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• W2012068051 abstract "In this report, we characterize GIV (Gα-interacting vesicle-associated protein), a novel protein that binds members of the Gαi and Gα subfamilies of heterotrimeric G proteins. The Gαs interaction site was mapped to an 83-amino acid region of GIV that is enriched in highly charged amino acids. BLAST searches revealed two additional mammalian family members, Daple and an uncharacterized protein, FLJ00354. These family members share the highest homology at the Gα binding domain, are homologous at the N terminus and central coiled coil domain but diverge at the C terminus. Using affinity-purified IgG made against two different regions of the protein, we localized GIV to COPI, endoplasmic reticulum (ER)-Golgi transport vesicles concentrated in the Golgi region in GH3 pituitary cells and COS7 cells. Identification as COPI vesicles was based on colocalization with β-COP, a marker for these vesicles. GIV also codistributes in the Golgi region with endogenous calnuc and the KDEL receptor, which are cis Golgi markers and with Gαi3-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with β-COP and Gαi3 on vesicles found in close proximity to ER exit sites and to cis Golgi cisternae. In cell fractions prepared from rat liver, GIV is concentrated in a carrier vesicle fraction (CV2) enriched in ER-Golgi transport vesicles. β-COP and several Gα subunits (Gαi1–3, Gαs) are also most enriched in CV2. Our results demonstrate the existence of a novel Gα-interacting protein associated with COPI transport vesicles that may play a role in Gα-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi. In this report, we characterize GIV (Gα-interacting vesicle-associated protein), a novel protein that binds members of the Gαi and Gα subfamilies of heterotrimeric G proteins. The Gαs interaction site was mapped to an 83-amino acid region of GIV that is enriched in highly charged amino acids. BLAST searches revealed two additional mammalian family members, Daple and an uncharacterized protein, FLJ00354. These family members share the highest homology at the Gα binding domain, are homologous at the N terminus and central coiled coil domain but diverge at the C terminus. Using affinity-purified IgG made against two different regions of the protein, we localized GIV to COPI, endoplasmic reticulum (ER)-Golgi transport vesicles concentrated in the Golgi region in GH3 pituitary cells and COS7 cells. Identification as COPI vesicles was based on colocalization with β-COP, a marker for these vesicles. GIV also codistributes in the Golgi region with endogenous calnuc and the KDEL receptor, which are cis Golgi markers and with Gαi3-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with β-COP and Gαi3 on vesicles found in close proximity to ER exit sites and to cis Golgi cisternae. In cell fractions prepared from rat liver, GIV is concentrated in a carrier vesicle fraction (CV2) enriched in ER-Golgi transport vesicles. β-COP and several Gα subunits (Gαi1–3, Gαs) are also most enriched in CV2. Our results demonstrate the existence of a novel Gα-interacting protein associated with COPI transport vesicles that may play a role in Gα-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi. Heterotrimeric G proteins are well known to act as intracellular transducers to propagate a variety of signals across the plasma membrane (1Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4728) Google Scholar). Over the last 15 years it has become evident that trimeric G proteins are also present at intracellular locations such as the Golgi apparatus (2Ercolani L. Stow J.L. Boyle J.F. Holtzman E.J. Lin H. Grove J.R. Ausiello D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4635-4639Crossref PubMed Scopus (92) Google Scholar, 3Stow J.L. de Almeida J.B. Narula N. Holtzman E.J. Ercolani L. Ausiello D.A. J. Cell Biol. 1991; 114: 1113-1124Crossref PubMed Scopus (240) Google Scholar, 4Wilson B.S. Komuro M. Farquhar M.G. Endocrinology. 