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Originally published In Press as doi:10.1074/jbc.M501833200 on March 4, 2005

J. Biol. Chem., Vol. 280, Issue 23, 22012-22020, June 10, 2005
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Identification and Characterization of GIV, a Novel G{alpha}i/s -interacting Protein Found on COPI, Endoplasmic Reticulum-Golgi Transport Vesicles*

Helen Le-Niculescu{ddagger}, Ingrid Niesman{ddagger}, Thierry Fischer{ddagger}, Luc DeVries§, and Marilyn G. Farquhar{ddagger}

From the {ddagger}Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093 and the §Departement de Biologie Cellulaire et Moleculaire, Institut de Recherche Pierre Fabre CRPF, Castres Cedex, France 81106

Received for publication, February 17, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this report, we characterize GIV (G{alpha}-interacting vesicle-associated protein), a novel protein that binds members of the G{alpha}i and G{alpha} subfamilies of heterotrimeric G proteins. The G{alpha}s 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 G{alpha} 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 {beta}-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 G{alpha}i3-yellow fluorescent protein expressed in COS7 cells. By immunoelectron microscopy, GIV colocalizes with {beta}-COP and G{alpha}i3 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. {beta}-COP and several G{alpha} subunits (G{alpha}i1–3, G{alpha}s) are also most enriched in CV2. Our results demonstrate the existence of a novel G{alpha}-interacting protein associated with COPI transport vesicles that may play a role in G{alpha}-mediated effects on vesicle trafficking within the Golgi and/or between the ER and the Golgi.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterotrimeric G proteins are well known to act as intracellular transducers to propagate a variety of signals across the plasma membrane (1). Over the last 15 years it has become evident that trimeric G proteins are also present at intracellular locations such as the Golgi apparatus (2–5), the endoplasmic reticulum (6), secretory granules (7), endosomes (8, 9), the cytoskeleton (10–12), and even the nucleus (13). 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 G{alpha}-interacting proteins. Within the last 5–10 years, a remarkable array of novel G{alpha}-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) (14, 15) that act as GTPase-activating proteins; 2) a group of proteins containing G protein regulatory or GoLoco motifs, such as AGS3 (16, 17), LGN (18, 19), PCP2 (20), and RapIGAP (21) 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 (22).

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 G{alpha}13 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 (14). RGSPX1, which serves as a GTPase-activating protein for G{alpha}s 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 (9, 23). LGN and its Drosophila ortholog Pins play an essential role in the assembly and organization of the mitotic spindle (24, 25), which is a G{alpha}o-mediated process in Drosophila.

In this report, we describe the discovery and molecular characterization of GIV (G{alpha}-interacting vesicle-associated protein), which has a novel G{alpha}-interacting domain and is found on vesicles concentrated in the Golgi region where it colocalizes with {beta}-COP, a marker for COPI, ER-Golgi transport vesicles. Previously, heterotrimeric G proteins have been implicated in regulation of ER-Golgi transport (3, 26), but the mechanisms involved are not yet understood. The discovery of a G{alpha}-interacting protein located on these transport vesicles provides a new tool that may provide insights into the role of G{alpha} subunits in vesicular trafficking. The localization and structure of GIV suggest that it may serve to connect COPI transport vesicles to G{alpha} subunits and microtubules.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 (pGBT9G{alpha}i3) as described (27, 28). 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 1 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 G{alpha}i1, G{alpha}i2, G{alpha}i3, G{alpha}o, and G{alpha}z, mouse G{alpha}12, G{alpha}13, and G{alpha}q, rat G{alpha}s, and G{alpha}s(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 (29). No background color was detected after 20 h.

BLAST Searches—Online BLAST searches were performed via the National Center for Biotechnology Information (NCBI) website (30). Homologous human EST clones were purchased from Incyte (Palo Alto, CA). Protein alignments were carried out with the ClustalW program (31) 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 G{alpha}i3 cDNA was subcloned into pBluescript SK+ (Stratagene) (27). In vitro transcription/translation of G{alpha}i3 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 G{alpha}i3 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 (32). 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-G{alpha}i1 (LD), anti-G{alpha}i1/2 (AS), anti-G{alpha}i3 (EC), and anti-G{alpha}s 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-{beta}-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 (33) and calnexin (34) were obtained from Dr. John Bergeron (McGill University, Montréal, Canada). Rabbit antiserum to {alpha}-mannosidase II (Man II) was prepared as described (35), 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 G{alpha} 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/G{alpha}i3-YFP (Weiss et al. (44)), 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 (36).

