RGS17/RGSZ2, a Novel Regulator of Gi/o, Gz, and Gq Signaling
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m401800200
ISSN1083-351X
AutoresHelen Mao, Qingshi Zhao, Mireille Daigle, Mohammad H. Ghahremani, Peter Chidiac, Paul R. Albert,
Tópico(s)Receptor Mechanisms and Signaling
ResumoTo identify novel regulators of Gαo, the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Gαo as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Gα-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Gαi1–3, Gαo, Gαz, or Gαq but not Gαs were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Gαi2 and Gαo under pre-steady-state conditions, and on M2-muscarinic receptor-activated Gαi1, Gαi2, Gαi3, Gαz, and Gαo in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for Gz, RGS17 exhibited limited selectivity for Go among Gi/Go proteins. All RZ family members reduced dopamine-D2/Gαi-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Gαq-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via Gi/o, Gz, and Gq over Gs to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors. To identify novel regulators of Gαo, the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Gαo as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Gα-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Gαi1–3, Gαo, Gαz, or Gαq but not Gαs were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Gαi2 and Gαo under pre-steady-state conditions, and on M2-muscarinic receptor-activated Gαi1, Gαi2, Gαi3, Gαz, and Gαo in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for Gz, RGS17 exhibited limited selectivity for Go among Gi/Go proteins. All RZ family members reduced dopamine-D2/Gαi-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Gαq-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via Gi/o, Gz, and Gq over Gs to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors. Regulators of G-protein signaling (RGS) 1The abbreviations used are: RGS, regulator of G-protein signaling; GAP, GTPase activating protein; PMSF, phenylmethylsulfonyl fluoride; TRHR1, rat thyrotropin-releasing hormone receptor; RACE, rapid amplification of cDNA ends; β-ME, β-mercaptoethanol; Ni-NTA, nickel-nitrilotriacetic acid; PTX, pertussis toxin; GST, glutathione S-transferase; GS, Gly → Ser mutation; QL, Gln → Leu mutation; RC, Arg → Cys mutation; TRH, thyrotropin-releasing hormone. proteins accelerate the intrinsic GTPase activity of heterotrimeric G-protein Gα subunits. All RGS proteins contain a conserved RGS core domain, which is an interaction site for the Gα subunits (1Burchett S.A. J. Neurochem. 2000; 75: 1335-1351Crossref PubMed Scopus (98) Google Scholar, 2De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (508) Google Scholar, 3Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (929) Google Scholar). There are more than 30 human RGS or RGS-like proteins that are classified into six subfamilies based upon their sequence homology and that have conserved functional and targeting domains outside of the RGS domain. For instance, a membrane-targeting domain immediately proximal to the RGS core domain directs small RGS proteins such as RGS1–5 and -16 to the cell membrane (4Heximer S. Lim H. Bernard J. Blumer K. J. Biol. Chem. 2001; 276: 14195-14203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Putative nuclear localization signals have been found within (5Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2000; 275: 24013-24021Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) and also outside of the RGS core domain (6Dulin N.O. Pratt P. Tiruppathi C. Niu J. Voyno-Yasenetskaya T. Dunn M.J. J. Biol. Chem. 2000; 275: 21317-21323Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and this may direct certain RGS subtypes to the nucleus (7Zmijewski J.W. Song L. Harkins L. Cobbs C.S. Jope R.S. Biochim. Biophys. Acta. 2001; 1541: 201-211Crossref PubMed Scopus (25) Google Scholar, 8Burchett S.A. J. Neurochem. 