RGS12 and RGS14 GoLoco Motifs Are GαiInteraction Sites with Guanine Nucleotide Dissociation Inhibitor Activity
2001; Elsevier BV; Volume: 276; Issue: 31 Linguagem: Inglês
10.1074/jbc.m103208200
ISSN1083-351X
AutoresRandall J. Kimple, Luc De Vries, Hélène Tronchère, Cynthia I. Behe, Rebecca A. Morris, Marilyn G. Farquhar, David P. Siderovski,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein α subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single Gαi/o-Loco (GoLoco) motif predicted to represent a second Gα interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with Gαi1, Gαi2, and Gαi3 in their GDP-bound forms. In GTPγS binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by Gαi1. Both regions also stabilize Gαi1 in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF 4−. Our results indicate that both RGS12 and RGS14 harbor two distinctly different Gα interaction sites: a previously recognized N-terminal RGS box possessing Gαi/o GAP activity and a C-terminal GoLoco region exhibiting Gαi GDI activity. The presence of two, independent Gα interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple Gα GAP activity. The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein α subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single Gαi/o-Loco (GoLoco) motif predicted to represent a second Gα interaction site. Here, we describe functional characterization of the GoLoco motif regions of RGS12 and RGS14. Both regions interact exclusively with Gαi1, Gαi2, and Gαi3 in their GDP-bound forms. In GTPγS binding assays, both regions exhibit guanine nucleotide dissociation inhibitor (GDI) activity, inhibiting the rate of exchange of GDP for GTP by Gαi1. Both regions also stabilize Gαi1 in its GDP-bound form, inhibiting the increase in intrinsic tryptophan fluorescence stimulated by AlF 4−. Our results indicate that both RGS12 and RGS14 harbor two distinctly different Gα interaction sites: a previously recognized N-terminal RGS box possessing Gαi/o GAP activity and a C-terminal GoLoco region exhibiting Gαi GDI activity. The presence of two, independent Gα interaction sites suggests that RGS12 and RGS14 participate in a complex coordination of G-protein signaling beyond simple Gα GAP activity. G-protein-coupled receptor AGS3 GoLoco motif consensus peptide GTPase-activating protein guanine nucleotide dissociation inhibitor guanine nucleotide exchange factor Gαi/o-Loco interaction guanosine triphosphatase RGS12 GoLoco motif peptide RGS12 GoLoco motif scrambled peptide RGS14 GoLoco motif peptide Ras-binding domain regulator of G-protein signaling guanosine 5′-O-(3-thiotriphosphate) polymerase chain reaction amino acid(s) surface plasmon resonance glutathione S-transferase response units In the standard model of heterotrimeric G-protein signaling, cell surface receptors (GPCRs)1are coupled to a membrane-associated heterotrimer composed of Gα, Gβ, and Gγ subunits (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4711) Google Scholar, 2Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (938) Google Scholar). Gβ and Gγ form an obligate heterodimer that binds tightly to GDP-bound Gα subunits, enhancing Gα coupling to receptor and inhibiting its release of GDP (i.e. Gβγ dimers exhibit "guanine nucleotide dissociation inhibitor" (GDI) activity; Refs. 3Brandt D.R. Ross E.M. J. Biol. Chem. 1985; 260: 266-272Abstract Full Text PDF PubMed Google Scholar, 4Higashijima 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, 5Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (701) Google Scholar). Upon agonist binding, the GPCR becomes a guanine nucleotide exchange factor (GEF) and promotes replacement of bound GDP for GTP on the Gα subunit. The binding of GTP changes the conformation of three "switch" regions within Gα, allowing Gβγ dissociation. GTP-bound Gα and free Gβγ subunits both initiate signals by interactions with downstream effector proteins until the intrinsic guanosine triphosphatase (GTPase) activity of Gα returns the protein to the GDP-bound state. Reassociation of Gβγ with GDP-bound Gα obscures critical effector contact sites and terminates all effector interactions (6Ford C.E. Skiba N.P. Bae H. Daaka Y. Reuveny E. Shekter L.R. Rosal R. Weng G. Yang C.S. Iyengar R. Miller R.J. Jan L.Y. Lefkowitz R.J. Hamm H.E. Science. 