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• W2083996981 abstract "We previously reported that substitution of Arg258 within the switch 3 region of Gsα impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976–23983). The adjacent residue (Glu259) is strictly conserved in G protein α-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsα-E259A), Gln (Gsα-E259Q), Asp (Gsα-E259D), or Val (Gsα-E259V), and the properties of in vitrotranslation products were examined. The Gsα-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsα-E259D stimulated adenylyl cyclase normally in the presence of GTPγS but was less efficient with isoproterenol or AlF4−. The other mutants had more severely impaired effector activation, particularly in response to AlF4−. In trypsin protection assays, GTPγS was a more effective activator than AlF4− for all mutants, with Gsα-E259D being the least severely impaired. For Gsα-E259D, the AlF4−-induced activation defect was more pronounced at low Mg2+ concentrations. Gsα-E259D and Gsα-E259A purified fromEscherichia coli had normal rates of GDP release (as assessed by the rate GTPγS binding). However, for both mutants, the ability of AlF4− to decrease the rate of GTPγS binding was impaired, suggesting that they bound AlF4− more poorly. GTPγS bound to purified Gsα-E259D irreversibly in the presence of 1 mm free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis ofin vitro translates demonstrated that all mutants except Gsα-E259V bind to βγ at 0 °C and were stable at higher temperatures. In the active conformation Glu259interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4− in the guanine nucleotide binding pocket. Although both Gsα Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsα protein activation are distinct. We previously reported that substitution of Arg258 within the switch 3 region of Gsα impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976–23983). The adjacent residue (Glu259) is strictly conserved in G protein α-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsα-E259A), Gln (Gsα-E259Q), Asp (Gsα-E259D), or Val (Gsα-E259V), and the properties of in vitrotranslation products were examined. The Gsα-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsα-E259D stimulated adenylyl cyclase normally in the presence of GTPγS but was less efficient with isoproterenol or AlF4−. The other mutants had more severely impaired effector activation, particularly in response to AlF4−. In trypsin protection assays, GTPγS was a more effective activator than AlF4− for all mutants, with Gsα-E259D being the least severely impaired. For Gsα-E259D, the AlF4−-induced activation defect was more pronounced at low Mg2+ concentrations. Gsα-E259D and Gsα-E259A purified fromEscherichia coli had normal rates of GDP release (as assessed by the rate GTPγS binding). However, for both mutants, the ability of AlF4− to decrease the rate of GTPγS binding was impaired, suggesting that they bound AlF4− more poorly. GTPγS bound to purified Gsα-E259D irreversibly in the presence of 1 mm free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis ofin vitro translates demonstrated that all mutants except Gsα-E259V bind to βγ at 0 °C and were stable at higher temperatures. In the active conformation Glu259interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4− in the guanine nucleotide binding pocket. Although both Gsα Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsα protein activation are distinct. Mutagenesis of the conserved residue Glu259 of Gsα demonstrates the importance of interactions between switches 2 and 3 for activation.Journal of Biological ChemistryVol. 