-designated αs*) for stimulation of adenylyl cyclase. To establish conditions for this experiment, increasing amounts of αs* cDNA were transfected with a fixed amount of TSH-R cDNA into COS-1 cells and cAMP levels were measured in the absence or presence of TSH (Fig. 2A). As the amount of αs* cDNA increased, the difference between hormone-stimulated and basal cAMP levels decreased. At 450 ng of αs* transfected with the TSH-R, there was little hormone-stimulated activity, suggesting that the adenylyl cyclase in these cells was near maximally stimulated by the activated αs mutant (Fig. 2A). To examine whether the N54 mutant could inhibit activated αs* from stimulating adenylyl cyclase, αs* cDNA (450 ng) was co-transfected alone or with an equal amount of either wild type or N54-αs (Fig. 2B). As seen in Fig. 2B, a representative experiment conducted in COS-1 cells, the expression of αs* increased basal cAMP levels to a greater extent than did wild type or N54-αs. The co-expression of N54-αs with αs* failed to appreciably inhibit αs* from stimulating adenylyl cyclase under basal conditions or under hormone stimulation (Fig. 2B, compare αs* to αs* + N54-αs). A summary of experiments conducted in COS-1 cells is shown in Fig. 2C. Control immunoblots showed that mutant and wild type αs were actually expressed at ∼10 times the level of the activated αs* protein in these experiments (Fig. 2D). Thus, at least at a 10-fold excess, N54-αs does not block the stimulation of adenylyl cyclase by αs*. These studies did not support the idea that N54-αs blocks the ability of wild type αs to stimulate adenylyl cyclase. Interestingly, it was observed that transfection of αs* in COS-1 cells resulted in an increase in the endogenous long form of αs (Fig. 2D). Although this is a potentially interesting result, we have not yet followed it up. Because it was not clear how this induction affected the results of these experiments, we conducted the same experiment in HEK 293 cells, which do not show an increase in the expression of endogenous long form of αs upon expression of αs* (Fig. 2D). The effect of transfection of αs* alone compared with αs* plus N54-αs on basal and TSH-stimulated cAMP levels in HEK 293 cells demonstrated that N54-αs did not prevent αs* from stimulating adenylyl cyclase in these cells either (Fig. 2C). Co-expression of Wild type or N54-αs with Adenylyl Cyclase II and III in COS-1 Cells—Another possible mechanism for the dominant negative effect of N54-αs might involve its interference with downstream effects of βγ. This could result from N54-αs irreversibly binding to βγ, as has been suggested to explain the phenotype of the analogous mutation in Gαi (25Slepak V.Z. Katz A. Simon M.I. J. Biol. Chem. 1995; 270: 4037-4041Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To determine whether the mutant interferes with the βγ regulation of downstream effectors, experiments were conducted with co-expressed adenylyl cyclase isotypes (AC II or AC III) that differ with respect to βγ regulation. AC II can be activated directly by βγ dimers in the presence of activated αs, whereas AC III is not regulated directly by βγ (5Iyengar R. FASEB J. 1993; 7: 768-775Crossref PubMed Scopus (266) Google Scholar, 6Taussig R. Tang W.J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar). AC II or AC III were co-expressed with the TSH-R and with αs (wild type or N54 mutant) in COS cells. Co-expression of AC II with the TSH-R increased TSH stimulation of cAMP accumulation 5-fold compared with transfection of the TSH-R alone (Fig. 3), whereas co-expression of AC III with the TSH-R only increased cAMP accumulation 3-fold in response to TSH. It is not clear whether these differences are due to differences in the level of expression of these two adenylyl cyclase isoforms, to differences in their inherent activities, or to differences in their regulation. Nevertheless, the differences in the inherent regulation of these two enzymes allowed us to test whether they differed also in their response to the presence of the αs mutants. When wild type αs was transfected with AC II, TSH-stimulated cAMP accumulation was decreased. This is consistent with the idea that TSH maximally stimulated AC II by a dual mechanism involving both αs and βγ and that expressed αs inhibited this maximal stimulation by binding βγ. In contrast, the expression of wild type αs with AC III increased cAMP accumulation in response to TSH, compatible with the idea that αs is rate-limiting for activation of AC III under these conditions. However, regardless of the response to wild type αs, transfection of the N54-αs mutant with either AC II or AC III decreased cAMP accumulation in response to TSH (Fig. 3, insets). This finding suggests that the effects of N54-αs on receptor regulation of cAMP are independent of the properties of the downstream adenylyl cyclase isoform producing the cAMP. This makes it unlikely that the mutant inhibits TSH stimulation of adenylyl cyclase by simply sequestering βγ and preventing the effects of βγ on downstream effectors. This idea is further supported by the fact that the mutant can inhibit TSH stimulation of adenylyl cyclase in HEK 293 cells, which reportedly lack an adenylyl cyclase that is stimulated by βγ (38Federman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (482) Google Scholar). Effect of Wild type αk or N54-αs on Signaling to Phospholipase-β through the α1B-Adrenergic Receptor in COS-7 Cells—In the case of the Ser47 mutant of αi, the analogous site to Ser54 of αs, it interferes not only with receptor regulation of αi-mediated signaling but also with signaling mediated through other G proteins, even for receptors that do not regulate Gi (25Slepak V.Z. Katz A. Simon M.I. J. Biol. Chem. 1995; 270: 4037-4041Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). This is one argument that the mutant works by sequestering βγ and preventing upstream receptor heterotrimer coupling. The β-adrenergic receptor, the TSH receptor, and the VIP receptor all couple to Gs. Presumably, a dominant negative effect of αs mediated by its binding to receptor would not directly interfere with signaling through receptors that do not couple to Gs. Therefore, we tested whether N54-αs blocked α1B-adrenergic receptor (α1B-AR) signaling to phospholipase Cβ through Gq. The α1B-adrenergic receptor is not known to couple to Gs. COS-7 cells were used for these experiments, because they reportedly lack a phospholipase Cβ isoform that is stimulated by βγ (39Jiang H. Kuang Y. Wu Y. Smrcka A. Simon M.I. Wu D. J. Biol. Chem. 1996; 271: 13430-13434Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The α1B-AR when expressed in COS-7 cells (Fig. 4A) had an EC50 for phenylephrine of ∼1 μm (Fig. 4B). Increasing the expression of wild type αs slightly decreased the ability of the α1B-AR to increase inositol phosphate levels (Fig. 4C). In contrast, increasing the expression of N54-αs had no effect on the ability of the α1B-AR to stimulate phospholipase C (Fig. 4C). The expression of αs* failed to alter phenylephrine stimulation of PI turnover in COS-7 cells (Fig. 4D), indicating that the effects of wild type αs cannot be accounted for by elevations in cAMP levels. Although these data are complicated by small effects of wild type αs protein on signaling through phospholipase (discussed below), they clearly demonstrate that N54-αs does not block α1B-AR stimulation of phospholipase C through Gq. This result supports the idea that N54-αs does not nonspecifically interfere with receptor regulation of downstream processes by sequestering βγ dimers necessary for receptor G protein coupling. Co-" @default.
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• W2064507764 date "2004-08-01" @default.
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• W2064507764 title "A Dominant Negative Gαs Mutant That Prevents Thyroid-stimulating Hormone Receptor Activation of cAMP Production and Inositol 1,4,5-Trisphosphate Turnover" @default.
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• W2064507764 doi "https://doi.org/10.1074/jbc.m406232200" @default.
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