1998; 280: 1271-1274Crossref PubMed Scopus (373) Google Scholar, 7Li Y. Sternweis P.M. Charnecki S. Smith T.F. Gilman A.G. Neer E.J. Kozasa T. J. Biol. Chem. 1998; 273: 16265-16272Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Hence, the duration of heterotrimeric G-protein signaling is controlled by the guanine nucleotide state of the Gα subunit. We and others have identified a family of GTPase-activating proteins (GAPs) for Gα subunits, the "regulators of G-protein signaling" or RGS proteins (8De Vries L. Mousli M. Wurmser A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11916-11920Crossref PubMed Scopus (266) Google Scholar, 9Siderovski D.P. Hessel A. Chung S. Mak T.W. Tyers M. Curr. Biol. 1996; 6: 211-212Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 10Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar, 11Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). These proteins all contain a hallmark "RGS box," which accelerates the intrinsic GTPase rate of Gα subunits by binding avidly to the transition state for GTP hydrolysis (12Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar). Discovery of RGS box-mediated GAP activity finally resolved the paradox that GPCR-stimulated signals terminate much faster in vivothan predicted from the slow GTP hydrolysis rates exhibited by purified Gα subunits in vitro (13Arshavsky V.Y. Pugh Jr., E.N. Neuron. 1998; 20: 11-14Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). However, RGS proteins are clearly more than just Gα GAPs (14Siderovski D.P. Strockbine B. Behe C.I. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 215-251Crossref PubMed Scopus (97) Google Scholar, 15De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (506) Google Scholar). For example, additional functional domains outside the RGS box have been identified that extend the roles of specific RGS proteins into assembly of novel Gβγ heterodimers (16Snow B.E. Krumins A.M. Brothers G.M. Lee S.-F. Wall M.A. Chung S. Mangion J. Arya S. Gilman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (229) Google Scholar, 17Sondek J. Siderovski D.P. Biochem. Pharmacol. 2001; 61: 1329-1337Crossref PubMed Scopus (110) Google Scholar), cross-talk between heterotrimeric and Ras superfamily G-proteins (18Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (674) Google Scholar, 19Traver S. Bidot C. Spassky N. Baltauss T. De Tand M.F. Thomas J.L. Zalc B. Janoueix-Lerosey I. Gunzburg J.D. Biochem. J. 2000; 350: 19-29Crossref PubMed Scopus (79) Google Scholar), and coordination between heterotrimeric G-protein and tyrosine-kinase signaling pathways (20Schiff M.L. Siderovski D.P. Jordan J.D. Brothers G. Snow B. De Vries L. Ortiz D.F. Diverse-Pierluissi M. Nature. 2000; 408: 723-727Crossref PubMed Scopus (125) Google Scholar). In 1997, we identified two RGS box-containing proteins, RGS12 and RGS14 (21Snow B.E. Antonio L. Suggs S. Gutstein H.B. Siderovski D.P. Biochem. Biophys. Res. Commun. 1997; 233: 770-777Crossref PubMed Scopus (101) Google Scholar). Recent, independent analyses of the primary amino acid sequences of RGS12 and RGS14 have led us (22Siderovski D.P. Diversé-Pierluissi M.A. De Vries L. Trends Biochem. Sci. 1999; 24: 340-341Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and Ponting (23Ponting C.P. J. Mol. Med. 1999; 77: 695-698Crossref PubMed Scopus (53) Google Scholar) to predict the existence of a novel Gα-subunit interaction module within both RGS and non-RGS proteins, the GoLoco motif. Lanier and colleagues (24Takesono A. Cismowski M.J. Ribas C. Bernard M. Chung P. Hazard III, S. Duzic E. Lanier S.M. J. Biol. Chem. 1999; 274: 33202-33205Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) independently