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 x g. Membrane pellets were prepared by centrifugation of the postnuclear supernatant at 100,000 x 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 (37, 38). 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 (39), 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.



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  		FIG. 1.
GIV is a member of a novel coiled coil family of proteins. A partial sequence (724 aa) of rat GIV (aa 1174–1898) was identified by two-hybrid screening to interact with G{alpha}i3. By BLAST search of the nucleotide and EST data base with this rat cDNA sequence we found full-length rat GIV (1898 aa) and its human ortholog (1843 aa). A BLAST search with full-length human GIV revealed two additional family members, mouse Daple and human FLJ00354 and an incomplete, uncharacterized protein with similar characteristics. Members of this family share a conserved N-terminal and central coiled coil domain and diverge at the C terminus. GIV differs in that it does not contain the GCV, PDZ-binding motif through which Daple binds the protein Dvl. At the N terminus, this family has 29% identity (52% similarity) to the N-terminal microtubule-binding domain of the HOOK family.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of GIV—To identify proteins that interact with the heterotrimeric G protein, G{alpha}i3, we screened a rat GC pituitary cell cDNA library as described previously (27, 28). 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 (GenBankTM XP_223709 [GenBank] ), which we named GIV (Fig. 1). The human ortholog, KIAA1212, has 1843 aa residues (GenBankTM NP_060554 [GenBank] ), and the mouse ortholog (GenBankTM CAI24878 [GenBank] 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 {alpha}-helical conformation that is a coiled coil, which has been shown to facilitate dimerization (40, 41). By performing yeast two-hybrid 1 to 1 interactions, we found that GIV can homodimerize within the coiled coil domain (data not shown).



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  		FIG. 2.
Characterization of a novel G{alpha}i3 binding motif that specifically interacts with G{alpha}i/s subunits. A, schematic representation of the various GST-GIV proteins used for the two-hybrid 1 to 1 {beta}-galactosidase ({beta}-Gal) assay and for in vitro interactions. B, the deletion mutants of GST-GIV shown in A were bound to glutathione-agarose beads and incubated with in vitro translated, 35S-labeled G{alpha}i3 as described under "Experimental Procedures." [35S]G{alpha}i3 bound to GST-GIV(1174–1898) (lane 2), GST-GIV(1399–1546) (lane 3), and GST-GIV(1399–1481) (lane 5) but not to GST alone (lane 1), GST-GIV(1174–1404) (lane 4), or GST-GIV(1480–1546) (lane 6). C, alignment of the highly charged, G{alpha}i3 binding region (aa 1399–1481) of rat GIV with mouse and human GIV, Daple (human, KIAA1509), FLJ00354(human), and the A. gambiae, Drosophila, and C. elegans orthologs. This G{alpha} binding region is the most homologous within the different orthologs and family members. Daple has 66% identity (81% similarity) and FLJ00354has 48% identity (80% similarity) to GIV in this region.

 



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  		FIG. 3.
Yeast two-hybrid 1 to 1 interaction assays of GIV with G{alpha} family members. GIV interacts with G{alpha}i1, G{alpha}2, G{alpha}3, G{alpha}0, G{alpha}z, Gs, and the yeast G{alpha}i homolog GPA1, but it does not interact with G{alpha}q, G{alpha}12, and G{alpha}13 and reacts very weakly with the activated mutant, G{alpha}s (Q226L). Interactions were analyzed qualitatively by a colony lift assay on three independent clones using 5-bromo-4-chloro-3-indolyl D-galactoside (14), and the appearance of blue colonies was assessed after 2, 4, and 8 h. No background color was detected after 20 h.