2003; 87: 551-559Crossref PubMed Scopus (44) Google Scholar). RZ family members, such as RGSZ1 and RGS-Gα-interacting protein (GAIP), contain a cysteine-rich motif that may serve as a palmitoylation site for membrane association (9Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 10De Vries L. Elenko E. Hubler L. Jones T. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (156) Google Scholar). A more recent study showed that RZ family members also serve as adapter proteins for Gα subunit degradation (11Fischer T. De Vries L. Meerloo T. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8270-8275Crossref PubMed Scopus (65) Google Scholar). The cysteine-rich motif interacts with the leucine-rich region of GAIP-interacting protein N terminus, an E3 ubiquitin ligase responsible for Gαi3 degradation. Recently RGSZ1 and RGS6 have been shown to associate with SCG-10, a protein involved in neuronal differentiation (12Nixon A.B. Grenningloh G. Casey P.J. J. Biol. Chem. 2002; 277: 18127-18133Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 13Liu Z. Chatterjee T.K. Fisher R.A. J. Biol. Chem. 2002; 24: 30261-30271Google Scholar), and the Gz GTPase-activating protein (GAP) effects of RGSZ1 (and other RGS proteins) were found to be negatively regulated by synapsin-1a (14Tu Y. Nayak S.K. Woodson J. Ross E.M. J. Biol. Chem. 2003; 278: 52273-52281Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Thus, RZ proteins may play diverse and important roles in the regulation of signaling and cytoskeletal events in the brain. G-protein specificity of RGS proteins has been investigated in various studies. Some RGS proteins display a preference for Gα sub-families (15Posner B.A. Mukhopadhyay S. Tesmer J.J. Gilman A.G. Ross E.M. Biochemistry. 1999; 38: 7773-7779Crossref PubMed Scopus (68) Google Scholar), with the majority regulating Gαi/o and a subset of these also acting on Gαq (16Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 17Mukhopadhyay S. Ross E.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9539-9544Crossref PubMed Scopus (154) Google Scholar, 18Zeng W. Xu X. Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K.A. Fisher S.L. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 25Cladman W. Chidiac P. Mol. Pharmacol. 2002; 62: 654-659Crossref PubMed Scopus (42) Google Scholar). Other RGS members are more selective regulators of specific Gα subunits. For instance, p115RhoGEF has a preference for Gα12/13 subunits (19Kozasa T. Jiang X. Hart M.J. Sternweis P.M. Singer W.D. Gilman A.G. Bollag G. Sternweis P.C. Science. 1998; 280: 2109-2111Crossref PubMed Scopus (740) Google Scholar). RGSZ1 seems to be selective for Gαz (20Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Glick J.L. Meigs T.E. Miron A. Casey P.J. J. Biol. Chem. 1998; 273: 26008-26013Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), although at higher concentrations it has been shown to interact with and negatively regulate Gαi subunits (22Wang Y. Ho G. Zhang J.J. Nieuwenhuijsen B. Edris W. Chanda P.K. Young K.H. J. Biol. Chem. 2002; 277: 48325-48332Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Similarly, a series of R7 family RGS proteins displayed a relative hierarchical preference in the regulation of Gαi proteins rather than an absolute specificity for one subtype (23Hooks S.B. Waldo G.L. Corbitt J. Bodor E.T. Krumins A.M. Harden T.K. J. Biol. Chem. 2003; 278: 10087-10093Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The most abundant G-protein expressed in the brain is Gαo. However, many actions of Gαo are mediated by means of Gβγ subunits, and little is known of the effectors for the Gαo subunit. To identify Gαo-dependent regulators or effectors, we screened human brain cDNA library for interacting proteins using a yeast two-hybrid system with constitutively active GαoR179C as bait. RGS17 was identified among a number of clones. RGS17 contains a cysteine-rich string at its N terminus followed by the highly conserved RGS domain, and is most homologous to RGSZ1 and other RZ family members. We have addressed the G-protein specificity of RGS17 by GAP interaction and functional assays. Our results indicate that RGS17, as well as RGSZ1, negatively regulates the functions of Gαi/o, Gz, and Gαq subunits. Plasmid Constructs—Constitutively active rat GαoR179C (24Pace A.M. Wong Y.H. Bourne H.