1994; 134: 233-244Crossref PubMed Scopus (76) Google Scholar, 5Denker S. McCaffery J.M. Palade G.E. Insel P.A. Farquhar M.G. J. Cell Biol. 1996; 133: 1037-1040Crossref Scopus (117) Google Scholar), the endoplasmic reticulum (6Audigier Y. Nigam S.K. Blobel G. J. Biol. Chem. 1988; 263: 16352-16357Abstract Full Text PDF PubMed Google Scholar), secretory granules (7Toutant M. Aunis D. Bockaert J. Homburger V. Rouot B. FEBS Lett. 1987; 215: 339-344Crossref PubMed Scopus (96) Google Scholar), endosomes (8Ali N. Milligan G. Evans W.H. Biochem. J. 1989; 261: 905-912Crossref PubMed Scopus (30) Google Scholar, 9Zheng B. Lavoie C. Tang T.D. Ma P. Meerloo T. Beas A. Farquhar M.G. Mol. Biol. Cell. 2004; 15: 5538-5550Crossref PubMed Scopus (52) Google Scholar), the cytoskeleton (10Rasenick M.M. Wang N. Yan K. Adv. Second Messenger Phosphoprotein Res. 1990; 24: 381-386PubMed Google Scholar, 11Roychowdhury S. Panda D. Wilson L. Rasenick M.M. J. Biol. Chem. 1999; 274: 13485-13490Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 12Chen N.F. Yu J.Z. Skiba N.P. Hamm H.E. Rasenick M.M. J. Biol. Chem. 2003; 278: 15285-15290Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), and even the nucleus (13Crouch M.F. Davy D.A. Willard F.S. Berven L.A. Immunol. Cell Biol. 2000; 78: 408-414Crossref PubMed Scopus (25) Google Scholar). Because classical receptors and effectors had not been identified at intracellular sites, investigators have attempted to gain understanding of the role of trimeric G proteins on intracellular organelles by identifying and characterizing Gα-interacting proteins. Within the last 5–10 years, a remarkable array of novel Gα-binding proteins have been identified and shown to play various roles in regulating heterotrimeric G protein signaling. These include 1) the RGS proteins (regulators of G protein signaling) (14De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (513) Google Scholar, 15Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (937) Google Scholar) that act as GTPase-activating proteins; 2) a group of proteins containing G protein regulatory or GoLoco motifs, such as AGS3 (16De Vries L. Fischer T. Tronchère H. Brothers G.M. Strockbine B. Siderovski D.P. Farquhar M.G. Proc. Natl. Acad. Sci. 2000; 97: 14364-14369Crossref PubMed Scopus (134) Google Scholar, 17Takesono 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), LGN (18Mochizuki N. Cho G. Wen B. Insel P.A. Gene (Amst.). 1996; 181: 39-43Crossref PubMed Scopus (97) Google Scholar, 19Natochin M. Gasimov K.G. Artemyev N.O. Biochemistry. 2001; 40: 5322-5328Crossref PubMed Scopus (79) Google Scholar), PCP2 (20Natochin M. Gasimov K.G. Artemyev N.O. Biochemistry. 2002; 41: 258-265Crossref PubMed Scopus (22) Google Scholar), and RapIGAP (21Jordan J.D. Carey K.D. Stork P.J. Iyengar R. J. Biol. Chem. 1999; 274: 21507-21510Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) that act as guanine dissociation inhibitors; and 3) Ric-8A and Ric-8B, mammalian homologs of Ric-8/synembryn, which are potent guanine nucleotide exchange factors (22Tall G.G. Krumins A.M. Gilman A.G. J. Biol. Chem. 2003; 278: 8356-8362Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The discovery and characterization of these proteins has implicated heterotrimeric G proteins in a surprisingly diverse variety of cell processes including assembly of the actin cytoskeleton, growth