Identification and Characterization of Human Glucose Transporter-like Protein-9 (GLUT9)

The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of expressed sequence tags, we have cloned an alternative splice variant of GLUT9 from human kidney cDNA. The RNA of this splice variant consists of 13 exons and codes for a putative protein of 512 amino acids (GLUT9ΔN). The predicted proteins differ only in their N terminus, suggesting a different subcellular localization and possible physiological role. Screening human tissue RNA by reverse transcription-PCR showed that GLUT9 is expressed mainly in kidney, liver, placenta, and leukocytes, whereas GLUT9ΔN was detected only in kidney and placenta. The GLUT9 protein localized by immunohistochemistry to human kidney proximal tubules, and subcellular fractionation of human kidney revealed the GLUT9 protein in plasma membranes and high density microsomal membranes. Treatment of kidney membrane proteins with peptide N-glycosidase F showed that GLUT9 and GLUT9ΔN are expressed in vivo. Localization of GLUT9 and GLUT9ΔN in three kidney-derived cell lines revealed a plasma membrane distribution for GLUT9 in COS-7 and HEK293 cells, whereas GLUT9ΔN showed a perinuclear pattern and plasma membrane staining in COS-7 and HEK293 cells, respectively. In polarized Madin-Darby canine kidney cells, GLUT9 trafficked to the basolateral membrane, whereas GLUT9ΔN localized to the apical membrane. Using heterologous expression of GLUT9 in Xenopus oocytes, GLUT9 appears to be a functional isoform with low affinity for deoxyglucose. Deoxyglucose transport mediated by GLUT9 was not inhibited by cytochalasin B. GLUT9 did not bind cytochalasin B as shown by a cytochalasin B binding assay, indicating a similar behavior of GLUT9 compared with GLUT5. The recently cloned human GLUT9 gene, which maps to chromosome 4p15.3-p16, consists of 12 exons coding for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of expressed sequence tags, we have cloned an alternative splice variant of GLUT9 from human kidney cDNA. The RNA of this splice variant consists of 13 exons and codes for a putative protein of 512 amino acids (GLUT9ΔN). The predicted proteins differ only in their N terminus, suggesting a different subcellular localization and possible physiological role. Screening human tissue RNA by reverse transcription-PCR showed that GLUT9 is expressed mainly in kidney, liver, placenta, and leukocytes, whereas GLUT9ΔN was detected only in kidney and placenta. The GLUT9 protein localized by immunohistochemistry to human kidney proximal tubules, and subcellular fractionation of human kidney revealed the GLUT9 protein in plasma membranes and high density microsomal membranes. Treatment of kidney membrane proteins with peptide N-glycosidase F showed that GLUT9 and GLUT9ΔN are expressed in vivo. Localization of GLUT9 and GLUT9ΔN in three kidney-derived cell lines revealed a plasma membrane distribution for GLUT9 in COS-7 and HEK293 cells, whereas GLUT9ΔN showed a perinuclear pattern and plasma membrane staining in COS-7 and HEK293 cells, respectively. In polarized Madin-Darby canine kidney cells, GLUT9 trafficked to the basolateral membrane, whereas GLUT9ΔN localized to the apical membrane. Using heterologous expression of GLUT9 in Xenopus oocytes, GLUT9 appears to be a functional isoform with low affinity for deoxyglucose. Deoxyglucose transport mediated by GLUT9 was not inhibited by cytochalasin B. GLUT9 did not bind cytochalasin B as shown by a cytochalasin B binding assay, indicating a similar behavior of GLUT9 compared with GLUT5. Transport of hexoses across plasma membranes of mammalian cells is mediated by active as well as passive mechanisms, represented by the protein families of Na+ glucose symporters (1Wood I.S. Trayhurn P. Br. J. Nutr. 2003; 89: 3-9Crossref PubMed Scopus (687) Google Scholar) and facilitative glucose carriers (2Joost H.G. Bell G.I. Best J.D. Birnbaum M.J. Charron M.J. Chen Y.T. Doege H. James D.E. Lodish H.F. Moley K.H. Moley J.F. Mueckler M. Rogers S. Schurmann A. Seino S. Thorens B. Am. J. Physiol. 