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7031-7035Crossref PubMed Scopus (136) Google Scholar) cDNA was subcloned into EcoRI-cut pAS2–1 (Clontech) for two-hybrid screening. GST-RGS17, GST-RGSZ1, His-RGS17, and His-GαoR179C for bacterial expression were subcloned into pGEX-4T-1 (Amersham Biosciences) and pET-30a(+) (Novagen) using EcoRI or HindIII/EcoRI sites. All pcDNA3-FLAG and pcDNA3-myc constructs were made using PCR products of RGS17 or Gα subunits subcloned into EcoRI/XhoI-cut vectors. The Gly → Ser (GS) mutants of all Gα subunit cDNAs were constructed using the Stratagene QuikChange site-directed mutagenesis protocol. All constructs were confirmed by DNA sequencing. Human Gαi1-Q204L, Gαi2-Q205L, Gαi3-Q204L, Gαz-Q205L, Gαz-wt, Gαq-Q209L, GαsL-Q227L, Gαcone-Q204L, RGSZ1 and RGS-GAIP fragment in pcDNA3.1+ were purchased from the Guthrie Institute (www.guthrie.org). Rat Gαo-wt, Gαi1-wt, Gαi2-wt, and Gαi3-wt for expression in mammalian cells were constructed in pcDNA3. For the purification of proteins from Escherichia coli for GTPase experiments, constructs for histidine-tagged RGS4, Gαi2, and Gαo were kindly provided by Dr. John Hepler (Emory University). Baculoviruses for Gαi1 and Gαi3 were kindly provided by Dr. Jim Garrison (University of Virginia). Baculoviruses for Gαo and Gαz, respectively, were gifts from Dr. John Hepler (Emory University) and Dr. Pat Casey (Duke University). Other baculoviruses were obtained as noted previously (25Cladman W. Chidiac P. Mol. Pharmacol. 2002; 62: 654-659Crossref PubMed Scopus (42) Google Scholar). Rat thyrotropin-releasing hormone receptor (TRHR1) cDNA was kindly provided by Dr. Armen H. Tashjian, Jr. (Harvard University, Boston, MA). Yeast Two-hybrid Screening—Rat GαoR179C was used as a bait to screen 5 × 106 clones of a human brain cDNA library (HL4004AH, Clontech) in AH109 yeast strain using the LiAc method (27Ito M. Yasui A. Komamine A. FEBS Lett. 1993; 320: 125-129Crossref PubMed Scopus (8) Google Scholar). The yeast cells were grown at 30 °C for 5–7 days. Transformants (1.4 × 105) were selected on S.D.-Leu-Trp-His- plates with 40 mm 3-amino-1,2,4-trizole and screened by X-galactosidase assay. DNA from positive clones was extracted, re-transformed into MH6 cells, and selected on M9-Leu- plates for sequencing. For liquid β-galactosidase assay, pAS2-Gα subunits and pACT2 constructs (positive clones) were transformed into Y187 and AH109 strains and plated onto S.D.-Trp- and S.D.-Leu- plates, respectively. Resultant colonies were mated, selected on S.D.-Leu-Trp-His- plates and subcultured in S.D.-Leu-Trp-His- medium (30 °C, overnight). Yeast cells were lysed by three freeze-thaw cycles and re-suspended in Z buffer (60 mm Na2HPO4, 40mm NaH2PO4, 10mm KCl, 1 mm MgSO4, pH 7.0). For quantitative assay, O-nitrophenyl β-d-galactopyranoside (Sigma) and yeast lysate were incubated at 30 °C; absorbance at 420 nm was measured to determine β-galactosidase activity units (28Schneider S. Buchert M. Hovens C.M. BioTechniques. 1996; 20: 960-962Crossref PubMed Scopus (72) Google Scholar), which was normalized by the β-galactosidase unit of the positive control vector pCL1 in each assay. The X-gal assay was performed by using the mated yeast cells described above. Briefly, the yeast cells grew in S.D.-Leu-Trp- medium at 30 °C overnight and were diluted to A600 = 0.7, from which 10 μl of the yeast cell medium was spotted on S.D.