identified this polypeptide sequence as the "G-protein-regulatory" or GPR motif within AGS3, a protein first isolated in a yeast-based screen for receptor-independent "activators of G-proteinsignaling." We have speculated that the GoLoco/GPR motif may possess receptor-independent GEF activity (22Siderovski D.P. Diversé-Pierluissi M.A. De Vries L. Trends Biochem. Sci. 1999; 24: 340-341Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), based on the report of Luo and Denker (25Luo Y. Denker B.M. J. Biol. Chem. 1999; 274: 10685-10688Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) demonstrating in vitro guanine nucleotide exchange activity by the GoLoco motif-containing Purkinje-cell protein 2 (Pcp2). We and others have since shown that the four GoLoco motifs of AGS3 possess GDI activity on Gαisubunits (26De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (133) Google Scholar, 27Natochin M. Lester B. Peterson Y.K. Bernard M.L. Lanier S.M. Artemyev N.O. J. Biol. Chem. 2000; 275: 40981-40985Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In this report, we describe results from yeast two-hybrid and biophysical analyses designed to address whether the single GoLoco motifs within RGS12 and RGS14 are capable of interacting with Gα subunits and affecting their guanine nucleotide cycle. BODIPY FL-GTPγS was purchased from Molecular Probes, Inc. (Eugene, OR). Peptides corresponding to the GoLoco region of rat RGS14 ("R14GL";496DIEGLVELLNRVQSSGAHDQRGLLRKEDLVLPEFLQ531), a scrambled version of the minimal rat RGS12 GoLoco motif ("R12Scr"; AQLRFISAEAREDNGSFKDEQ), and the consensus sequence (28Peterson Y.K. Bernard M.L. Ma H. Hazard III, S. Graber S.G. Lanier S.M. J. Biol. Chem. 2000; 275: 33193-33196Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) from the four GoLoco motifs of AGS3 ("AGS3Con"; TMGEEDFFDLLAKSQSKRMDDQRVDLAG) were synthesized using conventional Fmoc (N-(9-fluorenyl)methoxycarbonyl) blocking group chemistry by the University of North Carolina Peptide Chemistry Group (Chapel Hill, NC). A peptide corresponding to the GoLoco region of rat RGS12 (1186EAEEFFELISKAQSNRADDQRGLLRKEDLVLP- EFLR1221) was purchased from New England Peptide Inc. (Fitchburg, MA). All peptides were synthesized with free amine N termini and amide-blocked C termini; peptide purity was confirmed by mass spectrometry and amino acid analyses. A panel of Gα subunit baits constructed as Gal4p-DNA binding domain fusions in the vector pGBT9 has previously been described (26De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (133) Google Scholar). A cDNA fragment encoding amino acids 1093–1259 of rat RGS12 was amplified from the full-length ratRgs12 cDNA (21Snow B.E. Antonio L. Suggs S. Gutstein H.B. Siderovski D.P. Biochem. Biophys. Res. Commun. 1997; 233: 770-777Crossref PubMed Scopus (101) Google Scholar) by PCR (sense primer, 5′-CGAATTCTAAGTCTGGATGGACAGCGGGTC-3′; antisense primer, 5′-TCTCGAGTTAGCTCTCCTCTGTCTGAACTGCTC-3′), trapped in the pCR2.1-Topo vector (Invitrogen), sequence-verified, and subcloned in frame downstream of the Gal4p activation domain using theEcoRI and XhoI sites of pACT2. In a similar fashion, a cDNA fragment encoding amino acids 496–544 of rat RGS14 was PCR-amplified from the full-length rat Rgs14 cDNA (sense primer, 5′-CGAATTCCTGACATTGAAGGCCTAGTGGAG-3′; antisense primer, 5′-GGTCGACGGGAGGGGCAAACAACAG-3′) and subcloned into the same sites of pACT2. Bait and prey plasmid pairs were cotransformed into yeast strain SFY526 (CLONTECH), and interactions were analyzed by a qualitative colony lift assay for β-galactosidase expression using 5-bromo-4-chloro-3-indolyl β-d-galactoside (29Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (873) Google Scholar). Myristoylated, recombinant rat Gαi3 protein was purchased from Calbiochem. His6-tagged mouse Gαo1, expressed and purified from a pET15b-based Escherichia coli expression vector, was provided as a kind gift from Drs. Laurie Betts and John Sondek (University