2002; 282: E974-E976Crossref PubMed Scopus (332) Google Scholar, 3Joost H.G. Thorens B. Mol. Membr. Biol. 2001; 18: 247-256Crossref PubMed Scopus (572) Google Scholar), respectively. The family of facilitative transporters currently comprises 14 isoforms that differ in their tissue distribution, kinetic properties, and substrate specificity. A common structural feature shared among all members of the glucose transporter family (SLC2A family) is the presence of 12 transmembrane helices with N and C termini facing the cytoplasm and an N-linked glycosylation site. Highly conserved motifs, also referred to as sugar transporter signatures, are located in helices 6 and 12 (PESPR/PETK) and loops 2 and 8 (GRR/GRK) and further specify the sequence characteristics of SLC2A family members. Amino acid residues that are required for glucose transport/function as well as binding of the specific transport inhibitor cytochalasin B have been identified by site-directed mutagenesis analysis of GLUT1, the most intensively characterized isoform (4Hruz P.W. Mueckler M.M. Mol. Membr. Biol. 2001; 18: 183-193Crossref PubMed Scopus (139) Google Scholar). Previous studies suggested the presence of additional isoforms (5Katz E.B. Stenbit A.E. Hatton K. DePinho R. Charron M.J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (396) Google Scholar, 6Kayano T. Burant C.F. Fukumoto H. Gould G.W. Fan Y.S. Eddy R.L. Byers M.G. Shows T.B. Seino S. Bell G.I. J. Biol. Chem. 1990; 265: 13276-13282Abstract Full Text PDF PubMed Google Scholar), and the availability of expressed sequence tag data bases and the sequencing and mapping of the human genome allowed the identification of several new members during the last few years, leading to a rather complex picture of glucose/hexose transport and whole body sugar homeostasis. To date, only few functional data are available for the new isoforms. However, based on sequence characteristics and phylogenetic alignments, three subclasses in the family can be distinguished (7Uldry M. Thorens B. Pfluegers Arch. 2003; 447: 480-489Crossref PubMed Scopus (387) Google Scholar). This suggests a broader range of possible substrates and transport characteristics among these new isoforms that is not restricted to hexoses, as shown by the cloning of the H+/myo-inositol transporter isoform or GLUT13 (8Uldry M. Ibberson M. Horisberger J.D. Chatton J.Y. Riederer B.M. Thorens B. EMBO J. 2001; 20: 4467-4477Crossref PubMed Scopus (185) Google Scholar). Here, we describe the characterization of human GLUT9 (9Phay J.E. Hussain H.B. Moley J.F. Genomics. 2000; 66: 217-220Crossref PubMed Scopus (173) Google Scholar) and demonstrate that alternative splicing leads to differential targeting, suggesting possible new mechanisms in the regulation of hexose transport in mammalian cells. RNA Extraction, Reverse Transcription-PCR, and PCR Cloning Total RNA from human tissues was prepared using TRIzol® reagent (Invitrogen) according to the manufacturer's instructions. DNase (DNA-free™, Ambion Inc., Austin, TX)-treated total RNA was primed with random hexamers (Roche Applied Science) and reverse-transcribed using Superscript™ (Invitrogen). The human GLUT9 splice variant (GLUT9ΔN) was cloned by reverse transcription-PCR using Klentaq LA polymerase (Wayne Barnes, Washington University) from human kidney cDNA based on the expressed sequence tag clone IMAGE:3949549 and NCBI accession number BC018897. The GLUT9ΔN coding sequence was amplified using the following primers: 5′-CGG GGT ACC CCC GCC ATG AAG CTC AGT AAA AAG GAC-3′ and 5′-GCC CTC TAG ATT AAG GCC TTC CAT TTA TCT TAC CAT. The PCR