Identification and Characterization of GIV, a Novel Gαi/s -interacting Protein Found on COPI, Endoplasmic Reticulum-Golgi Transport Vesicles
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. 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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. 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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 (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). which serves as a GTPase-activating protein for Gαs and binds through its domain, of the growth factor receptor and links heterotrimeric G protein to trafficking 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, B. Ma Lavoie C. Insel P.A. Farquhar M.G. 2001; PubMed Scopus Google Scholar). LGN and its play an role in the assembly and of the Y. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. A. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which is a in In this report, we the discovery and 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 it colocalizes with β-COP, a marker for COPI, ER-Golgi transport vesicles. heterotrimeric G proteins have been implicated in of ER-Golgi transport 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, J.B. FEBS Lett. PubMed Scopus Google Scholar), but the are not The discovery of a Gα-interacting protein on these transport vesicles a that may the role of Gα subunits in The and of GIV that it may to COPI transport vesicles to Gα subunits and of a rat cell in was as Vries L. M. A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. H. R. McCaffery J.M. M. H. T. Vries L. Farquhar M.G. J. Cell Biol. PubMed Scopus Google Scholar). by and from the or these for the of the amino fluorescent expressed fluorescent carrier ER-Golgi For to rat GIV in was in with the G protein subunits rat and and rat and and by a and the of was and Vries L. Farquhar M.G. 2002; PubMed Scopus Google Scholar). was BLAST BLAST searches the for J. 1996; PubMed Google Scholar). from with the J.D. 1996; PubMed Scopus Google Scholar) for domain and for protein for the of and for the of coiled coil the of GIV was to of GIV from the GIV The was by for of of from rat or to a rat to rat GIV The was by with to a of was for to the and in 15 at for at and of GIV the of proteins, various of rat GIV to and the and proteins affinity-purified from on In rat Gαi3 was Vries L. M. A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In of Gαi3 from the was the in the of to the proteins or was on and with in Gαi3 in binding and as N. M. Y. Insel P.A. FEBS Lett. PubMed Scopus Google Scholar). The was by for at The with binding in of and for and the proteins on and for and from of from the from from from receptor from and from was from the of was by S. J. to N. C. R. T. N. P. G. J. Cell Biol. PubMed Scopus Google Scholar) and D. D. D. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) from to was prepared as A. L. Farquhar M.G. J. Cell Biol. PubMed Scopus Google Scholar), and was by was prepared by Lin of against central region of rat GIV containing a of the coiled coil domain the Gα binding domain and the C terminus of GIV which has to known mammalian proteins, expressed in as proteins, and the proteins and For protein was to to the and with and of affinity-purified GIV by By also endogenous GIV in cell prepared from COS7 and cells. Cell and and cells as by the COS7 cells on and in with T. Lin P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google cells to the and for and immunoelectron as E. Fischer T. T. T. M. Farquhar M.G. Mol. Pharmacol. 2003; PubMed Scopus Google Scholar). of and at and a and rat with and cell and fractions by on and with in for the with IgG or with by with IgG in and by or COS7 cells containing and the at by 15 through a and a was prepared by for at prepared by of the at for at The membrane was in a of to that of the of prepared from rat by and as L. Palade G.E. J. Cell Biol. 1994; PubMed Scopus Google Scholar, M. L. Farquhar M.G. Palade G.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The protein of fraction was by and of protein of fraction was in and by and cells in in for with in and for with or by an with highly or in with and with a for or by with the to a For of with a to the of the was on an and as For at the Vries L. Elenko E. McCaffery J.M. Fischer T. L. T. N. Farquhar M.G. Mol. Biol. Cell. Scholar), cells in in and in at a with an on on at and with by or or IgG in with in with and Identification of proteins that with the heterotrimeric G protein, we a rat pituitary cell as Vries L. M. A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. H. R. McCaffery J.M. M. H. T. Vries L. Farquhar M.G. J. Cell Biol. PubMed Scopus Google Scholar). of in for the of a novel protein BLAST of the nucleotide with this rat revealed to a rat protein of which we GIV The has and the has searches also that are of and GIV have of and based amino acid with a of and GIV a of a of shown in GIV is of coiled coil regions of the protein to to and for GIV a of that are in at which is the of The an that is a coiled which has been shown to R. 1989; PubMed Scopus Google Scholar, R. D. T. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). By to we found that GIV within the coiled coil domain not BLAST searches with GIV that are two additional mammalian family members Daple which is in the and its and an uncharacterized protein from which not a protein, the is the at the C terminus. The to GIV is a protein that with the domain of a protein in the A. S. H. T. T. M. A. 2003; PubMed Scopus Google Scholar). Daple as a of the by of and of A. S. H. T. T. M. A. 2003; PubMed Scopus Google Scholar). Daple and to and and to and a family are on and These proteins share a domain and a central coiled coil domain, diverge at the C terminus The N terminus of members of this family also homology to the domain of the and the coiled coil domain in GIV and Daple homology to family members also several within the coiled coil domain as by GIV also was by to an binding site within the found of this family in in and in with the highest homology to rat is S. The of the for with and of the from the it was evident that GIV with Gαi3 within the the region of rat GIV and mapped the domain by a of and which we in an in binding with various of to and in found that Gαi3 to and and but not to or and the region amino was to with Gαi3 in this in binding Gαi3 region of rat GIV that with Gαi3 is highly C. A. and region of highly charged amino and is also the region in Daple and that is most homologous to Daple is and to GIV at this Gα binding domain of Daple containing this region also with Gαi3 in an in binding not a region that also has homology to the Gα binding domain of GIV but it not with Gαi3 in the or in a to not GIV with the Gαi and Gαs of G the to GIV with G protein on the GIV with members of the Gαi and as well as Gαs and of but not with or GIV with the of Gαs) with Gαs in this These results that GIV with members of the Gαi and Gαs subfamilies of heterotrimeric G proteins. for the regulatory of GIV on Gαi3 subunits by classical guanine nucleotide exchange factor and guanine dissociation the through but we to effects of GIV not GIV in the C terminus of rat GIV with homology to known protein was to a rat The highest was in the and in with in the and GIV protein we affinity-purified GIV to the central region and to the C terminus of GIV and on a variety of and cell the of was in the with the and the highest and the and the In the and an was also GIV in cell with COS7 and the highest and and cells the and cells also the GIV in and we endogenous GIV in membrane and fractions prepared from COS7 and cells. GIV was found in membrane and fractions in these cell but at different is in in COS7 cells it is between and GIV on and to the Golgi with β-COP, and the KDEL the intracellular of GIV we on GH3 pituitary cells and COS7 cells. for GIV and markers for the cell vesicles COPI vesicles or Golgi KDEL by a or GIV was localized in a the cells and was most concentrated in the Golgi region it with β-COP in GH3 cells and COS7 cells GIV also with the KDEL receptor and calnuc a protein that with and colocalizes with Gαi3 in the cis Golgi region P. H. R. McCaffery J.M. M. H. T. Vries L. Farquhar M.G. J. Cell Biol. PubMed Scopus Google Scholar). calnuc and the KDEL receptor J. J. D. J. J. Cell Biol. PubMed Scopus Google Scholar) are cis Golgi GIV not with of the markers including a marker for vesicles. that GIV is found on vesicles the but is most concentrated in COPI vesicles in the cis Golgi GIV with in the we to GIV colocalizes with which is also found in cis Golgi 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). found this is the as a between Gαi3 and GIV was by in COS7 cells with was at by Gαi3 also codistributes with the KDEL receptor β-COP and in the Golgi on the of GIV and Gαi3 at we immunoelectron and on found that GIV and Gαi3 on vesicles in the Golgi region that concentrated on the cis of the Golgi and GIV also colocalizes with β-COP on COPI vesicles found in the Golgi region and in close with the ER and that GIV is localized on COPI transport vesicles in close proximity to ER exit sites or associated with cis Golgi cisternae. GIV in from on the of GIV we the of GIV in fractions prepared from rat in which carrier vesicles from Golgi and by L. Palade G.E. J. Cell Biol. 1994; PubMed Scopus Google Scholar, M. L. Farquhar M.G. Palade G.