 
BLAST searches with full-length human GIV indicated that there are two additional mammalian family members (Fig. 1): Daple (GenBankTM MP_080957), which is 2009 aa in the mouse, and its human ortholog KIAA1509 (1898 aa), and an uncharacterized human protein FLJ00354(GenBankTM NP_115627 [GenBank] ) (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 (42). Daple acts as a negative regulator of the Wnt signaling pathway by inhibiting Wnt-3a-dependent accumulation of {beta}-catenin and transcriptional activation of Tcf-4 (42). Daple possesses 48% identity and 67% similarity overall to GIV, and FLJ00354shows 36% identity and 58% similarity to GIV. Together, GIV, Daple, and FLJ00354constitute 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 [GenBank] and one in Caenorhabditis elegans (T27075 [GenBank] ), 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 G{alpha}i3—From sequencing and analysis of the three clones isolated from the yeast two-hybrid screen it was evident that GIV interacts with G{alpha}i3 within the 724-aa fragment spanning the C-terminal region of rat GIV (aa 1174–1898) (Figs. 1 and 2A). We further mapped the G{alpha}i3-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 G{alpha}i3. We found that G{alpha}i3 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 G{alpha}i3 in this in vitro binding assay.



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  		FIG. 4.
GIV is widely expressed among tissues and cell lines. A, analysis of a rat multiple tissue Northern blot with a rat GIV probe (aa 1174–1499) shows an 8.5-kb mRNA most highly expressed in the testis (T) with a larger 9-kb transcript in the brain (B), which is also detected in the heart (H), lung (Lu), spleen (Sp), and kidney (K). B, Western blot (20 µg of protein/lane) with affinity-purified GIV IgG (anti-CC, 8.5 µg/ml in TBST + 5% milk) showing that GIV is widely expressed in rat tissues. The ~200 kDa, GIV protein can be detected in all tissues tested, with testis (T), ovary (Ov), and brain (B) having the highest, and heart (H) and lung (Lu) the lowest levels of expression. In brain and ovary, two bands, ~200 and ~180 kDa are detected. Li, liver; St, stomach; Si, small intestine; Sk, skeletal muscle; Th, thymus. (C), expression of GIV in various cell lines. Western blot (30 µg protein/lane) with affinity-purified GIV IgG (anti-CT) showing that GIV is widely expressed. The ~200-kDa protein can be detected in all the cell lines tested. COS7 and PC12 cells have the highest expression and CHO-K1, REF52, and HEK293 cells have the lowest. PC12 and AtT-20 cells also have an additional smaller band at ~180 kDa.

 



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  		FIG. 5.
GIV is found in both membrane and cytosolic fractions. Postnuclear supernatant (PNS) was centrifuged at 100,000 x g to yield membrane pellet (P) and supernatant (S) fractions. Pellets were resuspended in homogenization buffer to the same volume as the supernatants. These fractions (normalized by volume) were separated by 8% SDS-PAGE and immunoblotted with affinity-purified anti-GIV IgG or anti-calnexin (control (CN)) and detected by ECL. GIV is associated primarily with membranes (95%) in HeLa cells, whereas in COS7 cells it is equally divided (50%) between membrane (P) and cytosolic (S) fractions. Calnexin, an integral membrane protein is found in the pellet.

 
A Novel G{alpha}i3 Binding "Motif"—The 83-aa region of human rat GIV that interacts with G{alpha}i3 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 FLJ00354that is most homologous to GIV. Daple is 66% identical and 81% similar to GIV at this G{alpha} binding domain (Fig. 2C). A deletion mutant of Daple containing this region also interacts with G{alpha}i3 in an in vitro binding assay (data not shown). FLJ00354contains a region that also has homology to the G{alpha} binding domain of GIV (48% identity, 80% similarity) (Fig. 2C), but it does not interact with G{alpha}i3 in the same assay or in a two-hybrid 1 to 1 assay (data not shown).

GIV Interacts with the G{alpha}i and G{alpha}s Subfamilies of G Proteins—We used the two-hybrid system to test whether GIV interacts with other G protein {alpha}-subunits. Based on the semiquantitative {beta}-galactosidase filter assay (Fig. 3), GIV interacted with all members of the G{alpha}i subfamily (G{alpha}i1, G{alpha}i2, G{alpha}i3, G{alpha}o, and G{alpha}z) as well as wild-type G{alpha}s and GPA1 (yeast homolog of G{alpha}i) but not with G{alpha}q, G{alpha}12, or G{alpha}13. Interestingly, GIV interacts only weakly with G{alpha}sQ226L (which mimics the GTP-bound form of G{alpha}s) compared with wild-type G{alpha}s in this assay. These results suggest that GIV specifically interacts with members of the G{alpha}i and G{alpha}s subfamilies of heterotrimeric G proteins. We tested for the regulatory activity of GIV on G{alpha}i3 subunits by performing classical single turnover GTPase assays, guanine nucleotide exchange factor assays, and guanine dissociation inhibitor assays (both the latter through GTP{gamma}S loading), but we were unable to show any effects of GIV (data not shown).