primers included the restriction sites for KpnI and XbaI for subsequent cloning into pcDNA3.1+ (Invitrogen). The resulting GLUT9ΔN clone was confirmed by bidirectional sequencing. To study the expression pattern of GLUT9 splice variants in human tissues, cDNAs were amplified with GLUT9- and GLUT9ΔN-specific forward primers (5′-ACT GAG ACC CAT GGC AAG GAA A-3′ and 5′-ATG AAG CTC AGT AAA AAG GAC-3′, respectively). A common reverse primer for both splice variants was used (5′-GAG TGT CTG GGT CTA TTG GA-3′), resulting in PCR amplicons of 326 and 229 bp for GLUT9 and GLUT9ΔN, respectively. To ensure that an equal amount of cDNA was used for PCR amplification from different tissues, the housekeeping gene actin was amplified as an internal standard at a linear range of the PCR (forward primer, 5′-TGC GTG ACA TTA AGG AGA AG-3′ and reverse primer, 5′-CTG CAT CCT GTC GGC AAT G-3′). Human kidney biopsy samples were homogenized in buffer A (20 mm Tris-HCl, 1 mm EDTA, and 255 mm sucrose, pH 7.4) containing a protease inhibitor mixture (Sigma) using a Potter-Elvehjem tissue grinder (Kimble/Kontes, Vineland, NJ). Nuclei, mitochondria, and plasma membranes were obtained by centrifugation at 14,000 × g for 15 min (Beckman rotor JA-20, Beckman Coulter, Fullerton, CA). The resulting pellet was resuspended in buffer A, homogenized, and layered on a 38.7% sucrose cushion in Tris/EDTA. After centrifugation at 100,000 × g for 1 h, the plasma membrane containing interphase was pelleted by centrifugation at 20,000 × g for 1 h. High density microsomes were obtained by centrifugation from the first supernatant at 50,000 × g for 1 h. The second supernatant was centrifuged at 200,000 × g for 75 min to sediment the low density microsomal membrane fraction. Membrane pellets were resuspended in homogenization buffer and stored at -80 °C prior to further analysis. For preparation of membrane fractions from cell lines stably expressing GLUT9, cells were washed twice with phosphate-buffered saline (PBS). 1The abbreviations used are: PBS, phosphate-buffered saline; MDCK, Madin-Darby canine kidney; BSA, bovine serum albumin. After rinsing in cold (4 °C) buffer A, cells were scraped into buffer A containing proteinase inhibitors. All subsequent steps were carried out as described above. Plasma membrane and high and low density microsomal protein fractions were separated on 10% polyacrylamide gels, transferred onto nitrocellulose, blocked with 5% dry milk in Tris-buffered saline/Tween 20, and probed with an antibody raised against a C-terminal peptide of human GLUT9 (KIDSAVTDGKINGRP). For detection of GLUT9 in human kidney membrane extracts, IgG-purified antiserum (HiTrap™ IgG, Amersham Biosciences) was applied at a concentration of 5 μg/ml (in 1% dry milk in Tris-buffered saline/Tween 20). Western blots were probed with a horseradish peroxidase-coupled goat anti-rabbit secondary antibody (Pierce) and developed using the SuperSignal Dura Western kit (Pierce). For detection of human GLUT9 in overexpressing cell lines, anti-human GLUT9 serum (1:1000) and enhanced chemiluminescence (ECL, Amersham Biosciences) were used. To determine the extent of glycosylation and the molecular masses of human GLUT9 and GLUT9ΔN, 10 μg of plasma membrane extracts from transfected HEK293 cells were treated for 1 h at 37 °C with peptide N-glycosidase F (New England Biolabs Inc., Beverly, MA) following the manufacturer's instructions. After enzyme incubation, the reaction was stopped adding SDS-PAGE loading buffer, and Western blot analysis was carried out as described above. To show in vivo expression of GLUT9 and GLUT9ΔN, 20 μg of human kidney high density microsomes were treated with peptide N-glycosidase F, and the protein samples were separated on a standard 10% SDS-polyacrylamide gel (20 × 20 cm). Western blotting was carried out as described