274Issue 13PreviewPage 4978, Table I: The four treatments are: GTP (100 μm), isoproterenol (10 μm) + GTP (100 μm), GTPγS (100 μm), and AlF4−. Full-Text PDF Open Access Heterotrimeric guanine nucleotide-binding proteins (G proteins) 1The abbreviations G proteinguanine nucleotide-binding proteinGsstimulatory G proteinGsαGs α-subunitGsα-E259D-E259A, -E259Q, and -E259V, Gsα mutant with Glu259 substituted to aspartate, alanine, glutamine, and valine, respectivelyAlF4−mixture of 10 μm AlCl3 and 10 mmNaFGTPγSguanosine-5′-O-(3-thiotriphosphate)WTwild type 1The abbreviations G proteinguanine nucleotide-binding proteinGsstimulatory G proteinGsαGs α-subunitGsα-E259D-E259A, -E259Q, and -E259V, Gsα mutant with Glu259 substituted to aspartate, alanine, glutamine, and valine, respectivelyAlF4−mixture of 10 μm AlCl3 and 10 mmNaFGTPγSguanosine-5′-O-(3-thiotriphosphate)WTwild typecouple heptahelical receptors to intracellular effectors and are composed of three subunits (α, β, and γ) (reviewed in Refs. 1Spiegel A.M. Shenker A. Weinstein L.S. Endocr. Rev. 1992; 13: 536-565Crossref PubMed Scopus (309) Google Scholar, 2Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1286) Google Scholar, 3Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta Rev. Biomembr. 1990; 1031: 163-224Crossref PubMed Scopus (961) Google Scholar). The α-subunits, which are distinct for each G protein, bind guanine nucleotide and modulate the activity of specific downstream effectors. For Gs, these include the stimulation of adenylyl cyclase and modulation of ion channels (4Yatani A. Codina J. Imoto Y. Reeves J.P. Birnbaumer L. Brown A.M. Science. 1987; 238: 1288-1292Crossref PubMed Scopus (362) Google Scholar, 5Schreibmayer W. Dessauer W. Vorobiev D. Gilman A.G. Lester H.A. Davidson N. Dascal N. Nature. 1996; 380: 624-627Crossref PubMed Scopus (97) Google Scholar). In the inactive state, GDP-bound α-subunit is associated with a βγ-dimer. Upon receptor activation, the α-subunit undergoes a conformational change resulting in the exchange of GTP for GDP and dissociation from βγ. While GTP is bound, the α-subunit interacts with and regulates specific effectors. An intrinsic GTPase activity within the α-subunit hydrolyzes bound GTP to GDP, returning the G protein to the inactive state. Analogs of GTP, such as GTPγS and GDP-AlF4−, lock the G protein in the active state. guanine nucleotide-binding protein stimulatory G protein Gs α-subunit -E259A, -E259Q, and -E259V, Gsα mutant with Glu259 substituted to aspartate, alanine, glutamine, and valine, respectively mixture of 10 μm AlCl3 and 10 mmNaF guanosine-5′-O-(3-thiotriphosphate) wild type guanine nucleotide-binding protein stimulatory G protein Gs α-subunit -E259A, -E259Q, and -E259V, Gsα mutant with Glu259 substituted to aspartate, alanine, glutamine, and valine, respectively mixture of 10 μm AlCl3 and 10 mmNaF guanosine-5′-O-(3-thiotriphosphate) wild type X-ray crystal structures reveal that G protein α-subunits have two domains, a ras-like GTPase domain, which includes the regions for guanine nucleotide binding and effector interaction, and a helical domain, which may prevent release of GDP in the inactive state (6Noel J.P. Hamm H.E. Sigler P.B. Nature. 1993; 366: 654-663Crossref PubMed Scopus (707) Google Scholar, 7Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (530) Google Scholar, 8Coleman D.E. Berghuis A.M. Lee E. Linder M.E. Gilman A.G. Sprang S.R. Science. 1994; 265: 1405-1412Crossref PubMed Scopus (752) Google Scholar, 9Wall M.A. Coleman D.E. Lee E. Iñiguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1009) Google Scholar, 10Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (267) Google Scholar, 11Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 297-299Crossref PubMed Scopus (1047) Google Scholar, 12Sunahara R.K. Tesmer J.J.G. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (264) Google Scholar). Comparison of the crystal structures of inactive (GDP-bound) and activated (GTPγS- or AlF4−-bound) α-subunits demonstrates three regions (named switches 1, 2, and 3), the conformation of which changes upon switching from the inactive to active state. The movement of switches 1 and 2 is directly related to the presence of the γ-phosphate group, whereas switch 3 has no direct contact with bound guanine nucleotide. Upon activation, switches 2 and 3 move toward each other, and the two regions form multiple interactions that presumably stabilize the active state (7Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (530) Google Scholar, 10Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (267) Google Scholar). Switch 3 residues also make contacts with the helical domain that are important for high affinity guanine nucleotide binding (10Mixon M.B. Lee E. Coleman D.E. Berghuis A.M. Gilman A.G. Sprang S.R. Science. 1995; 270: 954-960Crossref PubMed Scopus (267) Google Scholar, 15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). At least for transducin, this region may also be important in effector activation (13Li Q. Cerione R.A. J. Biol. Chem. 1997; 272: 21673-21676Crossref PubMed Scopus (21) Google Scholar). We have previously shown that substitutions of the switch 3 residue Arg258 impairs activation by receptor or AlF4−(15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 2All numbering is based on the Gsα-1 sequence reported by Kozasa et al. (17Kozasa T. Itoh H. Tsukamoto T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2081-2085Crossref PubMed Scopus (348) Google Scholar). 2All numbering is based on the Gsα-1 sequence reported by Kozasa et al. (17Kozasa T. Itoh H. Tsukamoto T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2081-2085Crossref PubMed Scopus (348) Google Scholar). The latter effect was the direct result of decreased GDP binding due to loss of contacts between the Arg258 side chain and residues within the helical domain. The adjacent residue (Glu259) is invariant in all known G protein α-subunits and is predicted to be important in activation, because it makes interactions with switch 2 residues in the active state (7Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (530) Google Scholar, 12Sunahara R.K. Tesmer J.J.G. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (264) Google Scholar). Moreover, this residue is mutated to a valine in a patient with Albright hereditary osteodystrophy (16Ahmed S.F. Dixon P.H. Bonthron D.T. Stirling H.F. Barr D.G.B. Kelnar C.J.H. Thakker R.V. Clin. Endocrinol. 1998; 49: 525-531Crossref PubMed Google Scholar). In the present report, we provide evidence that substitution of Glu259also leads to impaired activation, particularly by receptor or AlF4−. However, impaired activation of these mutants by AlF4− is not the result of decreased GDP binding (as is the case for the Arg258mutants) but rather is the result of a decreased ability to bind the AlF4− moiety. The crystal structure of GTPγS-bound Gsα reveals interactions between the acidic side chain of Glu259 and basic residues within switch 2 that are important in maintaining the active state and in binding of AlF4− (12Sunahara R.K. Tesmer J.J.G. Gilman A.G. Sprang S.R. Science. 1997; 278: 1943-1947Crossref PubMed Scopus (264) Google Scholar). Although adjacent switch 3 residues in Gsα (Arg258 and Glu259) are both critical for activation, the mechanisms by which mutations of these residues result in defective activation are distinct. To generate Gsα Glu259 mutants, polymerase chain reaction was performed as described previously (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) using linearized vector containing wild type Gsα cDNA as template. The upstream primer was 5′-GACAAAGTCAACTTCCACATGTTTGACGTGGGTGGCCAGCGCGATGAACG-3′, and the downstream mutagenic primers were as follows: 5′-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCACCCGGATGACCATGTTG-3′ for E259V, 5′-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCGCCCGGATGACCATGTTG-3′ for E259A, 5′-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCCTGCCGGATGACCATGTTG-3′ for E259Q, and 5′-GAGCCTCCTGCAGGCGGTTGGTCTGGTTGTCGTCCCGGATGACCATGTTG-3′ for E259D. Each polymerase chain reaction product was digested withHincII andSse8387I and ligated into the transcription vector pBluescript II SK (Stratagene, La Jolla, CA) that contained wild type human Gsα cDNA (splice variant Gsα-1, Ref. 17Kozasa T. Itoh H. Tsukamoto T. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2081-2085Crossref PubMed Scopus (348) Google Scholar) in which the sameHincII-Sse8387I restriction fragment had been removed. Mutations were verified by DNA sequencing, and synthesis of full-length Gsα from each construct was confirmed by immune precipitation of in vitro translated products with RM antibody, directed against the carboxyl-terminal decapeptide of Gsα (18Simonds W.F. Goldsmith P.K. Woodard C.J. Unson C.G. Spiegel A.M. FEBS Lett. 1989; 249: 189-194Crossref PubMed Scopus (147) Google Scholar). In vitro transcription/translation was performed on Gsα plasmids as described previously (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Warner D.R. Gejman P.V. Collins R.M. Weinstein L.S. Mol. Endocrinol. 1997; 11: 1718-1727PubMed Google Scholar) using the TNT-coupled transcription/translation system from Promega, with the exception that in most experiments, no RNase inhibitor was added. Wild type and mutant Gsα in vitro transcription/translation products (10 μl of translation medium) were reconstituted into 25 μg of purified S49 cyc plasma membranes and tested for stimulation of adenylyl cyclase in the presence of various agents as indicated in Table I (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Warner D.R. Gejman P.V. Collins R.M. Weinstein L.S. Mol. Endocrinol. 1997; 11: 1718-1727PubMed Google Scholar, 20Sternweis P.C. Northup J.K. Smigel M.D. Gilman A.G. J. Biol. Chem. 1981; 256: 11517-11526Abstract Full Text PDF PubMed Google Scholar). Reactions were incubated for 15 min at 30 °C, and the amount of [32P]cAMP produced was measured as described previously (21Salomon Y. Londos C. Rodbell M. Anal. Biochem. 1974; 58: 541-548Crossref PubMed Scopus (3374) Google Scholar).Table IAdenylyl cyclase stimulation by Gsα mutantsGsα mutationIsoproterenol (10 μm)GTP (100 μm)+ GTP (100 μm)GTPγS (100 μm)AlF4−a10 mm NaF, 10 μmAlCl3, and 100 μm GDP.pmol of cAMP/ml of translation product/15 min (% of WT)WTbThe results for Gsα-WT are the same as previously published (15) because these were generated simultaneously with those obtained for the Gsα-Arg258 mutants.20 ± 6231 ± 6167 ± 7359 ± 41E259V3 ± 38 ± 7 (3 ± 3)18 ± 3 (11 ± 2)5 ± 5 (1 ± 1)E259A8 ± 337 ± 9 (16 ± 4)65 ± 10 (39 ± 6)10 ± 10 (3 ± 3)E259Q2 ± 126 ± 15 (11 ± 6)61 ± 5 (37 ± 3)12 ± 12 (3 ± 3)E259D15 ± 6160 ± 33 (69 ± 14)164 ± 6 (98 ± 6)118 ± 11 (33 ± 5)In vitro transcription/translation products were mixed with purified cyc- membranes and assayed for adenylyl cyclase stimulation as described under “Experimental Procedures.” Results are expressed as the mean ± S.D. (ςn − 1) of triplicate determinations and are corrected for the relative level of synthesis of each mutant to Gsα-WT. Gsα-E259V, -E259A, -E259Q, and -E259D were synthesized to 73, 78, 74, and 91% of Gsα-WT levels, respectively, as determined by in vitro translation with [35S]methionine, SDS-PAGE, and phosphorimaging. Background values determined from mock transcription/translation reactions (in pmol of cAMP/ml of translation medium/15 min: GTP, 29 ± 1; isoproterenol, 39 ± 5; GTPγS, 39 ± 2; and AlF4−, 64 ± 6) were subtracted from each determination.a 10 mm NaF, 10 μmAlCl3, and 100 μm GDP.b The results for Gsα-WT are the same as previously published (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) because these were generated simultaneously with those obtained for the Gsα-Arg258 mutants. Open table in a new tab In vitro transcription/translation products were mixed with purified cyc- membranes and assayed for adenylyl cyclase stimulation as described under “Experimental Procedures.” Results are expressed as the mean ± S.D. (ςn − 1) of triplicate determinations and are corrected for the relative level of synthesis of each mutant to Gsα-WT. Gsα-E259V, -E259A, -E259Q, and -E259D were synthesized to 73, 78, 74, and 91% of Gsα-WT levels, respectively, as determined by in vitro translation with [35S]methionine, SDS-PAGE, and phosphorimaging. Background values determined from mock transcription/translation reactions (in pmol of cAMP/ml of translation medium/15 min: GTP, 29 ± 1; isoproterenol, 39 ± 5; GTPγS, 39 ± 2; and AlF4−, 64 ± 6) were subtracted from each determination. Limited trypsin digestion ofin vitro translated Gsα was performed as described previously (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Warner D.R. Gejman P.V. Collins R.M. Weinstein L.S. Mol. Endocrinol. 