-Leu-Trp- plates. X-gal assay was performed on the plates after the yeast cells grew for 3–4 days at 30 °C. Northern Blot and 5′-RACE—RGS17 cDNA coding sequence was labeled with [32P]dATP using Strip-EZ™ DNA labeling kit (Ambion) and purified. Clontech blots (MTN blots 7780–1, 7755–1, and 7793–1) were pre-incubated with salmon sperm DNA in ULTRAhyb (Ambion) for 2 h at 42 °C and then hybridized with the 32P-labeled probe overnight. The blots were washed three times with 2× SSC plus 1% SDS, followed by 0.2× SSC in 1% SDS. The blots were exposed to x-ray film for 2–4 days. For 5′-RACE, a Marathon-ready human brain cDNA library (BD Biosciences) was amplified with AP1 primer (CCA TCC TAA TAC GAC TCA CTA TAG GGC) and RGS17AS 1S-R primer (GCA TCT TCA TAC ATG TGA GGA TTG GGA) using the Advantage 2 PCR kit (BD Biosciences). To perform the 5′-RACE, we used touchdown PCR with the following cycles: 30 s at 94 °C, 5 cycles at 94 °C (5 s)/72 °C (4 min), 5 cycles at 94 °C (5 s)/70 °C (4 min), and 25 cycles at 94 °C (5 s)/72 °C (4 min). PCR products were diluted in tricine-EDTA buffer (BD Biosciences) for nested PCR using RGS17AS 2S (GAC AAG ACT TCC TCT GCA GTG GGG T) and AP2 primers (ACT CAC TAT AGG GCT CGA GCG GC) with the following cycles: 30 s at 94 °C, 20 cycles 94 °C (5 s)/68 °C (4 min). The products were analyzed by agarose gel electrophoresis, the major band was excised, and DNA was recovered and subcloned into the pGEMT-easy vector for DNA sequencing. Purification of Proteins for GTPase Assays—RGS proteins and G-proteins were grown individually in transformed E. coli BL21/DE3 cells in LB medium (His-RGS17, His-RGS4) or enriched medium (others) (2% tryptone, 1% yeast extract, 0.5% NaCl, 0.2% glycerol, and 50 mm KH2PO4, pH 7.2) containing antibiotics at 37 °C in a shaking incubator at 200 rpm until mid-log phase (optical density at 600 nm of 0.6). A 3-h induction was commenced by adding 30 μm (G-proteins) or 200 μm (RGS proteins) isopropylthio-B-d-galactoside. For purification, frozen bacteria expressing N-terminally histidine-tagged RGS17 were resuspended in lysis buffer (100 mm Tris, pH 8.0, 20 mm EDTA, 10% sucrose (w/v), 0.2 mg/ml lysozyme, and protease inhibitors (10 μg/ml leupeptin, 1 μg/ml aprotinin, 0.1 mm PMSF)), stirred for 30 min in the cold room, and centrifuged at 20,000 × g for 30 min at 4 °C. The expressed protein was predominantly insoluble, and the pelleted material was resuspended in a buffer containing 100 mm Tris (pH 8.0), 10 mm EDTA, and protease inhibitors, passed three times through a French press, and centrifuged as above. The resultant pellet was resuspended and centrifuged again two times in a buffer containing 100 mm Tris (pH 8.0), 2% Triton X-100, 5 mm EDTA, 1 m guanidine hydrochloride, and protease inhibitors, and then resuspended once again in 100 mm Tris (pH 8.0). The resultant pellet was resuspended in an extraction buffer containing 10 mm Tris (pH 8.0), 0.5 mm EDTA, 6 m guanidine HCl, and 0.1 mm PMSF and centrifuged again as above. The supernatant containing the denatured RGS17 protein was diluted with two volumes of a buffer containing 100 mm Tris (pH 7.5), 1 m NaCl, 0.5 mm EDTA, 40 mm β-ME, 10% glycerol, 6 m guanidine HCl, and protease inhibitors and dialyzed twice against a buffer containing 50 mm HEPES (pH 7.5), 500 mm NaCl, 20 mm β-ME, 10% glycerol, 0.5 mm PMSF, and either 2 mm (first dialysis) or 0.5 mm (second dialysis) urea. The dialysate was cleared by centrifugation at 27,000 × g for 60 min at 4 °C, and the resultant supernatant was equilibrated with Ni-NTA affinity resin (Qiagen) for 90 min in the cold room. The resin was packed into a column and then washed with ∼10 volumes of a buffer containing 50 mm HEPES (pH 7.5), 500 mm NaCl, 1% Triton X-100, 20 mm β-ME, 0.5 m urea, protease inhibitors, plus 20 mm imidazole. This first washing was followed by a second wash using the same buffer but minus Triton X-100 and with imidazole increased to 35 mm. The protein was eluted by further increasing the imidazole concentration to 500 mm. The chromatographic removal of imidazole and urea (Superdex 75 column) resulted in precipitation of the purified protein; therefore, RGS17 was stored in the elution buffer and added directly into assays (residual imidazole concentration in assays ranged from 3–6 mm, which was held equal for all conditions within a given experiment). N-terminally histidine-tagged RGS4 was purified as described previously (25Cladman W. Chidiac P. Mol. Pharmacol. 