above, and combined plasma membrane fractions from GLUT9- and GLUT9ΔN-transfected HEK293 cells were used as positive controls. Three kidney-derived cell lines were used to investigate the localization of human GLUT9 and GLUT9ΔN. COS-7, HEK293, and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's with and 10% to cells were and for h, and was carried out using 6 (Roche Applied Science) according to the manufacturer's instructions. transfected cells were for expression by and Western blotting h expression was by the cells h into with 1 or for HEK293 and cells, respectively. GLUT9- and cell lines were obtained by To investigate the localization of GLUT9 and GLUT9ΔN in polarized cells, expressing cells were at high density × onto cell number Biosciences) and for 5 After human kidney were in 10 mm buffer, pH for using a After with PBS, antibody binding was blocked for 1 h with goat serum and bovine serum in IgG-purified human GLUT9 antiserum in the buffer was applied at After with PBS, were developed using an secondary antibody kit Inc., CA). were with IgG-purified serum was used as a of human kidney were used for staining of stored at -80 °C were to °C for 20 by at with in After with PBS, were blocked with in for min and to IgG-purified antibody for 1 h at GLUT9 staining was detected using a goat anti-rabbit antibody Inc., and (Sigma) were used to and actin respectively. were in and by using a with an or stably transfected cells expressing GLUT9 and GLUT9ΔN were on in the of polarized cells, on membranes. were washed twice with PBS, for 10 min in and by three with mm in were with for by three with PBS, and antiserum binding was blocked with in for were for 1 h with GLUT9 antiserum in BSA, washed with PBS, and probed with goat anti-rabbit antibody in in were washed with PBS, and were with for 10 After a cells were using cells on membranes from cell were using were by using a or into Xenopus transport by GLUT9 was as of into Xenopus Transport were carried out as described in by K. M. Mueckler M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and were with of RNA obtained from in and GLUT9 cDNAs cloned into the was a were with of in and 1 in for min at in the presence or of cytochalasin B. was stopped by three with with were in 1% and with and was were as of min and compared with GLUT9 expression in Xenopus was by Western blot analysis of total membrane Total membrane proteins were obtained by in 1 of buffer A by 10 a on a and an centrifugation at × g for 10 membranes were pelleted from the supernatant at 100,000 × g for To determine the localization of GLUT9 in Xenopus oocytes, whole was carried staining of was as described for cell were on using to the cells B of cytochalasin B to GLUT9 was by a cytochalasin B binding Plasma membrane extracts from and HEK293 cells were as described A. Doege H. H. A. H.G. PubMed Scopus Google Scholar). binding was by adding 15 μg of plasma membranes of to a reaction mixture of of 20 mm Tris-HCl, pH of cytochalasin and 20 of mixture B and were on for 10 and membranes were pelleted at × g for The supernatant was and proteins were in of tissue (Beckman After adding 10 of mixture (Beckman samples were in a for and for cytochalasin B were using were by to determine using and of Human GLUT9 human GLUT9 gene, which maps to chromosome 4p15.3-p16, codes for alternative The GLUT9 gene described by (9Phay J.E. Hussain H.B. Moley J.F. Genomics. 