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Golgi Golgi and and membrane fractions for GIV with β-COP, and GIV was found to concentrated in and membrane fractions and was not these in the Golgi Golgi and and fractions a of ER-Golgi transport Golgi and vesicles from and endosomes L. Palade G.E. J. Cell Biol. 1994; PubMed Scopus Google Scholar, M. L. Farquhar M.G. Palade G.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the with the results the that GIV is associated with COPI, ER-Golgi transport vesicles. In this we have identified a novel protein, which we and have mapped the interaction site with Gαi3 to a highly domain at the of the coiled coil domain of found that GIV is of a protein family that has members in and an uncharacterized protein The family is by a N terminus with a central coiled coil domain homology to the a Gα binding domain, and a C terminus that is homologs have been identified in A. and C. but not in The amino that the Gα binding domain is the most region in and between family homology was found to Gα-interacting proteins, and this a novel Gα-interacting has a domain with homology to the Gα-interacting domain, but it not with Gαi3 in this to for Gα interaction or the that with different subfamilies of Gα subunits found that GIV is expressed and with and Gαs but not with or that members of the GIV family may play a role in G protein signaling. we have been to demonstrate that GIV has in GTPase-activating protein, guanine nucleotide exchange or guanine dissociation for T. L. and M. G. is to that of an binding protein that with Gαi3 in P. H. R. McCaffery J.M. M. H. T. Vries L. Farquhar M.G. J. Cell Biol. PubMed Scopus Google Scholar), and T. Lin P. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar) The interaction between calnuc and Gαi3 is not on the of Gαi3 but is and P. Y. R. Farquhar M.G. J. Cell Biol. 1999; PubMed Scopus Google Scholar). between GIV and calnuc which is is to with Gαi and Gαs subunits in a is that Gα-interacting proteins have regulatory in intracellular processes of or classical effectors in G receptor and share homology at N terminus to the domain of the family of proteins, which have been to various organelles to H. J. Cell Biol. 2001; PubMed Scopus Google Scholar). The that Daple and have of homology to the family in this region that these proteins also with we have been to demonstrate such an interaction in heterotrimeric G proteins of the Gαi and Gαs have been to to of cells T. T. Rasenick M.M. J. 2003; PubMed Scopus Google Scholar). between the GIV and is that the family members are homologous the central coiled coil domain but diverge at the C terminus. In the family it was that the N terminus binds to and the C terminus binds to organelles H. J. Cell Biol. 2001; PubMed Scopus Google Scholar, A. H. Mol. Biol. Cell. 1999; PubMed Scopus Google Scholar). For example, the C terminus of is for its to the and to that and not with or markers H. J. Cell Biol. 2001; PubMed Scopus Google Scholar). the proteins, and GIV also through its coiled coil domain, which an of its the family to is a protein that a binding at its C terminus through which it with a protein in regulating the A. S. H. T. T. M. A. 2003; PubMed Scopus Google Scholar). of Daple to of the The that the GIV family members may have including Gα binding to and/or the to coiled coil domain but have different by the C terminus. Identification of proteins that with the C terminus of GIV to the of its Using two affinity-purified made against we localized GIV to the cis Golgi region it with β-COP and the cis Golgi markers and the KDEL β-COP has been shown to associated with ER-Golgi transport vesicles and with cis Golgi McCaffery M. Palade G.E. Farquhar M.G. Mol. Biol. Cell. PubMed Scopus Google Scholar, A. R. J. Cell Biol. PubMed Scopus Google Scholar). P. H. R. McCaffery J.M. M. H. T. Vries L. Farquhar M.G. J. Cell Biol. PubMed Scopus Google Scholar) and the KDEL receptor J. J. D. J. J. Cell Biol. PubMed Scopus Google Scholar) are concentrated in cis Golgi and the ER-Golgi on and cell GIV is associated with COPI as it colocalizes and codistributes with β-COP, a marker for COPI vesicles. COPI vesicles are well to in transport of proteins from the Golgi to the have also been implicated in transport and in transport between and the cis Golgi R. Mol. 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