2002; 62: 654-659Crossref PubMed Scopus (42) Google Scholar). GST-RGSZ1 was purified essentially as described previously (20Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and assayed in the uncleaved form, which exhibited GAP activity comparable with that of untagged RGSZ1. 2J. Wang, personal communication. For purification of histidine-tagged Gαi2 and Gαo proteins, transformed E. coli BL21/DE3 cells stored in buffer A (50 mm HEPES, pH 8.0, 100 mm NaCl, 20 mm β-ME, 1% Triton X-100, 5 μm GDP, and protease inhibitors) were thawed, 0.2 mg/ml lysozyme was added, and the cells were incubated on ice for 30 min, followed by the addition of 25 μg/ml DNase I in the presence of 0.5 mm MgCl2 for 20 min. The mixture was centrifuged at 140,000 × g for 30 min at 4 °C, and the volume of the supernatant was increased to 25 ml/liter culture, with buffer A supplemented with glycerol and imidazole (final concentrations of 20% and 0.02 m, respectively). Nickel-NTA (1.5 ml) was added, gently rotated in the cold for 90 min, and then loaded onto a 5-ml column, washed with 30 ml of buffer A supplemented with 0.5 m NaCl, 5% glycerol, and 20 mm imidazole, and then washed with 30 ml of buffer A without Triton X-100 and supplemented with 5% glycerol and 20 mm imidazole. G-protein was eluted with the final wash buffer plus 200 mm imidazole. The elution buffer was removed by dialysis or size-exclusion chromatography using a buffer of 50 mm HEPES, pH 8.0, 100 mm NaCl, 0.1 mm PMSF, 5% glycerol, 2 μm GDP, and 1 mm EDTA, 0.5 mm dithiothreitol. The purified protein was aliquoted and stored at -80 °C. Steady-state GTPase Assay—Sf9 insect cells at a density of 2 × 106/ml were infected with baculoviruses encoding the following: Gαi1, Gαi2, Gαi3, Gαo, or Gαz, plus N-terminally c-myc-tagged M2 muscarinic receptors, Gβ1 and Gγ2. After 48 h of infection, cell membranes were prepared as described previously (25Cladman W. Chidiac P. Mol. Pharmacol. 2002; 62: 654-659Crossref PubMed Scopus (42) Google Scholar) and stored at -80 °C in a buffer containing 20 mm Tris, pH 8.0, 0.1 mm PMSF, 1 μg/ml leupeptin, and 10 μg/ml aprotinin. The steady-state hydrolysis of [γ-32P]GTP by Sf9 membranes was measured in the absence and presence of purified RGS proteins essentially as described previously (25Cladman W. Chidiac P. Mol. Pharmacol. 2002; 62: 654-659Crossref PubMed Scopus (42) Google Scholar). Reaction mixtures (50 μl) containing 20 mm HEPES (pH 7.5), 1 mm EDTA, 1 mm dithiothreitol, 0.1 mm PMSF, 1 μg/ml leupeptin, 10 μg/ml aprotinin, 10–50 mm NaCl, and 10 mm MgCl2 (calculated free Mg2+ = 7.5 mm), were incubated at 30 °C for 5 min with 1 μm GTP, 500 μm ATP, [32P]GTP (1 × 106 cpm/assay), either 100 μm carbachol or 10 μm atropine, and cell membranes (2 μg/assay). The assay was stopped by adding 950 ml of ice cold 5% (w/v) Norit in 0.05 m NaH2PO4 (pH 3) and centrifuging. Radioactivity of [32Pi] in the resulting supernatant was determined by liquid scintillation counting. The nonspecific membrane GTPase signal was estimated by adding 1 mm of unlabeled GTP to the above assay mix. Agonist- and RGS-dependent GTPase activity was calculated as described (25Cladman W. Chidiac P. Mol. Pharmacol. 