factor receptor down-regulation, and mitosis. For example, the RGS protein p115RhoGEF serves as a GTPase-activating protein for Gα13 proteins, through its RGS domain, and a guanine nucleotide exchange factor for Rho, through its DH/PH domain, and links G proteins to Rho signaling (14De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (513) Google Scholar). RGSPX1, which serves as a GTPase-activating protein for Gαs and binds phosphoinositides through its PX domain, delays degradation of the epidermal growth factor receptor and thus links heterotrimeric G protein signaling to vesicular trafficking (9Zheng B. Lavoie C. Tang T.D. Ma P. Meerloo T. Beas A. Farquhar M.G. Mol. Biol. Cell. 2004; 15: 5538-5550Crossref PubMed Scopus (52) Google Scholar, 23Zheng B. Ma Y.C. Ostrom R.S. Lavoie C. Gill G.N. Insel P.A. Huang X.Y. Farquhar M.G. Science. 2001; 294: 1939-1942Crossref PubMed Scopus (196) Google Scholar). LGN and its Drosophila ortholog Pins play an essential role in the assembly and organization of the mitotic spindle (24Yu F. Morin X. Cai Y. Yang X. Chia W. Cell. 2000; 100: 399-409Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 25Schaefer M. Shevchenko A. Knoblich J.A. Curr. Biol. 2000; 10: 353-362Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), which is a Gαo-mediated process in Drosophila. In this report, we describe the discovery and molecular characterization of GIV (Gα-interacting vesicle-associated protein), which has a novel Gα-interacting domain and is found on vesicles concentrated in the Golgi region where it colocalizes with β-COP, a marker for COPI, ER-Golgi transport vesicles. Previously, heterotrimeric G proteins have been implicated in regulation of ER-Golgi transport (3Stow J.L. de Almeida J.B. Narula N. Holtzman E.J. Ercolani L. Ausiello D.A. J. Cell Biol. 1991; 114: 1113-1124Crossref PubMed Scopus (240) Google Scholar, 26Helms J.B. FEBS Lett. 1995; 369: 84-88Crossref PubMed Scopus (77) Google Scholar), but the mechanisms involved are not yet understood. The discovery of a Gα-interacting protein located on these transport vesicles provides a new tool that may provide insights into the role of Gα subunits in vesicular trafficking. The localization and structure of GIV suggest that it may serve to connect COPI transport vesicles to Gα subunits and microtubules. Yeast Two-hybrid Interactions—For yeast two-hybrid screening 50 μg of a rat GC cell (pituitary) cDNA library in pACT2 was transformed into yeast HF7c (pGBT9Gαi3) as described (27De 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, 28Lin P. Le-Niculescu H. Hofmeister R. McCaffery J.M. Jin M. Hennemann H. McQuistan T. De Vries L. Farquhar M.G. J. Cell Biol. 1998; 141: 1515-1527Crossref PubMed Scopus (140) Google Scholar). Twenty-four positive clones, grouped by insert size and restriction pattern, were sequenced from the 5′- or 3′-end. Among these were three partial clones for GIV, grossly encoding the C-terminal third of the molecule (aa 1The abbreviations used are: aa, amino acid; GST, glutathione S-transferase; GFP, green fluorescent protein; mAb, monoclonal antibody; EST, expressed sequence tag; CHO, Chinese hamster ovary; YFP, yellow fluorescent protein; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-O-(thiotriphosphate); CV, carrier vesicle; ERGIC, ER-Golgi intermediate compartment. 1174–1898, see below). For 1 to 1 two-hybrid interactions, rat GIV in pACT2 vector was cotransformed in yeast strain SFY526 (BD Biosciences) with the following G protein subunits subcloned into pGBT9 vector: rat Gαi1, Gαi2, Gαi3, Gαo, and Gαz, mouse Gα12, Gα13, and Gαq, rat Gαs, and Gαs(Q226L), and Saccharomyces cerevisiae GPA1. Interactions were analyzed qualitatively by a colony lift assay using 5-bromo-4-chloro-3-indolyl d-galactoside, and the appearance of blue colonies was assessed after 2, 4, and 8 h (29De Vries L. Farquhar M.G. Methods Enzymol. 