1997; 11: 1718-1727PubMed Google Scholar). Briefly, 1 μl of in vitrotranslated [35S]methionine-labeled Gsα was incubated in incubation buffer (20 mm HEPES, pH 8.0, 10 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol) with or without 100 μmGTPγS or 10 mm NaF/10 μm AlCl3at various temperatures for 1 h and then digested with 200 μg/ml tosyl-l-phenylyalanine chloromethyl ketone-trypsin for 5 min at 20 °C. In some experiments, GDP was also included in the preincubation, and in other experiments the MgCl2concentration was varied. Reactions were terminated by boiling in Laemmli buffer. Digestion products were separated on 10% SDS-polyacrylamide gels, and the amount of 38-kDa protected fragment was measured by phosphorimaging. The percentage of protection is the signal in 38-kDa protected band divided by the signal in the undigested full-length Gsα band × 100. [35S]Methionine-labeled Gsα was synthesized, and rate zonal centrifugation was performed on linear 5–20% sucrose gradients (200 μl) as described previously (19Warner D.R. Gejman P.V. Collins R.M. Weinstein L.S. Mol. Endocrinol. 1997; 11: 1718-1727PubMed Google Scholar, 22Basi N.S. Rebois R.V. Anal. Biochem. 1997; 251: 103-109Crossref PubMed Scopus (5) Google Scholar). Gradients were prepared in 20 mmHEPES, pH 8.0, 1 mm MgCl2, 1 mmEDTA, 1 mm dithiothreitol, 100 mm NaCl, 0.1% Lubrol-PX. Six-μl fractions were obtained and analyzed by SDS-polyacrylamide gel electrophoresis, and the relative amount of Gsα in each fraction was quantified as described previously (19Warner D.R. Gejman P.V. Collins R.M. Weinstein L.S. Mol. Endocrinol. 1997; 11: 1718-1727PubMed Google Scholar). To assess the ability of Gsα to bind to Gβγ, in vitro translation products were preincubated for 1 h at 0 °C in the presence or absence of Gβγ (20 μg/ml) prior to centrifugation. In order to optimize separation between free α-subunit and heterotrimer, 0.15% (w/v) CHAPS was substituted for Lubrol-PX in the preincubations and gradients, and the samples were centrifuged at 120,000 rpm (627,000 × g at the maximum radial distance from the center of rotation (R max) in a TLA-120.2 rotor (Beckman). Gβγ was isolated from bovine brain (23Roof D.J. Applebury M.L. Sternweis P.C. J. Biol. Chem. 1985; 260: 16242-16249Abstract Full Text PDF PubMed Google Scholar). Plasmid pQE60, containing the long form of bovine Gsα cDNA with a hexa-histidine extension at the carboxyl terminus, was a generous gift of A. G. Gilman and R. K. Sunahara. The Glu259 residue was mutated by site-directed mutagenesis using the Quickchange kit (Statagene). Each mutated cDNA was sequenced to confirm the presence of the desired mutation and to rule out polymerase chain reaction artifacts. After transformation into E. coli strain JM109, cultures were grown, Gsα expression was induced, and Gsα proteins were purified as described previously (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar,24Lee E. Linder M.E. Gilman A.G. Methods Enzymol. 1994; 237: 146-163Crossref PubMed Scopus (247) Google Scholar), except that [GDP] was only 10 μm in the storage buffer. Assays measuring the rate of binding of GTPγS were performed as described previously (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 25Carty D.J. Iyengar R. Methods Enzymol. 