of North Carolina). The open reading frame of human Gαi1 was amplified by PCR (sense primer, 5′-ACCATGGGCTGCACGCTGAGCGCCGAGGAC-3′; antisense primer, 5′-AGCGGCCGCACTGCAAAACTTAAAAGAGAC-3′) from Marathon™ human brain cDNA (CLONTECH), digested withNcoI and NotI, subcloned into theNcoI/NotI sites of pProEX-HTb (Life Technologies, Inc.), sequence-verified, and transformed into E. colistrain BL21(DE3). Expression of His6-Gαi1protein was induced with 1 mmisopropyl-β-d-thiogalactopyranoside for 4 h at 37 °C in 1-liter bacterial cultures at anA 600 of 0.9. Bacterial pellets were frozen at −80 °C, thawed on ice, and resuspended in lysis buffer (100 mm NaCl, 10 mm imidazole, 10 mm Na2HPO4, 10 mmNaH2PO4, pH 7.5). Cell suspensions were lysed using an AMINCO French press (SLM Instruments Inc., Urbana, IL) and clarified by centrifugation at 100,000 × g for 25 min. Supernatant was then loaded onto a nickel-nitrilotriacetic acid resin FPLC column (HisTrap; Amersham Pharmacia Biotech), and His6-Gαi1 protein was eluted using a gradient of 10 mm to 1 m imidazole in lysis buffer. Eluted protein was cleaved with tobacco etch virus protease (Life Technologies) overnight at 4 °C to remove the His6 tag, diluted into low salt buffer (25 mm NaCl, 20 mmTris-HCl, pH 8.0), and loaded onto a 6-ml Source 15Q column (Amersham Pharmacia Biotech). Gαi1 protein was eluted with an 80-ml gradient of 25–400 mm NaCl, and peak fractions were pooled and resolved using a calibrated 150-ml size exclusion column (Sephacryl S200; Amersham Pharmacia Biotech). Protein was buffer-exchanged into storage buffer (200 mm NaCl, 1 mmMgCl2, 10 µm GDP, 1 mmdithiothreitol, 20 mm Tris, pH 7.5, 5% glycerol) and concentrated in a Centriprep Centrifugal Filter Device, YM-30 (Millipore Corp.). The concentrations of all proteins purified in this study were determined by A 280 measurements upon denaturation in guanidine hydrochloride, and calculation of concentration was based on predicted extinction coefficients. To create the expression plasmid pGEX4T1-rRGS14H6, the full-length open reading frame of rat Rgs14 cDNA (aa 1–544; Ref. 21Snow B.E. Antonio L. Suggs S. Gutstein H.B. Siderovski D.P. Biochem. Biophys. Res. Commun. 1997; 233: 770-777Crossref PubMed Scopus (101) Google Scholar) was amplified by PCR with primers designed to add a 5′-end EcoRV site and a 3′-end His6 tag/stop codon/NotI site (sense primer, 5′-CGATATCGATGCCAGGGAAGCCCAAGCAC-3′; antisense primer, 5′-TGCGGCCGCTAGTGATGATGGTGGTGATGTGGTGGAGCCCTCCTGAGA-3′), subcloned into the SmaI and NotI sites of pGEX4T1 (Amersham Pharmacia Biotech). pGEX4T1-rRGS14ΔGoLoco, encoding a C-terminally truncated open reading frame (aa 1–450) lacking the GoLoco region, was created by introducing a stop codon at codon 451 within pGEX4T1-rRGS14H6 using the QuikChange site-directed mutagenesis system (Stratagene). pGEX4T2-rRGS14496–531, encoding amino acids 496–531 of rat RGS14 spanning the GoLoco motif, was amplified by PCR (sense primer, 5′-CGAATTCCTGACATTGAAGGCCTAGTGGAG-3′; antisense primer, 5′-GGTCGACTACTGCAGAAATTCTGGAAGGAC-3′) and subcloned into the EcoRI and SalI sites of pGEX4T2. pGEX4T3-rRGS14ΔRGS, encoding amino acids 263–544 of rat RGS14, was PCR-amplified (sense primer, 5′-CGAATTCTTCCGCGAGTCTGGACCTG-3′; antisense primer, 5′-GGTCGACTATGGTGGAGCCTCCTGAGAACCTG-3′) and subcloned into the EcoRI and XhoI sites of pGEX4T3. pGEX4T2-rRGS121093–1259 was created by cloning theEcoRI/XhoI fragment (encoding amino acids 1093–1259) of pACT2-rRGS12GoLoco into the EcoRI andSalI sites of pGEX4T2. pGEX4T2-rRGS121093–1228and pGEX4T2-rRGS121184–1228 plasmids were created based on pGEX4T2-rRGS121093–1259 by sequential rounds of site-directed mutagenesis to introduce a stop codon at codon 1229 and delete codons 1093–1183 (QuikChange system; Stratagene). All expression plasmids were sequence-verified prior to transformation intoE. coli strain BL21(DE3). Bacteria were grown to anA 600 of 0.6–0.8 at 37 °C before induction with 1 mmisopropyl-β-d-thiogalactopyranoside. After