2002; 344: 657-673Crossref PubMed Scopus (4) Google Scholar). No background color was detected after 20 h. BLAST Searches—Online BLAST searches were performed via the National Center for Biotechnology Information (NCBI) website (30Madden T.L. Tatusov R.L. Zhang J. Methods Enzymol. 1996; 266: 131-141Crossref PubMed Google Scholar). Homologous human EST clones were purchased from Incyte (Palo Alto, CA). Protein alignments were carried out with the ClustalW program (31Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol. 1996; 266: 383-402Crossref PubMed Scopus (1288) Google Scholar) InterProScan (EMBL-EBI) for domain and motif searches, PSORTII for protein topology predictions, TMpred for the prediction of transmembrane, and Coils for the prediction of coiled coil domains via the ExPASy proteomics tools server website. Isolation of GIV cDNA—PCR was used to isolate 5.25 kb of GIV cDNA from human Fetal Brain QUICK-Clone cDNA (BD Biosciences) using Pfu turbo polymerase (Stratagene), encoding the near-complete GIV open reading fame (aa 56–1843). The 5.25-kb insert was sequenced by automated sequencing (Center for AIDS Research, DNA sequencing facility, University of California San Diego (UCSD)). Northern Blot Analysis—Multiple tissue blots of poly(A)+ RNA from rat (Seegene, Seoul, Korea) or human (BD Biosciences) tissues were hybridized to a 976-bp rat cDNA fragment (corresponding to rat GIV aa 1174–1499). The probe was labeled by random priming with [32P]dCTP (3000 Ci/mmol) (Amersham Biosciences) to a specific activity of 109 cpm/μg. ExpressHyb solution (BD Biosciences) was used under high stringency conditions for hybridization (68 °C) according to the manufacturer's guidelines, and high stringency washes were performed in 0.1% SSC (150 mm NaCl, 15 mm sodium citrate, pH 7) plus 0.1% SDS at 65 °C. Autoradiographs were exposed for 1–3 days at –70 °C. Expression and Purification of Glutathione S-Transferase GIV Fusion Proteins—For the production of recombinant glutathione S-transferase (GST) fusion proteins, various deletion mutants of rat GIV (corresponding to aa 1174–1404, 1399–1546, 1399–1481, and 1480–1546) were subcloned into the pGEX-KG vector (Pharmacia Biotechnology, Inc.) and transformed into Escherichia coli (BL21DE3). GST-GIV fusion proteins were affinity-purified from bacterial lysates on glutathione-Sepharose beads (Amersham Biosciences). In Vitro Interactions—Wild-type rat Gαi3 cDNA was subcloned into pBluescript SK+ (Stratagene) (27De 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). In vitro transcription/translation of Gαi3 from the T7 promoter was performed using the TnT-coupled reticulocyte lysate system (Promega) in the presence of [35S]methionine (Amersham Biosciences) according to the manufacturer's instructions. GST-GIV fusion proteins (6 μg) or GST alone (6 μg) was immobilized on glutathione-Sepharose beads and incubated with 15,000 cpm 35S-labeled, in vitro translated Gαi3 in binding buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 3 mm EDTA, 0.1% Nonidet P-40, 1 mm dithiothreitol, and protease inhibitors) as described previously (32Mochizuki N. Hibi M. Kanai Y. Insel P.A. FEBS Lett. 1995; 373: 155-158Crossref PubMed Scopus (43) Google Scholar). The mixture was incubated by rotating for 2 h at 4 °C. The beads were washed three times with binding buffer, resuspended in 25 μl of Laemmli buffer, and boiled for 5 min, and the proteins were loaded on 10% SDS gels and exposed for autoradiography. Antibodies—Anti-Gαi1 (LD), anti-Gαi1/2 (AS), anti-Gαi3 (EC), and anti-Gαs were gifts from Dr. Allen Spiegel (NIDDK, National Institutes of Health). Mouse mAbs were obtained from the following sources: AP-2-adaptin from Dr. Sandra Schmid (The Scripps Research Institute, La Jolla, CA), anti-early endosome antigen 1 from BD Transduction Laboratories, anti-β-COP from Novis Biochemicals, anti-KDEL receptor from Stressgene, and anti-caveolin 1 from Zymed Laboratories Inc. Anti-LAMP2 (H4B4) was from the Developmental Studies Hybridoma Bank (University of Iowa). Sheep anti-PMP-70 was provided by Dr. S. J. Gould, Johns Hopkins. Rabbit antisera to GM130 (33Nakamura N. Rabouille C. Watson R. Nilsson T. Hui N. Slusarewicz P. Kreis T.E. Warren G. J. Cell Biol. 1995; 131: 1715-1726Crossref PubMed Scopus (679) Google Scholar) and calnexin (34Wada I. Rindress D. Cameron P.H. Ou W.-J. Doherty J.J. Louvard D. Bell A.W. Dignard D. Thomas D.Y. Bergeron J.J.M. J. Biol. Chem. 1991; 266: 19599-19610Abstract Full Text PDF PubMed Google Scholar) were obtained from Dr. John Bergeron (McGill University, Montréal, Canada). Rabbit antiserum to α-mannosidase II (Man II) was prepared as described (35Velasco A. Hendricks L. Moremen K.W. Tulsiani D.R. Touster O. Farquhar M.G. J. Cell Biol. 1993; 122: 39-51Crossref PubMed Scopus (281) Google Scholar), and polyclonal anti-ERGIC53 was kindly provided by Dr. Hans Peter Hauri (Biocentrum, Basel, Switzerland). Anti-Calnuc IgY (chicken) was prepared by Dr. Ping Lin (UCSD). Preparation of Antibodies against GIV—The central region of rat GIV (CC, aa 1174–1499) containing a portion of the coiled coil domain without the Gα binding domain and the C terminus of human GIV (CT, aa 1574–1843), which has no homologies to other known mammalian proteins, were expressed in bacteria as GST-fusion proteins, and the recombinant proteins were purified and injected into rabbits. For affinity purification, purified recombinant fusion protein was coupled to Affi-Gel 10 (Bio-Rad). Antibodies were then bound to the coupled beads and eluted sequentially with 0.1 m glycine, HCl, pH 2.5, and 0.1 m triethylamine, pH 11.5. Both antisera recognized 10 pg of affinity-purified GST-tagged GIV by immunoblotting (1:5000 dilution). By immunoblotting they also recognized endogenous GIV (180–200 kDa) in cell lysates prepared from COS7 and NRK cells. Cell Culture and Transfection—COS7, NRK, HeLa, PC12, Madin-Darby canine kidney, AtT-20, GH3, CHO-K1, Rat1, REF52, NIH3T3, and HEK293 cells were grown as recommended by the American Type Culture Collection. COS7 cells were grown on coverslips and maintained in Dulbecco's high glucose medium supplemented with 10% (v/v) fetal calf serum (Invitrogen). To transiently overexpress pcDNA3/Gαi3-YFP (Weiss et al. (44Weiss T.S. Chamberlain C.E. Takeda T. Lin P. Hahn K.M. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14961-14966Crossref PubMed Scopus (50) Google Scholar)), cells were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions and processed for immunofluorescence and immunoelectron microscopy 24 h after transfection as described (36Elenko E. Fischer T. Niesman I. Harding T. McQuistan T. Von Zastrow M. Farquhar M.G. Mol. Pharmacol. 