1994; 237: 38-44Crossref PubMed Scopus (26) Google Scholar). Briefly, 1–2 pmol of purified Gsα was incubated at 37 °C in a final volume of 2 ml containing 1 μm[35S]GTPγS (5,000–10,000 cpm/pmol) in 25 mm HEPES, pH 8.0, 1 mm EDTA, 100 mmNaCl, 10 mm MgCl2, 1 mmdithiothreitol, and 0.01% Lubrol-PX with or without 10 mmNaF/10 μm AlCl3. At various times, 50-μl aliquots were removed and diluted with 2 ml of ice-cold stop solution (25 mm Tris-HCl, 100 mm NaCl, 25 mmMgCl2, and 100 μm GTP) and maintained on ice until all samples were collected. Samples were then filtered under vacuum through nitrocellulose filters (Millipore) and washed twice with 10 ml of stop solution without GTP, and filters were dissolved in 10 ml of scintillation mixture. To determine the effect of Mg2+on the rate of GTPγS dissociation, ∼2.5 pmol of purified Gsα was loaded with [35S]GTPγS at 30 °C for 45 min in the presence of various free Mg2+concentrations. After addition of 100 μm cold GTPγS, bound [35S]GTPγS was determined at various time points as described above. k off for GTPγS dissociation was determined by fitting the data to the functiony = ae −kt +b using the software GraphPad Prism, version 2.01. Free Mg2+ concentrations were calculated as described (26Skooge D.A. West D.M. Fundamentals of Analytical Chemistry. Saunders College Publishing, Philadelphia, PA1982: 276-303Google Scholar). Gsα Glu259substitution mutants were cloned into the transcription vector pBluescript, and the in vitro transcription/translation products were compared with those of Gsα-WT in various biochemical assays. We substituted Glu259 with valine (Gsα-E259V) because a mutation encoding this substitution has been identified in a patient with Albright hereditary osteodystrophy (16Ahmed S.F. Dixon P.H. Bonthron D.T. Stirling H.F. Barr D.G.B. Kelnar C.J.H. Thakker R.V. Clin. Endocrinol. 1998; 49: 525-531Crossref PubMed Google Scholar), a human disorder associated with heterozygous loss-of-function mutations of Gsα (27Patten J.L. Johns D.R. Valle D. Eil C. Gruppuso P.A. Steele G. Smallwood P.M. Levine M.A. N. Engl. J. Med. 1990; 322: 1412-1419Crossref PubMed Scopus (369) Google Scholar, 28Weinstein L.S. Gejman P.V. Friedman E. Kadowaki T. Collins R.M. Gershon E.S. Spiegel A.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8287-8290Crossref PubMed Scopus (255) Google Scholar). Because the presence of an amino acid with a bulky and branched side chain (valine) may introduce nonspecific steric effects, we also generated and analyzed additional mutants in which Glu259 was replaced by alanine (Gsα-E259A), glutamine (Gsα-E259Q), or aspartate (Gsα-E259D). In Gsα-E259A, the acidic side chain was removed, whereas in Gsα-E259Q it is converted to a residue in which the carboxyl group is replaced by a neutral amide group. In Gsα-E259D, the charge of the residue at position Glu259 is maintained, but the length of the side chain is shortened by one methylene group. After reconstitution of translation products into purified S49 cyc membranes (which lack endogenous Gsα), Gsα-E259V had markedly decreased ability to stimulate adenylyl cyclase in the presence of GTPγS, AlF4−, or activated receptor (isoproterenol + GTP) (Table I). For Gsα-E259A and -E259Q, the ability to stimulate adenylyl cyclase was moderately reduced in the presence of GTPγS (∼40% of Gsα-WT) and more markedly reduced in the presence of AlF4− or activated receptor. Stimulation of adenylyl cyclase by Gsα-E259D was normal in the presence of GTPγS but moderately reduced in the presence of AlF4− or activated receptor. Although the severity of the defect varied depending on which specific residue replaced Glu259, for each Gsα-Glu259 mutant, GTPγS was the most effective activator and AlF4− the least effective activator. We next examined the ability of AlF4− or GTPγS to protect each mutant from trypsin digestion, which measures the ability of each agent to bind to Gsα and induce the active conformation (29Miller R.T. Masters S.B. Sullivan K.A. Beiderman B. Bourne H.R. Nature. 