an additional 4 h at 37 °C, cell pellets were lysed, and GST-fusion protein was purified by glutathione-Sepharose chromatography as previously described (30Snow B.E. Hall R.A. Krumins A.M. Brothers G.M. Bouchard D. Brothers C.A. Chung S. Mangion J. Gilman A.G. Lefkowitz R.J. Siderovski D.P. J. Biol. Chem. 1998; 273: 17749-17755Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). GST-RGS14496–531 and GST-RGS121184–1228 proteins were further purified by size exclusion chromatography over Sephacryl S200 resin prior to use in biosensor kinetics and GTPγS binding assays. SPR binding assays were performed at 25 °C on a BIAcore 2000 (BIAcore, Piscataway, NJ) at the University of North Carolina Macromolecular Interactions Facility. Carboxymethylated dextran (CM5) sensor chips with covalently bound anti-GST antibody surfaces were created as previously described (26De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (133) Google Scholar). Recombinant GST or GST fusion proteins were bound to separate flow cells of anti-GST surfaces to a density of ∼1000 response units (RU), except for the kinetic analyses in which GST-RGS14496–531 and GST-RGS121184–1228 were bound to a density of 50 RU. To test the GoLoco interaction with myristoylated Gαi3, GST, GST-RGS14ΔRGS, and GST-RGS121093–1259 proteins were directly coupled to the carboxymethylated dextran biosensor surfaces (to levels of 5700, 7100, and 6200 RU, respectively) using N-hydroxysuccinimide andN-ethyl-N′-(dimethylaminopropyl)carbodiimide according to the manufacturer's instructions (BIAcore). As recommended by Lenzen and colleagues (31Lenzen C. Cool R.H. Prinz H. Kuhlmann J. Wittinghofer A. Biochemistry. 1998; 37: 7420-7430Crossref PubMed Scopus (197) Google Scholar), binding analyses were performed using Buffer W (150 mm NaCl, 5 mmMgCl2, 0.005% Nonidet P-40, 20 mm HEPES, pH 7.4) as the running buffer to stabilize the anti-GST antibody surface. Recombinant Gα subunits were initially diluted to 1 µmin Buffer W containing either 32 µm GDP, 32 µm GDP plus 32 µm AlCl3 and 10 mm NaF, or 32 µm GTPγS, incubated for 90 min at 30 °C (or overnight at room temperature for GTPγS loading of Gαi1 and myristoylated Gαi3), and further diluted in the same Buffer W plus nucleotide combination prior to injection. 25 µl of Gα protein aliquots were injected at a flow rate of 5 µl/min over four flow cell surfaces simultaneously using the KINJECT command; for kinetic analyses, injections using the COINJECT command were employed to add a 1.5-fold molar excess of R14GL peptide to the running buffer during dissociation phases. Surface regeneration was performed by serial injections of 5 µl of 10 mm glycine, pH 2.2, and 5 µl of 0.05% SDS at a 20 µl/min flow rate. Background binding to a GST-coated surface (as acquired simultaneously with GST-GoLoco surface binding curves) was subtracted from all binding curves using BIAevaluation software version 3.0 (BIAcore) and plotted using GraphPad Prism version 3.0 (GraphPad Software Inc., San Diego, CA). Measurements of BODIPY fluorescence were performed with a PerkinElmer Life Sciences LS50B spectrometer with excitation at 485 nm and emission at 530 nm (slit widths each at 2.5 nm). BODIPY FL-GTPγS was diluted to 1 µm in 10 mm Tris, pH 8.0, 1 mmEDTA, and 10 mm MgCl2 and equilibrated to 30 °C in 2-ml cuvettes. 100 nm Gαo or Gαi1 protein was preincubated with 200 nm GST fusion protein or 400 nm GoLoco peptide at 25 °C for 10 min before the addition to the cuvette. Relative fluorescence levels were set to zero at the average fluorescence reading over the first 70 s, and Gα/GoLoco mixtures were added at the 100-s mark. Measurements of intrinsic tryptophan fluorescence were performed on the LS50B spectrometer with excitation at 292 nm and emission at 342 nm (slit widths 2.5 and 5.0 nm, respectively). Recombinant Gαo and Gαi1proteins were diluted in 2-ml cuvettes to 200 nm in preactivation buffer (100 mm NaCl, 100 µmEDTA, 2 mm MgCl2, 20 µm GDP, 20 mm Tris-HCl, pH 8.0) and