2002; 62: 654-659Crossref PubMed Scopus (42) Google Scholar). Pre-steady-state GTPase Assay—Measurement of pre-steady-state, single-turnover GTP hydrolysis by Gαi2 and Gαo was conducted as described (29Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (477) Google Scholar) with slight modifications. Purified Gαi2 or Gαo (1 μm) was incubated with 2 × 106 cpm [γ-32P]GTP at 20 °C(Gαo)or30 °C(Gαi2) for 15 min and then kept on ice. After 5 min, a single round of GTP hydrolysis was initiated by the addition of MgCl2 (10 mm), GTP (500 μm, to quench the binding reaction), and varying amounts of RGS protein. Reactions were terminated at various time points by mixing aliquots of sample with activated charcoal. The charcoal was pelleted, and the released 32Pi in the supernatant was measured by scintillation counting. GTP hydrolysis rates were determined by fitting to a single exponential function (Origin 6). In Vitro Pull-down Assay and Western Blotting—E. coli strain BL21DE3 was transformed with pET-GαoR179C, pGEX-4T-1, or pGEX-RGS17, induced with 0.5 mm isopropylthio-B-d-galactoside (37 °C, 4 h), harvested, and re-suspended in lysis buffer (50 mm HEPES, pH 7.4, 100 mm NaCl, 2 mm MgCl2, 1 mm dithiothreitol, 0.1% Triton X-100). The bacteria were sonicated, centrifuged at (12,000 × g for 15 min) and the supernatants were incubated with Ni-NTA resin on ice for 20 min. The resin was washed 3× with lysis buffer containing 40 mm imidazole, eluted in 200 mm imidazole, and resolved on an SDS-12% polyacrylamide gel. The gel was transferred onto a polyvinylidene difluoride membrane and blocked overnight at 4 °C in 5% skimmed milk in TTBS buffer (0.1% Tween 20, 50 mm Tris, pH 8, 100 mm NaCl). The membrane was blotted with anti-His (Covance Inc., Princeton, NJ) or anti-GST antibodies (Santa Cruz Biotechnology), and then horseradish peroxidase-linked secondary antibodies. The membrane was incubated with chemiluminescence substrate (peroxidase (POD), Roche Applied Science) and exposed to Kodak X-OMAT film. Co-immunoprecipitation—The pcDNA3FLAG-RGS17 construct was transiently co-transfected with individual pcDNA3myc-Gα mutants into HEK293 cells by the CaPO4 precipitation method (30Albert P.R. Sajedi N. Lemonde S. Ghahremani M.H. J. Biol. Chem. 1999; 274: 35469-35474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Forty-eight hours after transfection, the cells were scraped in Nonidet P-40 lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, protease inhibitor mixture, Roche Applied Science) and left on ice for 30 min to lyse. The cell lysate was centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatants of FLAG-RGS17 and mycGα subunits were incubated with ∼5 μl of anti-FLAG M2-agarose affinity gel (Sigma) at 4 °C overnight. The gel was washed three times with Nonidet P-40 lysis buffer, resuspended in 0.1 m glycine (pH 3.3) at room temperature for 5 min to elute the proteins. The eluted supernatants were resolved on an SDS-12% polyacrylamide gel and analyzed by Western blotting. cAMP Assay—HEK293 cells were co-transfected with dopamine-D1 (Gs-coupled) and -D2 (Gi/o-coupled) receptor expression plasmids with or without pcDNA3FLAG-RGS17, pcDNA3FLAG-GAIP, pcDNA3FLAG-RGSZ1, or PTX-insensitive rat Gα subunits as described (30Albert P.R. Sajedi N. Lemonde S. Ghahremani M.H. J. Biol. Chem. 1999; 274: 35469-35474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Plasmid pcDNA3-FLAG was added to equalize the total DNA content for each transfection. After 24 h, cells were plated into six-well dishes. Forty-eight hours after transfection, the cells were pre-treated with 100 ng/ml PTX (Sigma) for four hours and assayed in serum-free Dulbecco's modified Eagle's medium/10 μm isobutylmethylxanthine (IBMX) with compounds indicated in the figure legends (37 °C, 20 min). The medium was collected, floating cells were pelleted (10,000 × g for 30 s), and the supernatant was collected for cAMP radioimmunoassay (30Albert P.R. Sajedi N. Lemonde S. Ghahremani M.H. J. Biol. Chem. 1999; 274: 35469-35474Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Calcium Mobilization—Ltk- cells in 15-cm plates were transiently co-transfected with 5 μg of TRHR1 cDNA (26Zhao D. Yang J. Jones K.E. Gerald C. Suzuki Y. Hogan P. Chin W.W. Tashjian Jr., A.H. Endocrinology. 