Cloning and Characterization of a Novel Insulin-regulated Membrane Aminopeptidase from Glut4 Vesicles

Abstract

The insulin-regulated glucose transporter isotype GluT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle. The insulin-regulated glucose transporter isotype GluT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4. However, the tissue distribution of vp165 is much wider than that of GluT4; consequently, vp165 may also function in processes unrelated to insulin action and may serve as a ubiquitous marker for a specialized regulated secretory vesicle. INTRODUCTIONAdipose and muscle cells contain a specific type of glucose transporter GluT4(1James D.E. Strube M. Mueckler M. Nature. 1989; 338: 83-87Crossref PubMed Scopus (669) Google Scholar). As determined by immunoelectron microscopy, this isoform is almost exclusively found in intracellular vesicular and tubular structures and the trans-Golgi reticulum in untreated cells(2Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (709) Google Scholar, 3Rodnick K.J. Slot J.W. Studelska D.R. Hanpeter D.E. Robinson L.J. Geuze H.J. James D.E. J. Biol. Chem. 1992; 267: 6278-6285Abstract Full Text PDF PubMed Google Scholar). Insulin treatment leads to the rapid redistribution of GluT4 from this intracellular pool to the cell surface and thus to a large increase in glucose transport (reviewed in (4Birnbaum M.J. Int. Rev. Cytol. 1992; 137: 239-297Crossref PubMed Scopus (117) Google Scholar) and (5James D.E. Piper R.C. J. Cell Biol. 1994; 126: 1123-1126Crossref PubMed Scopus (104) Google Scholar)). An enhanced rate of fusion of the GluT4-containing vesicles with the plasma membrane is most likely largely responsible for this effect(6Holman G.D. Lo Leggio L. Cushman S.W. J. Biol. Chem. 1994; 269: 17516-17524Abstract Full Text PDF PubMed Google Scholar).Unique GluT4 vesicles have been isolated from the low density microsomal fraction of adipocytes and skeletal muscle cells by immunoadsorption and by gel filtration chromatography and sucrose gradient centrifugation, and further characterized(3Rodnick K.J. Slot J.W. Studelska D.R. Hanpeter D.E. Robinson L.J. Geuze H.J. James D.E. J. Biol. Chem. 1992; 267: 6278-6285Abstract Full Text PDF PubMed Google Scholar, 7Kandror K. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar, 8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar, 9Kandror K.V. Coderre L. Pushkin A.V. Pilch P.F. Biochem. J. 1995; 307: 383-390Crossref PubMed Scopus (95) Google Scholar, 10Zorzano A. Wilkinson W. Kotliar N. Thoidis G. Wadzinkski B.E. Ruoho A.E. Pilch P.F. J. Biol. Chem. 1989; 264: 12358-12363Abstract Full Text PDF PubMed Google Scholar). Members of the VAMP and SCAMP family, proteins first described in synaptic and secretory vesicles, respectively, were shown by immunoblotting to be concentrated in these vesicles(11Laurie S.M. Cain C.C. Lienhard G.E. Castle J.D. J. Biol. Chem. 1993; 268: 19110-19117Abstract Full Text PDF PubMed Google Scholar, 12Cain C.C. Trimble W.S. Lienhard G.E. J. Biol. Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar, 13Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar, 14Volchuk A. Sargeant R. Sumitani S. Liu Z. He L. Klip A. J. Biol. Chem. 1995; 270: 8233-8240Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Moreover, several additional polypeptides have been found by protein staining, prominent among which is a protein of molecular mass 165 kDa (referred to herein as vp165 for vesicle protein of 165 kDa)(3Rodnick K.J. Slot J.W. Studelska D.R. Hanpeter D.E. Robinson L.J. Geuze H.J. James D.E. J. Biol. Chem. 1992; 267: 6278-6285Abstract Full Text PDF PubMed Google Scholar, 7Kandror K. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar, 8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar, 9Kandror K.V. Coderre L. Pushkin A.V. Pilch P.F. Biochem. J. 1995; 307: 383-390Crossref PubMed Scopus (95) Google Scholar, 10Zorzano A. Wilkinson W. Kotliar N. Thoidis G. Wadzinkski B.E. Ruoho A.E. Pilch P.F. J. Biol. Chem. 1989; 264: 12358-12363Abstract Full Text PDF PubMed Google Scholar). Another group (Kandror and Pilch(7Kandror K. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar), who designated this protein gp160) and ourselves (8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar) have purified vp165 from rat adipocytes and obtained the sequences of tryptic peptides. Through detection of vp165 with antibodies against two of these peptides, it has been shown that vp165 is concentrated in GluT4 vesicles in basal adipocytes and redistributes to the plasma membrane in response to insulin(8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar, 15Kandror K.V. Pilch P.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8017-8021Crossref PubMed Scopus (158) Google Scholar). Subsequently, while this study was in progress, Kandror et al.