incubated at 30 °C. To activate Gα, 2 mm NaF and 30 µm AlCl3(final concentrations) were added after 400 and 500 s, respectively. To determine the effect of GoLoco-derived peptides on AlF 4−-induced Gα activation, a complex of 400 nm GoLoco peptide and 200 nmGα-GDP was preformed in the same buffer and then activated with NaF and AlCl3 additions as described above. Unlike our previous use of the tryptophan-containing GST-AGS3424–650 fusion protein (26De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (133) Google Scholar), the Gα fluorescence measurements in this present study were unaffected by peptide fluorescence, since none of the GoLoco peptides tested contains tryptophan residues. The yeast two-hybrid system was used to assess whether the GoLoco motifs of rat RGS12 (aa 1188–1220) and rat RGS14 (aa 498–530) are capable of binding Gα subunits. Yeast two-hybrid "prey" were constructed by fusing the Gal4p-activation domain with either a 167-amino acid span of rat RGS12 (aa 1093–1259) or a 49-amino acid span of rat RGS14 (aa 496–544); both regions are C-terminal to the tandem Ras-binding domains (19Traver S. Bidot C. Spassky N. Baltauss T. De Tand M.F. Thomas J.L. Zalc B. Janoueix-Lerosey I. Gunzburg J.D. Biochem. J. 2000; 350: 19-29Crossref PubMed Scopus (79) Google Scholar, 23Ponting C.P. J. Mol. Med. 1999; 77: 695-698Crossref PubMed Scopus (53) Google Scholar) and centered about the GoLoco motif (Fig. 1). The yeast reporter strain SFY526 was transformed with pairs of GoLoco-region prey and Gα protein "baits" and interactions identified by a qualitative, chromogenic β-galactosidase filter lift assay as previously described (26De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (133) Google Scholar). Of the Gα protein baits tested from all four subfamilies (αs, αi, αq, and α12; Ref. 32Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1585) Google Scholar), interaction was only detected between GoLoco-region prey and Gαi1, Gαi2, and Gαi3. In contrast to our previous yeast two-hybrid results with AGS3 (26De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (133) Google Scholar), no interaction was detected between Gαo and the GoLoco regions of either RGS12 or RGS14 in this assay (Table I).Table IYeast two-hybrid analysis of Gα interactions with RGS12 and RGS14 GoLoco motif regionsBait1-aGα baits constructed as Gal4p-DNA binding domain fusions in vector pGBT9 as described in Ref.26.RGS12-GoLoco prey1-bGoLoco region prey constructed as Gal4p-activation domain fusions in vector pACT2. (aa 1093–1259)RGS14-GoLoco prey1-bGoLoco region prey constructed as Gal4p-activation domain fusions in vector pACT2. (aa 496–544)Gαi1+++++Gαi2++Gαi3++++++Gαo−−Gαz−−Gαq−−Gαs−−Gα12−−Gα13ND−The β-galactosidase filter-lift assay was performed on Leu- and Trp-deficient media plates, and color intensity was scored after 8 h. −, no color; +, moderate color; ++, strong color; +++, very strong color; ND, not done. Yeast cotransfected with empty bait and prey vectors were assayed for background color development, and none was detected after 20 h of incubation.1-a Gα baits constructed as Gal4p-DNA binding domain fusions in vector pGBT9 as described in Ref.26De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14364-14369Crossref PubMed Scopus (133) Google Scholar.1-b GoLoco region prey constructed as Gal4p-activation domain fusions in vector pACT2. Open table in a new tab The β-galactosidase filter-lift assay was performed on Leu- and Trp-deficient media plates, and color intensity was scored after 8 h. −, no color; +, moderate color; ++, strong color; +++, very strong color; ND, not done. Yeast cotransfected with empty bait and prey vectors were assayed for background color development, and none was detected after 20 h of incubation. Although wild-type (and thus presumably GDP-bound) Gα subunits were used in the yeast two-hybrid analysis, one possibility is that the observed interactions occurred between GoLoco region prey and a fraction of GTP-bound