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  		FIG. 6.
Localization and deconvolution analysis of GIV in GH3 and COS7 cells. GIV is associated with discrete punctate, vesicular structures scattered throughout the cytoplasm and is most concentrated in the Golgi region where it partially overlaps with {beta}-COP, a marker for COPI ER-Golgi transport vesicles, in both GH3 cells (yellow in C) and COS7 cells (yellow in F). GIV staining also overlaps with the cis Golgi markers calnuc (G) and the KDEL receptor (H) but not with clathrin (I), a marker for clathrin coated vesicles. Cells were fixed in 2% paraformaldehyde, incubated with affinity-purified rabbit anti-GIV IgG (CC) (1:100) and mouse anti-{beta}-COP mAb (1:100), anti-KDEL receptor or chicken anti-calnuc, followed by donkey anti-rabbit Alexa Fluor-594 and Alexa Fluor-488 anti-mouse or anti-chicken F(ab')2 and examined by deconvolution analysis (A–F) or routine immunofluorescence (G–I). Bar = 2 µm.

 
GIV Is Widely Expressed in Mammalian Tissues—A 976-bp fragment spanning the C terminus (aa 1174–1499) of rat GIV with no homology to any other known protein was used to probe a multiple rat tissue Northern blot (Fig. 4A). The highest mRNA expression was detected in the testis and in brain, with lower expression levels in the heart, lung, spleen, and kidney.

To determine GIV protein expression we generated affinity-purified GIV antibodies directed to the central region (anti-GIV (CC)) and to the C terminus (anti-GIV (CT)) of GIV and performed Western blotting on a variety of tissues and cultured cell lines. A specific ~200-kDa band, slightly smaller than the theoretical molecular mass of GIV, was detected in all the tissues tested with the testis, ovary, and brain having the highest expression levels and the heart and lung the lowest (Fig. 4B). In the brain, ovary, and testis an additional, smaller (~180 kDa) band, was detected. We also detected GIV in all cell lines tested with COS7 and PC12 having the highest expression levels and HEK293, CHO-K1, and Ref52 cells the lowest (Fig. 4C). AtT-20 and PC12 (Fig. 3C) cells also express the additional, smaller ~180-kDa band.

GIV Is Distributed in Both Membrane and Cytosolic Fractions—Next we assessed endogenous GIV distribution in membrane (100,000 x g pellet) and cytosolic (100,000 x g supernatant) fractions prepared from COS7 and HeLa cells. GIV was found in both membrane and cytosolic fractions in these cell lines but at different levels. It is 95% membrane-associated in HeLa cells, whereas in COS7 cells it is evenly distributed between cytosol and membranes (Fig. 5).



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  		FIG. 7.
GIV colocalizes with G{alpha}i3-YFP in the cis Golgi region. G{alpha}i3-YFP is localized both on the cell membrane and in the Golgi region where there is a striking overlap with GIV (yellow in C and F). It also colocalizes with the KDEL receptor (G), {beta}-COP (H), and ERGIC53 (I). COS7 cells were transiently transfected with G{alpha}i3-YFP for 24–48 h followed by fixation and preparation for immunofluorescence as in Fig. 6. Incubation was with affinity-purified anti-GIV (CC) (1:50) (A–C) or anti-GIV (CT) (1:100) IgG (D–F) plus one of the following: mouse anti-GFP mAb (1:100) (B, E), rabbit anti-{beta}-COP (1:100) (H), anti-KDEL receptor (G), or rabbit anti-ERGIC53 (I) followed by appropriate secondary antibodies and examined by routine immunofluorescence (G–I) or deconvolution analysis (A–F) as in Fig. 6. Bar = 2 µm.