2000; 66: 217-220Crossref PubMed Scopus (173) Google Scholar) consists of 12 exons of the GLUT9 gene and codes for a 540-amino acid protein. Based on a sequence entry (NCBI accession number BC018897) and screening of human expressed sequence tags, we cloned an alternative splice variant of GLUT9 from human kidney referred to as GLUT9ΔN. The GLUT9ΔN splice variant consists of 13 exons of the GLUT9 gene and codes for a putative protein of 512 amino The predicted proteins differ only in their N termini The alternative splicing of the GLUT9 gene suggests that GLUT9 and GLUT9ΔN are by different that are of the 1 2002; Google Scholar). protein of the currently protein showed that GLUT9 to the family of facilitative sugar transporters of the protein sequence for GLUT9 showed several of the glucose transporter family as the and in helices and loops respectively. the members of the glucose transporter GLUT9 the in of and the to in GLUT9 not and at the to amino acids and in GLUT1, respectively. A structural feature of GLUT9 is the of residues in the with and in and the of with in for in and in are to and respectively. A specific of the family transporters is a in in the last to amino acids in Amino acids that were characterized to be in glucose transport and cytochalasin B binding by (4Hruz P.W. Mueckler M.M. Mol. Membr. Biol. 2001; 18: 183-193Crossref PubMed Scopus (139) Google Scholar) differ in GLUT9 as well as in and and of a of the human family members. All currently human glucose transporter proteins were using the NCBI accession number and hexose from NCBI accession number were included in the The three of glucose transporter are of human glucose transporter amino acid residues that have been characterized as or for (4Hruz P.W. Mueckler M.M. Mol. Membr. Biol. 2001; 18: 183-193Crossref PubMed Scopus (139) Google Scholar) of human glucose transporter amino acid residues that have been characterized as or for (4Hruz P.W. Mueckler M.M. Mol. Membr. Biol. 2001; 18: 183-193Crossref PubMed Scopus (139) Google in a new of GLUT9 RNA and in Human GLUT9 RNA was expressed mainly in liver, kidney, and and to a extent in leukocytes, and In for GLUT9ΔN was detected only in kidney and To demonstrate expression of the GLUT9 protein in human tissues, we Western blot analysis of kidney membrane fractions using IgG-purified GLUT9 A specific was detected in plasma membrane and high density microsomal fractions in the range of was detected membranes with Western blot analysis of kidney membrane fractions treated with peptide N-glycosidase F showed at the molecular masses of GLUT9 and GLUT9ΔN indicating that both of GLUT9 are expressed as proteins in human To determine the localization of human GLUT9 in human tissues, we immunohistochemistry on as well as kidney using an detection and respectively. were to show that GLUT9 is expressed in the proximal A and staining revealed that GLUT9 localized to the basolateral membrane in proximal cells and Localization of Human GLUT9 and GLUT9ΔN in three different kidney-derived cell lines HEK293, and we the of the different N termini of human GLUT9 and GLUT9ΔN on their subcellular first the of GLUT9 antiserum with the GLUT9 and GLUT9ΔN proteins in HEK293 cells. shown in GLUT9 and GLUT9ΔN were detected in plasma membrane and high density microsomal fractions of transfected cells, whereas cells did not show in the Treatment of plasma membrane fractions with peptide N-glycosidase F revealed that GLUT9 and GLUT9ΔN were as shown by the in their molecular masses from a to for GLUT9 and a from to for GLUT9ΔN To study the subcellular localization of GLUT9 and GLUT9ΔN, we transfected cells by In all three cell lines, GLUT9 localized to the plasma membrane whereas a cell pattern was for GLUT9ΔN. In COS-7 cells, GLUT9ΔN whereas in HEK293 cells, GLUT9ΔN localized to the plasma membrane In in transfected cells, GLUT9ΔN showed a pattern and as well as plasma membrane GLUT9 is localized in human kidney proximal cells, we GLUT9 and GLUT9ΔN are in polarized cells, a well cell for the study of basolateral apical of of polarized cells stably expressing GLUT9 or GLUT9ΔN showed a in localization for the GLUT9 was in the basolateral membrane, GLUT9ΔN trafficked to the apical membrane of polarized cells of GLUT9 expression of