Gαi bait. Therefore, to assess directly the dependence of GoLoco/Gα interactions on bound nucleotide and also to confirm the observed Gαi binding selectivity, real time binding assays were performed using the SPR technique. RGS12 and RGS14 polypeptides were purified as GST fusion proteins and bound to anti-GST antibody-coated biosensor surfaces at saturating levels. Recombinant Gαo and Gαi1 proteins were injected for 300 s over these biosensor surfaces, having first been incubated with one of the following guanine nucleotides: GTPγS (a nonhydrolyzable GTP analog) to mimic the activated GTP-bound form, GDP with AlF 4− to mimic the transition state of GTP hydrolysis, or GDP alone to preserve the Gα subunit in the GDP-bound, inactive state. Both Gαo and Gαi1, when preloaded with GDP and AlF 4−, bound avidly to full-length RGS14 protein (GST-RGS14; Fig. 2A,left panel), as predicted based on the preference of the RGS14 RGS box to exhibit GAP activity toward Gαi/osubunits (19Traver S. Bidot C. Spassky N. Baltauss T. De Tand M.F. Thomas J.L. Zalc B. Janoueix-Lerosey I. Gunzburg J.D. Biochem. J. 2000; 350: 19-29Crossref PubMed Scopus (79) Google Scholar, 33Cho H. Kozasa T. Takekoshi K. De Gunzburg J. Kehrl J.H. Mol. Pharmacol. 2000; 58: 569-576Crossref PubMed Scopus (78) Google Scholar). In addition, avid binding of full-length RGS14 to GDP-bound Gαi1, but not GDP-bound Gαo, was seen. Neither Gα subunit bound appreciably to full-length RGS14 when in the activated, GTPγS-bound form (data not shown). Deletion of the C-terminal 94 amino acids of RGS14 (aa 451–544), including the GoLoco motif, eliminated binding by GDP-bound Gαi1 but did not abrogate binding to Gαo-GDP/AlF 4− and Gαi1-GDP/AlF 4− subunits (GST-RGS14ΔGoLoco; Fig. 2 A, middle panel). Conversely, a GST fusion biosensor surface composed solely of the GoLoco motif region of RGS14 (aa 496–531) only interacted with GDP-bound Gαi1 (GST-RGS14496–531; Fig.2 A, right panel). Avid binding of GDP-bound Gαi1 was also observed with a GST fusion protein containing a 136-amino acid span of RGS12 (aa 1093–1228) that includes the GoLoco motif (Fig. 2 B,left panel). Deletion of amino acids 1093–1183, residues that are N-terminal to the GoLoco motif, did not inhibit Gαi1-GDP binding (GST-RGS121184–1228; Fig.2 B, right panel). In contrast with results using RGS14, much reduced but still significant binding was also observed between the AlF 4− form of Gαi1 and both forms of the RGS12 GoLoco region. To confirm this difference between RGS12 and RGS14 GoLoco regions, recombinant myristoylated Gαi3 was injected over highly saturated, directly conjugated surfaces of GST-RGS121093–1259 or GST-RGS14ΔRGS proteins. While appreciable binding to GST-RGS14ΔRGS was only seen upon injection of GDP-bound myristoylated Gαi3, and not the GDP/AlF 4−- or GTPγS-bound forms (Fig.2 C, left panel), all three forms of myristoylated Gαi3 bound to some detectable degree to the GST-RGS121093–1259 surface. The rank order of binding avidity to GST-RGS121093–1259 was clearly Gαi3-GDP ≫ Gαi3-GDP/AlF 4− ≫ Gαi3-GTPγS (Fig. 2 C, right panel). Table II summarizes quantitative kinetic measurements of Gαi1-GDP binding to the minimal GoLoco regions of RGS12 and RGS14. Separate, low density (50 RU) biosensor surfaces of either GST-RGS14496–531 or GST-RGS121184–1228 protein were prepared, and various concentrations of GDP-bound Gαi1 (25–4000 nm) were injected over each surface for 300 s. To eliminate biosensor rebinding events during the dissociation of bound Gα (i.e. during the 300–600-s time interval), a synthetic peptide encompassing the RGS14 GoLoco region ("R14GL"; see below) was injected immediately after the Gα association phase; R14GL peptide was injected at a concentration 1.5-fold greater than the preceding Gαi1 injection (e.g. 6 µm R14GL injection after 4 µm Gai1-GDP injection). The resultant association and dissociation curves were fit to a Langmuir 1:1 interaction model using
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