(16Kandror K.V. Yu L. Pilch P.F. J. Biol. Chem. 1994; 269: 30777-30780Abstract Full Text PDF PubMed Google Scholar) provided indirect evidence that vp165 is an aminopeptidase. We now report the cloning of vp165 and its definitive characterization as a novel membrane aminopeptidase. INTRODUCTIONAdipose and muscle cells contain a specific type of glucose transporter GluT4(1James D.E. Strube M. Mueckler M. Nature. 1989; 338: 83-87Crossref PubMed Scopus (669) Google Scholar). As determined by immunoelectron microscopy, this isoform is almost exclusively found in intracellular vesicular and tubular structures and the trans-Golgi reticulum in untreated cells(2Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (709) Google Scholar, 3Rodnick K.J. Slot J.W. Studelska D.R. Hanpeter D.E. Robinson L.J. Geuze H.J. James D.E. J. Biol. Chem. 1992; 267: 6278-6285Abstract Full Text PDF PubMed Google Scholar). Insulin treatment leads to the rapid redistribution of GluT4 from this intracellular pool to the cell surface and thus to a large increase in glucose transport (reviewed in (4Birnbaum M.J. Int. Rev. Cytol. 1992; 137: 239-297Crossref PubMed Scopus (117) Google Scholar) and (5James D.E. Piper R.C. J. Cell Biol. 1994; 126: 1123-1126Crossref PubMed Scopus (104) Google Scholar)). An enhanced rate of fusion of the GluT4-containing vesicles with the plasma membrane is most likely largely responsible for this effect(6Holman G.D. Lo Leggio L. Cushman S.W. J. Biol. Chem. 1994; 269: 17516-17524Abstract Full Text PDF PubMed Google Scholar).Unique GluT4 vesicles have been isolated from the low density microsomal fraction of adipocytes and skeletal muscle cells by immunoadsorption and by gel filtration chromatography and sucrose gradient centrifugation, and further characterized(3Rodnick K.J. Slot J.W. Studelska D.R. Hanpeter D.E. Robinson L.J. Geuze H.J. James D.E. J. Biol. Chem. 1992; 267: 6278-6285Abstract Full Text PDF PubMed Google Scholar, 7Kandror K. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar, 8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar, 9Kandror K.V. Coderre L. Pushkin A.V. Pilch P.F. Biochem. J. 1995; 307: 383-390Crossref PubMed Scopus (95) Google Scholar, 10Zorzano A. Wilkinson W. Kotliar N. Thoidis G. Wadzinkski B.E. Ruoho A.E. Pilch P.F. J. Biol. Chem. 1989; 264: 12358-12363Abstract Full Text PDF PubMed Google Scholar). Members of the VAMP and SCAMP family, proteins first described in synaptic and secretory vesicles, respectively, were shown by immunoblotting to be concentrated in these vesicles(11Laurie S.M. Cain C.C. Lienhard G.E. Castle J.D. J. Biol. Chem. 1993; 268: 19110-19117Abstract Full Text PDF PubMed Google Scholar, 12Cain C.C. Trimble W.S. Lienhard G.E. J. Biol. Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar, 13Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar, 14Volchuk A. Sargeant R. Sumitani S. Liu Z. He L. Klip A. J. Biol. Chem. 1995; 270: 8233-8240Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Moreover, several additional polypeptides have been found by protein staining, prominent among which is a protein of molecular mass 165 kDa (referred to herein as vp165 for vesicle protein of 165 kDa)(3Rodnick K.J. Slot J.W. Studelska D.R. Hanpeter D.E. Robinson L.J. Geuze H.J. James D.E. J. Biol. Chem. 1992; 267: 6278-6285Abstract Full Text PDF PubMed Google Scholar, 7Kandror K. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar, 8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar, 9Kandror K.V. Coderre L. Pushkin A.V. Pilch P.F. Biochem. J. 1995; 307: 383-390Crossref PubMed Scopus (95) Google Scholar, 10Zorzano A. Wilkinson W. Kotliar N. Thoidis G. Wadzinkski B.E. Ruoho A.E. Pilch P.F. J. Biol. Chem. 1989; 264: 12358-12363Abstract Full Text PDF PubMed Google Scholar). Another group (Kandror and Pilch(7Kandror K. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar), who designated this protein gp160) and ourselves (8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar) have purified vp165 from rat adipocytes and obtained the sequences of tryptic peptides. Through detection of vp165 with antibodies against two of these peptides, it has been shown that vp165 is concentrated in GluT4 vesicles in basal adipocytes and redistributes to the plasma membrane in response to insulin(8Mastick C.C. Aebersold R. Lienhard G.E. J. Biol. Chem. 1994; 269: 6089-6092Abstract Full Text PDF PubMed Google Scholar, 15Kandror K.V. Pilch P.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8017-8021Crossref PubMed Scopus (158) Google Scholar). Subsequently, while this study was in progress, Kandror et al.(16Kandror K.V. Yu L. Pilch P.F. J. Biol. Chem. 1994; 269: 30777-30780Abstract Full Text PDF PubMed Google Scholar) provided indirect evidence that vp165 is an aminopeptidase. We now report the cloning of vp165 and its definitive characterization as a novel membrane aminopeptidase.