 
GIV Is Localized on Discrete Punctate Vesicles and to the Golgi Region where It Colocalizes with {beta}-COP, Calnuc, and the KDEL receptor—To determine the intracellular distribution of GIV we carried out immunofluorescence on GH3 pituitary cells and COS7 kidney cells. We performed double immunofluorescence labeling for GIV and markers for the following cell organelles: clathrin-coated vesicles (clathrin, AP2), peroxisomes (PMP70), lysosomes (LAMP-2), caveolae (caveolin), COPI vesicles ({beta}-COP), or Golgi subcompartments (calnuc, KDEL receptor) followed by a routine immunofluorescence or deconvolution analysis. GIV was localized in a discrete punctate pattern distributed throughout the cells and was most concentrated in the Golgi region where it overlapped with {beta}-COP in both GH3 cells (Fig. 6, A–C) and COS7 cells (Fig. 6, D–F). GIV staining also overlapped with the KDEL receptor (Fig. 6H) and calnuc (Fig. 6G), a Ca2+-binding protein that interacts with and colocalizes with G{alpha}i3 in the cis Golgi region (28). Both calnuc and the KDEL receptor (43) are cis Golgi markers. GIV staining did not overlap with any of the other markers tested including clathrin (Fig. 6I), a marker for clathrin-coated vesicles. We concluded that GIV is found on vesicles throughout the cytoplasm but is most concentrated in COPI vesicles in the cis Golgi region.

GIV Codistributes with G{alpha}i3-YFP in the Cis Golgi—Next we set out to test whether GIV colocalizes with G{alpha}i3, which is also found in cis Golgi cisternae (3, 4). We found this is indeed the case, as a striking overlap between G{alpha}i3 and GIV was seen by routine immunofluorescence in COS7 cells transfected with G{alpha}i3-YFP. This was confirmed at higher resolution by deconvolution analysis (Fig. 7, A–F). Like GIV, G{alpha}i3 also codistributes with the KDEL receptor (Fig. 7G), {beta}-COP (Fig. 7H), and ERGIC53 (Fig. 7I) in the Golgi region.

To check on the distribution of GIV and G{alpha}i3 at higher resolution we carried out immunoelectron microscopy and immunogold labeling on ultrathin cryosections. We found that GIV and G{alpha}i3 colocalize on vesicles in the Golgi region that were more concentrated on the cis side of the Golgi (Fig. 8, A and D). GIV also colocalizes with {beta}-COP on COPI vesicles found both in the Golgi region (Fig. 8C) and more peripherally in close association with the ER (Fig. 8, B, E, and F). We conclude that GIV is localized on COPI transport vesicles located in close proximity to ER exit sites or associated with cis Golgi cisternae.



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  		FIG. 8.
GIV is found on COPI vesicles concentrated near the ER or in the Golgi region. A, GIV (small gold) and G{alpha}i3 (large gold) colocalize (arrowheads) on vesicles found on the cis side (cis) of the Golgi stack. B, GIV and {beta}-COP (large gold), a marker for COPI ER-Golgi transport vesicles, colocalize on vesicles (arrows) that are located in close proximity to the endoplasmic reticulum (ER). C, D, and F, vesicles that stain for GIV (small gold) are seen in association with {beta}-COP (C and D) or G{alpha}i3 (D). Other vesicles do not stain for any of these markers. E and F, additional fields illustrating the presence of GIV on vesicles (arrowheads) in close proximity to the ER. COS7 cells were transfected as above and incubated with anti-GIV (CT) IgG and either anti-GFP mAb (A, D) or anti-{beta}-COP mAb (B and C) followed by 5 nm gold, goat anti-rabbit, and 10 nm gold, goat anti-mouse IgG. Bar = 100 nm.