2003; 64: 11-20Crossref PubMed Scopus (28) Google Scholar). Preparation of Cells and Tissue Lysates—Rat tissues were isolated at 4 °C and homogenized using a POLYTRON MR2100. Cells and rat tissues were lysed with 0.5% Nonidet P-40, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, and protease inhibitors (Sigma). Immunoblotting—Tissue lysates (20 μg), cell lysates (30 μg), and liver fractions (40 μg) were resolved by SDS-PAGE on 8–12% polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Millipore Corp). After blocking with 5% milk in TBST (10 mm Tris, pH 7.5, 100 mm NaCl, 5 mm KCl, 0.1% Tween) for 1 h, the membranes were probed with 0.85 μg/ml affinity-purified, anti-GIV IgG or with other primary antibodies followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000 in TBST) (Amersham Biosciences) and detection by enhanced chemiluminescence ECL (Pierce). Subcellular Fractionation—HeLa or COS7 cells were scraped into ice-cold PBS containing phenylmethylsulfonyl fluoride (1 mm) and aprotinin (100 units/ml). All the following steps were performed at 4 °C. Cells were homogenized by 15 passages through a 27.5-gauge needle, and a postnuclear supernatant was prepared by centrifugation for 3 min at 600 × g. Membrane pellets were prepared by centrifugation of the postnuclear supernatant at 100,000 × g for 1 h at 4 °Cina Beckman TL-100 ultracentrifuge. The resulting pellet (crude membrane fraction) was resuspended in a volume of PBS equal to that of the supernatant. Preparation of Rat Liver Fractions—Fractions were prepared from rat liver by density gradient centrifugation and characterized as described previously (37Saucan L. Palade G.E. J. Cell Biol. 1994; 125: 733-741Crossref PubMed Scopus (80) Google Scholar, 38Jin M. Saucan L. Farquhar M.G. Palade G.E. J. Biol. Chem. 1996; 271: 30105-30113Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The protein concentration of each fraction was determined by BCA assay (Pierce), and 40 μg of protein of each fraction was solubilized in Laemmli sample buffer and separated by SDS-PAGE. Immunofluorescence and Immunoelectron Microscopy—For immunofluorescence, cells were fixed in 2% paraformaldehyde in 100 mm phosphate buffer, pH 7.4, for 25 min, permeabilized with 0.1% Triton X-100 in PBS (10 min), and incubated for 1 h with primary rabbit polyclonal or mouse mAbs followed by an incubation (1 h) with highly cross-absorbed Alexa Fluor-594 goat anti-mouse, Alexa Fluor-488 goat anti-rabbit, or Alexa Fluor-488 goat anti-chicken F(ab′)2 (Molecular Probes). Cells were mounted in 25% PBS, 75% glycerol with 1 mg/ml phenylenediamine and then examined with a Zeiss Axiophot microscope equipped for epifluorescence, or they were analyzed by deconvolution microscopy with the DeltaVision imaging system (Applied Precision, Issaquah, WA) coupled to a Zeiss S100 fluorescence microscope (Carl Zeiss, Thornwood, NY). For cross-sectional images of cells, stacks were obtained with a 150-nm step-width to optimize the reconstruction of the center plane image. Deconvolution was done on an SGI work station (Mountain View, CA) using Delta Vision reconstruction software, and images were processed as TIFF files using Photoshop 5.5 (Adobe Systems, Mountain View, CA). For immunogold labeling at the electron microscope level (39De Vries L. Elenko E. McCaffery J.M. Fischer T. Hubler L. McQuistan T. Watson N. Farquhar M.G. Mol. Biol. Cell. 1998; 9: 1124-1134Google Scholar), cells were fixed in 4% paraformaldehyde in 10 mm phosphate buffer, pH 7.4, cryoprotected, and frozen in liquid nitrogen. Ultrathin cryosections were cut at –100 °C using a Leica Ultracut UCT Microtome with an EMFCS cryoattachment (Leica), placed on glow-discharged nickel grids, stored on 2% gelatin, PBS at 4 °C, and incubated with primary antibodies followed by 5 or 10 nm gold, goat anti-rabbit or anti-mouse IgG (Amersham Biosciences) in PBS with in 10% fetal calf serum. Grids were absorption stained with 0.2% neutral uranyl acetate, 0.2% methyl cellulose, and 3.2% polyvinyl alcohol. Identification of GIV—To identify proteins that interact with the heterotrimeric G protein, Gαi3, we screened a rat GC pituitary cell cDNA library as described previously (27De 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, 28Lin P. Le-Niculescu H. Hofmeister R. McCaffery J.M. Jin M. Hennemann H. McQuistan T. De Vries L. Farquhar M.G. J. Cell Biol. 1998; 141: 1515-1527Crossref PubMed Scopus (140) Google Scholar). 