1988; 334: 712-715Crossref PubMed Scopus (123) Google Scholar). In the inactive, GDP-bound state, two arginine residues within switch 2 (most likely Arg228 and Arg231, based upon sequence homology with transducin) are sensitive to trypsin digestion, leading to the generation of low molecular weight fragments. When Gsα attains the active conformation, these residues are inaccessible to trypsin digestion (7Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Nature. 1994; 369: 621-628Crossref PubMed Scopus (530) Google Scholar) and therefore trypsinization of activated Gsα generates a partially protected 38-kDa product. Gsα-WT was well protected by AlF4− or GTPγS at temperatures up to 37 °C (Fig. 1, TableII). At 30 °C, Gsα-E259V, -E259A, and -E259Q showed little protection by GTPγS and no protection by AlF4−(Fig. 1). In contrast, both GTPγS and AlF4− were able to protect Gsα-E259D, with GTPγS being a more efficient activator than AlF4− (Fig. 1, Table II). Consistent with the results of the cyc reconstitution assays, AlF4− was less effective than GTPγS in protecting all Gsα-E259 mutants from trypsin digestion.Table IIEffect of temperature and GDP on AlF4−-induced trypsin protectionTemperatureTreatmentWTE259DaThe percentage of protection of Gsα-E259D was significantly less than that of Gsα-WT at all conditions except at 30 °C in the presence of AlF4−(Student's t test).n°C% protection% of wild type25AlF4−56 ± 885 ± 154AlF4− + 2 mm GDP59 ± 660 ± 6430AlF4−59 ± 549 ± 7bp < 0.05 versus GTPγS (Student's t test).8AlF4−+ 2 mm GDP70 ± 1036 ± 7bp < 0.05 versus GTPγS (Student's t test).4100 μm GTPγS60 ± 380 ± 5437AlF4−49 ± 57 ± 2bp < 0.05 versus GTPγS (Student's t test).,cp < 0.05 versusAlF4− + GDP (Student's t test).9AlF4− + 2 mm GDP63 ± 322 ± 2bp < 0.05 versus GTPγS (Student's t test).4100 μm GTPγS62 ± 572 ± 89These data were obtained from experiments of the type presented in Fig.1. The amount of the 38-kDa trypsin-stable Gsα fragment was determined by phosphorimaging, and for Gsα-WT, it is expressed as a percent of undigested Gsα (mean ± S.E.). No protection was observed when AlF4− and GTPγS were excluded. Maximum trypsin protection has a theoretical limit of 71%, based on the removal of 2 of 7 total methionine residues by trypsin. For Gsα-E259D, the data are expressed as percentage of wild type at each condition (mean ± S.E.). The number of experiments performed for each condition is shown in the right column.a The percentage of protection of Gsα-E259D was significantly less than that of Gsα-WT at all conditions except at 30 °C in the presence of AlF4−(Student's t test).b p < 0.05 versus GTPγS (Student's t test).c p < 0.05 versusAlF4− + GDP (Student's t test). Open table in a new tab These data were obtained from experiments of the type presented in Fig.1. The amount of the 38-kDa trypsin-stable Gsα fragment was determined by phosphorimaging, and for Gsα-WT, it is expressed as a percent of undigested Gsα (mean ± S.E.). No protection was observed when AlF4− and GTPγS were excluded. Maximum trypsin protection has a theoretical limit of 71%, based on the removal of 2 of 7 total methionine residues by trypsin. For Gsα-E259D, the data are expressed as percentage of wild type at each condition (mean ± S.E.). The number of experiments performed for each condition is shown in the right column. Because the Gsα-E259D encoded the most subtle structural change and had the smallest activation defect, we studied the ability of this mutant to be protected by GTPγS and AlF4− at various temperatures and in the presence or absence of excess GDP (Table II). For Gsα-R258 mutants, the activation defect in the presence of AlF4− was more severe at higher temperatures and was reversible in the presence of excess GDP (15Warner D.R. Weng G. Yu S. Matalon R. Weinstein L.S. J Biol. Chem. 1998; 273: 23976-23983Abstract Full Text Full" @default.
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• W2083996981 title "Mutagenesis of the Conserved Residue Glu259 of Gsα Demonstrates the Importance of Interactions between Switches 2 and 3 for Activation" @default.
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