 
GIV Is Concentrated in Carrier Vesicle (CV2)-enriched Fractions from Rat Liver—To obtain biochemical evidence on the localization of GIV we determined the distribution of GIV in fractions prepared from rat liver in which carrier vesicles can be separated from Golgi and other membranes by sucrose gradient centrifugation (37, 38). Golgi light, Golgi heavy, CV1 and CV2, and residual membrane fractions were immunoblotted for GIV along with {beta}-COP, G{alpha}i1, G{alpha}i1/2, G{alpha}i3, and G{alpha}s. GIV was found to be concentrated in CV2 and residual membrane fractions (Fig. 9) and was not detected under these conditions in the Golgi light, Golgi heavy, and CV1 fractions. CV1 and CV2 fractions typically contain a mixture of ER-Golgi transport vesicles, TGN (trans Golgi network)-derived vesicles, transcytotic vesicles, and vesicles derived from early and late endosomes (37, 38). Thus the biochemical evidence together with the localization results support the conclusion that GIV is associated with COPI, ER-Golgi transport vesicles.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have identified a novel G{alpha}i3-interacting protein, which we named GIV, and have mapped the interaction site with G{alpha}i3 to a highly charged, 83-aa domain at the C-terminal end of the coiled coil domain of GIV. We found that GIV is part of a conserved protein family that has three members in mammals: GIV, Daple, and an uncharacterized protein (FLJ00354. The family is characterized by a conserved N terminus with HOOK-domain homology, a large central coiled coil domain showing some homology to the myosin heavy chain, a G{alpha} binding domain, and a C terminus that is divergent. Evolutionary homologs have been identified in A. gambiae, Drosophila, and C. elegans but not in yeast. The 83 amino acids that make up the G{alpha} binding domain is the most conserved region in all species and between family members. No homology was found to other G{alpha}-interacting proteins, and thus this sequence represents a novel G{alpha}-interacting domain. FLJ00354has a domain with high homology to the G{alpha}-interacting domain, but it did not interact with G{alpha}i3 in our assays. Interestingly, this result points to key residues for G{alpha} interaction (see Fig. 2C) or raises the possibility that FLJ00354interacts with different subfamilies of G{alpha} subunits (G{alpha}s, G{alpha}q, G{alpha}12/13).



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  		FIG. 9.
Distribution of GIV and G{alpha} subunits in Rat liver fractions. GIV is concentrated exclusively in a fraction enriched in carrier vesicles (CV2). {beta}-COP is also most concentrated in CV2. G{alpha} subunits are spread more broadly across the gradient but are also most concentrated in CV2. GIV is not detected in Golgi light (GL), Golgi heavy (GH) or CV1 fractions. Rat liver was homogenized, and fractions were prepared by sucrose gradient centrifugation as described under "Experimental Procedures." 40 µg each of Golgi light, Golgi heavy, CV1 and CV2, and residual membrane (RM) fractions were solubilized in Laemmli buffer and immunoblotted for GIV (anti-GIV (CC), 1:1000), {beta}-COP (1:1000), G{alpha}i1 (1:1000), G{alpha}i1/2 (1:1000), G{alpha}i3 (1:1000), and G{alpha}s (1:2000).

 
We found that GIV is broadly expressed and can interact with G{alpha}i1, G{alpha}i2, G{alpha}i3, G{alpha}0, G{alpha}z, and G{alpha}s but not with G{alpha}q or G{alpha}12/13 subunits, suggesting that members of the GIV family may play a broad role in G protein signaling. However, we have been unable so far to demonstrate that GIV has any in vitro GTPase-activating protein, guanine nucleotide exchange factor- or guanine dissociation inhibitor activity for G{alpha}i3.2 This situation is similar to that of calnuc, an EF-hand, Ca2+ binding protein that interacts with G{alpha}i3 in two-hybrid, pull-down (28), and FRET (44) assays. The interaction between calnuc and G{alpha}i3 is not dependent on the activation state of G{alpha}i3 but is Ca2+- and Mg2+-dependent (45). Another similarity between GIV and calnuc which is rather unique is their ability to interact with both G{alpha}i and G{alpha}s subunits in a two-hybrid assay. There is growing evidence that some G{alpha}-interacting proteins have regulatory functions in intracellular processes other than being direct regulators of G{alpha}-GTPase activity or classical effectors in G protein-coupled receptor signaling pathways.

GIV, Daple, and FLJ00354share considerable homology at their N terminus to the microtubule-binding domain of the HOOK family of proteins, which have been proposed to link various organelles to microtubules (46). The fact that Daple and FLJ00354have relatively high levels of homology to the HOOK family in this region suggests that these proteins might also interact with microtubules. However, we have been unable to demonstrate such an interaction in microtubule sedimentation assays. Interestingly, heterotrimeric G proteins of the G{alpha}i and G{alpha}s subclass have been reported to translocate to microtubules upon differentiation of PC12 cells (47).

Another similarity between the GIV and HOOK families is that the family members are homologous throughout the central coiled coil domain but diverge at the C terminus. In the HOOK family it was demonstrated that the N terminus binds to microtubules, and the divergent C terminus binds to specific organelles (46, 48). For example, the C terminus of HOOK3 is responsible for its localization to the Golgi, whereas HOOK1 and HOOK2 localize to discrete subcellular structures that remain elusive and do not colocalize with lysosomal, mitochondrial markers, or other markers tested (46). Like Daple, the HOOK proteins, and myosins, GIV can also homodimerize through its coiled coil domain, which might be an essential part of its function.