3 of 24 positive, independent clones contained partially overlapping inserts, making up 3.2 kb in sequence, coding for the 724 C-terminal residues (aa 1174–1898) of a novel protein (Fig. 1). A BLAST search of the nucleotide data base with this rat cDNA sequence revealed identity to a hypothetical rat protein of 1898 aa (GenBank™ XP_223709), which we named GIV (Fig. 1). The human ortholog, KIAA1212, has 1843 aa residues (GenBank™ NP_060554), and the mouse ortholog (GenBank™ CAI24878) has 1845 residues. EST data base searches also indicated that there are numerous alternative splice variants of GIV. Rat and human GIV have predicted molecular masses of 220 and 213 kDa, respectively, based upon amino acid sequence, with a theoretical pI of 7.6 and 5.7. GIV Is a Member of a Novel Coiled Coil Family of Proteins— As shown in Fig. 1, human GIV is largely composed of coiled coil regions spanning more than two-thirds of the protein (from aa 240 to 1384, according to Coils, EMBnet-CH, and PSORTII SMART ExPaSy Molecular Biology server for proteomic tools). GIV contains a high frequency of leucine residues that are arranged in periodic repeats at every seventh position, which is the characteristic structure of leucine zipper motifs (InterPro Scan). The leucine zipper motif forms an α-helical conformation that is a coiled coil, which has been shown to facilitate dimerization (40O'Shea E.K. Rutkowski R. Kim P.S. Science. 1989; 243: 538-542Crossref PubMed Scopus (698) Google Scholar, 41Rasmussen R. Benvegnu D. O'Shea E.K. Kim P.S. Alber T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 561-564Crossref PubMed Scopus (64) Google Scholar). By performing yeast two-hybrid 1 to 1 interactions, we found that GIV can homodimerize within the coiled coil domain (data not shown). BLAST searches with full-length human GIV indicated that there are two additional mammalian family members (Fig. 1): Daple (GenBank™ MP_080957), which is 2009 aa in the mouse, and its human ortholog KIAA1509 (1898 aa), and an uncharacterized human protein FLJ00354 (GenBank™ NP_115627) (1214 aa) deduced from cDNA, which might not be a full-length complete protein, because the sequence is missing the stop codon at the C terminus. The closest member to GIV is Daple, a recently isolated protein that interacts with the PDZ domain of Dvl, a protein involved in the Wnt signaling pathway (42Oshita A. Kishida S. Kobayashi H. Michiue T. Asahara T. Asashima M. Kikuchi A. Genes Cells. 2003; 8: 1005-1017Crossref PubMed Scopus (51) Google Scholar). Daple acts as a negative regulator of the Wnt signaling pathway by inhibiting Wnt-3a-dependent accumulation of β-catenin and transcriptional activation of Tcf-4 (42Oshita A. Kishida S. Kobayashi H. Michiue T. Asahara T. Asashima M. Kikuchi A. Genes Cells. 2003; 8: 1005-1017Crossref PubMed Scopus (51) Google Scholar). Daple possesses 48% identity and 67% similarity overall to GIV, and FLJ00354 shows 36% identity and 58% similarity to GIV. Together, GIV, Daple, and FLJ00354 constitute a three-protein family whose genes are located on human chromosomes 2, 14, and 11, respectively. These three proteins share a conserved N-terminal domain and a central coiled coil domain, whereas they diverge at the C terminus (Fig. 1). The N terminus of members of this family also shows homology (29% identity, 52% similarity) to the N-terminal microtubule-binding domain of the HOOK family, and the coiled coil domain in GIV and Daple