1993; 130: 3529-3536Crossref Scopus (75) Google Scholar) and 10 μg of vector pcDNA3FLAG, pcDNA3FLAG-RGS17, pcDNA3FLAG-GAIP, or pcDNA3FLAG-RGSZ1 by DEAE-dextran method (31Lembo P.M. Ghahremani M.H. Morris S.J. Albert P.R. Mol. Pharmacol. 1997; 52: 164-171Crossref PubMed Scopus (43) Google Scholar, 32Ghahremani M.H. Cheng P. Lembo P.M. Albert P.R. J. Biol. Chem. 1999; 274: 9238-9245Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Forty-eight hours after transfection, the cells were trypsinized and washed in HBBS buffer (20 mm HEPES, 0.1 m NaCl, 4.6 mm KCl, 10 mm d-glucose, 1 mm CaCl2) and incubated with 2 μm Fura-2 AM in HBBS at 37 °C for 30 min with shaking (100 rpm). The cells were washed twice with HBBS, resuspended in 2 ml of HBBS, and subjected to fluorometric measurement. Experimental compounds were added directly to the cuvette from 100-fold concentrated solutions. RGS17, a Novel RZ-RGS—To identify novel Gαo-interacting proteins, we screened a human brain cDNA library with constitutively active GαoR179C for interacting proteins using a yeast two-hybrid system. Among eight positive clones, human RGS17 (GenBank accession no. AF202257.3) was identified. Its cDNA sequence encodes a protein of 210 amino acids, with a predicted molecular mass of 24.4 kDa (Fig. 1). RGS17 contains a core RGS domain (Fig. 1, underlined) and a cysteine-repeat motif (Fig. 1, dotted underlined) at the N-terminal region. There are six putative casein kinase 2 sites (Fig. 1, black arrowheads) and three potential PKC sites (Fig. 1, white arrowheads). hRGS17 shares 93% amino acid identity to the rat RGS17 (GenBank accession no. XM_217837), 92% identity with the Gallus gallus homolog (GenBank accession no. AF151968), and 91% identity to murine RGSZ2 (GenBank accession no. AF191555), indicating that these are species homologues of RGS17. Human RGS17 has the highest homology to RZ subfamily RGS proteins with amino acid identity of 61 and 57% compared with hRGSZ1 and hRGS-GAIP, respectively, suggesting that RGS17 is a new member of the RZ subfamily. Expression of RGS17—To verify that RGS17 is expressed in vivo, we examined the distribution of RGS17 mRNA in human tissues by Northern blotting. At least five RGS17 transcripts were found in various brain regions, with three dominant bands at 8, 3, and 2 kb (Fig. 1B). Throughout the central nervous system, the 8-kb RGS17 transcript was expressed most abundantly in the cerebellum (Fig. 1B, lane 1), whereas the 2-kb species was more abundant in cortex and medulla (lanes 2 and 3). The multiple RGS17 transcripts in brain tissues may represent alternately spliced mRNA products. In contrast, in a variety of human peripheral tissues, only the single 2-kb RGS17 mRNA transcript was weakly detected (data not shown). The size of this transcript is similar to the 1.8-kb RGSZ1 (20Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 21Glick J.L. Meigs T.E. Miron A. Casey P.J. J. Biol. Chem. 1998; 273: 26008-26013Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) and the 1.6-kb RGS-GAIP (33De 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). Thus, multiple RGS17 transcripts are predominantly expressed in the cerebellum and other brain regions. Because we detected several large RGS17 RNA species in brain, we performed 5′-RACE studies of a human brain cDNA library (Marathon, Clontech) to search for alternate translational start sites (Fig. 1C). We obtained a single major band which, when subcloned and sequenced, was identical to and no longer than the cDNA sequence. Thus, the human RGS17 RNA variants in brain appear to have the same coding sequence, and variations in RNA size may be due to different 3′-untranslated regions. Gα Specificity of RGS17 Interaction—To demonstrate direct interactions between RGS17 and GαoR179C, in vitro pull-down assays were done using recombinant proteins (Fig. 2)
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