Daple, the closest family member to GIV, is a newly characterized protein that contains a PDZ binding motif at its C terminus through which it interacts with Dvl, a protein involved in regulating the Wnt signaling pathway (42). Overexpression of Daple leads to negative regulation of the Wnt pathway. The above findings suggest that the GIV family members may have common functions, including G{alpha} binding, binding to microtubules, and/or the possibility to dimerize via their coiled coil domain but have different cellular locations, probably directed by the C terminus. Identification of proteins that interact with the C terminus of GIV will likely lead to the elucidation of its function.

Using two affinity-purified IgGs made against GIV, we localized GIV to the cis Golgi region where it overlaps with {beta}-COP and the cis Golgi markers G{alpha}i3, calnuc, and the KDEL receptor. {beta}-COP has been shown to be associated with both ER-Golgi transport vesicles and with cis Golgi cisternae (49, 50). Calnuc (28) and the KDEL receptor (43) are concentrated in cis Golgi cisternae and the ER-Golgi intermediate compartment (ERGIC). Based on our immunofluorescence and cell fractionation results, GIV is associated with COPI vesicles, as it colocalizes and codistributes with {beta}-COP, a marker for COPI vesicles. COPI vesicles are well accepted to be involved in retrograde transport of proteins from the Golgi to the ER. They have also been implicated in intraGolgi transport and in anterograde transport between ERGIC and the cis Golgi (51, 52). The recent discovery of distinct subpopulations of COPI vesicles (63) suggests that these vesicles function at several steps in transport both within the Golgi and between the ER and Golgi (51, 52).

Interestingly, over 14 years ago trimeric G proteins were implicated in binding of {beta}-COP and ADP ribosylation factor (ARF) to membranes based on the finding that binding was inhibited by {beta}{gamma}-subunits (53), but this finding was never extended beyond this initial observation. Previous studies have shown that G{alpha}i3 resides in the Golgi in several cell lines and tissues, and by cell fractionation and immunofluorescence techniques we found GIV and G{alpha}i3 co-distribute in the same compartments.

The presence of heterotrimeric G proteins on intracellular membranes has been well documented by work from a number of laboratories (54–57), including our own (4, 5, 44, 58). For example, we (4, 5) and others (57) have localized G{alpha}i3 and G{alpha}s to Golgi membranes in many cell types, including pituitary cells, and others have localized G{alpha}o to secretion granules (7, 59). We (9) and others have also localized G{alpha}s to endosomes. Indirect evidence, based on use of agents such as cholera toxin that activate G{alpha}s subunits and pertussis toxin, which inactivates G{alpha}i subunits, has implicated G proteins in the control of a number of trafficking events along the secretory and endocytic pathways. Of particular interest for the present study, it has been proposed that trimeric G proteins may regulate a variety of steps in vesicle trafficking, including coat assembly (53), budding of vesicles from the ER, ER-Golgi transport (58, 60), budding of secretory granules from the TGN (54, 61), vesicle or granule fusion (55, 56), endosome fusion (62), or protein sorting (9); however, there is little direct evidence to support any of these suggestions. It is not yet known whether G{alpha} subunits are active on intracellular membranes or whether the classical paradigm involving heptahelical receptors, G proteins, and effectors applies, because no specific receptors or effectors have been discovered to date on intracellular membranes (26).

In summary, we have identified a novel family of three proteins, GIV, Daple, and FLJ00354 which share the highest homology at the G{alpha}i/s-interacting domain. The localization and structure of GIV suggest it could provide a link between G{alpha} subunits, COPI vesicles, and microtubules.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK17780 and CA100768 (to M. G. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Dr., George Palade Laboratories of Cellular and Molecular Medicine, La Jolla, CA 92093-0651. Tel.: 858-534-7711; Fax: 858-534-8549; E-mail: mfarquhar{at}ucsd.edu.

1 The 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; GTP{gamma}S, guanosine 5'-O-(thiotriphosphate); CV, carrier vesicle; ERGIC, ER-Golgi intermediate compartment. Back

2 H. Le-Niculescu, I. Niesman, T. Fischer, L. DeVries, and M. G. Farquhar, unpublished results. Back



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