shows homology to myosin heavy chain. All three family members also contain several putative peroxisomal targeting signal consensus sequences (PTS2) within the coiled coil domain as predicted by PSORT. GIV also was predicted by PSORT to contain an ATP/GTP consensus binding site within the C-terminal domain. We found one ortholog of this family in Drosophila (NP 647780.1), one in Anopheles gambiae (EAA07494) and one in Caenorhabditis elegans (T27075), with Drosophila having the highest homology to rat GIV. There is no S. cerevisiae ortholog. The Region C-terminal of the Coiled Coil Domain Is Required for Interaction with Gαi3—From sequencing and analysis of the three clones isolated from the yeast two-hybrid screen it was evident that GIV interacts with Gαi3 within the 724-aa fragment spanning the C-terminal region of rat GIV (aa 1174–1898) (Figs. 1 and 2A). We further mapped the Gαi3-binding domain by generating a series of deletion mutant GST fusion constructs, GST-GIV(1174–1404), GST-GIV(1399–1546), GST-GIV(1399–1481), and GST-GIV(1480–1546), which we tested in an in vitro binding assay with various mutants of GST-GIV bound to glutathione-agarose beads and in vitro translated Gαi3. We found that Gαi3 bound specifically to GST-GIV(1174–1898), GST-GIV(1399–1546), and GST-GIV(1399–1481) (Fig. 2B, lanes 2, 3, and 5) but not to GST alone, GST-GIV(1174–1404), or GST-GIV(1480–1546) (Fig. 2A, lanes 1, 4, and 6). Thus, the 83-aa region spanning amino acids 1399–1481 was sufficient to interact with Gαi3 in this in vitro binding assay. A Novel Gαi3 Binding “Motif”—The 83-aa region of human rat GIV that interacts with Gαi3 is highly conserved among species, i.e. C. elegans, A. gambiae, Drosophila, mouse, rat, and human (Fig. 2C). This region consists of highly charged amino acids and is also the region in Daple and FLJ00354 that is most homologous to GIV. Daple is 66% identical and 81% similar to GIV at this Gα binding domain (Fig. 2C). A deletion mutant of Daple containing this region also interacts with Gαi3 in an in vitro binding assay (data not shown). FLJ00354 contains a region that also has homology to the Gα binding domain of GIV (48% identity, 80% similarity) (Fig. 2C), but it does not interact with Gαi3 in the same assay or in a two-hybrid 1 to 1 assay (data not shown). GIV Interacts with the Gαi and Gαs Subfamilies of G Proteins—We used the two-hybrid system to test whether GIV interacts with other G protein α-subunits. Based on the semiquantitative β-galactosidase filter assay (Fig. 3), GIV interacted with all members of the Gαi subfamily (Gαi1, Gαi2, Gαi3, Gαo, and Gαz) as well as wild-type Gαs and GPA1 (yeast homolog of Gαi) but not with Gαq, Gα12, or Gα13. Interestingly, GIV interacts only weakly with GαsQ226L (which mimics the GTP-bound form of Gαs) compared with wild-type Gαs in this assay. These results suggest that GIV specifically interacts with members of the Gαi and Gαs subfamilies of heterotrimeric G proteins. We tested for the regulatory activity of GIV on Gαi3 subunits by performing classical single turnover GTPase assays, guanine nucleotide exchange factor assays, and guanine dissociation inhibitor assays (both the latter through G" @default.
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• W2012068051 date "2005-06-01" @default.
• W2012068051 modified "2023-10-13" @default.
• W2012068051 title "Identification and Characterization of GIV, a Novel Gαi/s -interacting Protein Found on COPI, Endoplasmic Reticulum-Golgi Transport Vesicles" @default.
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• W2012068051 doi "https://doi.org/10.1074/jbc.m501833200" @default.
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