GLUT9 in Xenopus in a in compared with The transport mediated by GLUT9 was not inhibited by cytochalasin B. with oocytes, GLUT9 showed a of that was not inhibited by cytochalasin B staining of Xenopus showed plasma membrane localization of GLUT9 To further the functional of GLUT9, we a cytochalasin B binding to determine GLUT9 cytochalasin B. Plasma membrane proteins from and HEK293 cells were with cytochalasin and binding was by with of the in plasma membranes did not bind cytochalasin B and showed in the binding compared with membranes from cells. The binding obtained for by revealed a of from three with which is in the range of data H. A. A. H. J. H.G. Schurmann A. J. 2001; PubMed Scopus Google Scholar). GLUT9 is a transporter protein that to of the glucose transporter GLUT9 structural that are conserved among all glucose transporter isoforms. the and helices 6 and 12 and the and in loops 2 and respectively. A all human glucose transporter isoforms that the of GLUT9 are and of amino acid residues in all isoforms that were to be for glucose and cytochalasin B binding (4Hruz P.W. Mueckler M.M. Mol. Membr. Biol. 2001; 18: 183-193Crossref PubMed Scopus (139) Google Scholar) that members of the for residues characterization of GLUT9 that GLUT9 is a functional with a affinity compared with This transport was not by cytochalasin and using a binding assay, we showed that GLUT9 did not bind cytochalasin B. GLUT9 is to the plasma membrane in Xenopus and is not The of the in GLUT9 be for the low transport for similar to well characterized C.F. J. Bell G.I. J. Biol. Chem. Full Text PDF PubMed Google Scholar). described for the transport of is not inhibited by cytochalasin B C.F. J. Bell G.I. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the transporter is not of binding cytochalasin B K. H. K. T. M. H. M. M. 1995; PubMed Scopus Google Scholar). recently characterized of the transporter family glucose only at high glucose by of the and the transport is by suggesting that transport H. A. A. H. J. H.G. Schurmann A. J. 2001; PubMed Scopus Google Scholar). a for cytochalasin B binding compared with H. A. A. H. J. H.G. Schurmann A. J. 2001; PubMed Scopus Google Scholar). data that transporters similar functional characteristics and to that hexose glucose be the substrate of studies on further the transport of A specific feature of GLUT9, only for A. Mol. 2002; PubMed Scopus Google Scholar, T. S. A. A. A. S. K. 2001; PubMed Scopus Google Scholar) and Genomics. 2002; PubMed Scopus Google Scholar), is the presence of alternative splice variants expressed in a for and alternative splicing of GLUT9 in proteins that differ only in their Using an antibody against the C-terminal of GLUT9 that not GLUT9 and GLUT9ΔN, we showed that GLUT9 was expressed in human kidney proximal tubules, with a staining of basolateral membranes. the expression of GLUT9 to for basolateral Western blot analysis of peptide N-glycosidase human kidney membrane fractions showed GLUT9 and GLUT9ΔN. the are expressed as proteins in human the localization of GLUT9 and GLUT9ΔN in three kidney-derived cell lines showed that GLUT9 trafficked to the plasma membrane in COS-7, HEK293, and cells. In GLUT9ΔN was in COS-7 cells, whereas plasma membrane localization was in HEK293 cells in cells. COS-7 cells are HEK293 and cells are indicating that GLUT9ΔN a that plasma membrane in cells. overexpressing both splice variants in polarized cells, we showed that GLUT9 and GLUT9ΔN were to apical basolateral respectively. the in GLUT9 not the protein in an as for and Schurmann A. H.G. H. J. 2001; PubMed Scopus Google Scholar, M. M. Thorens B. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, S. James D.E. Best J.D. Am. J. Physiol. 2002; 282: PubMed Google Scholar, S. Mol. Membr. Biol. 2001; 18: PubMed Scopus Google Scholar), indicating that of GLUT9 plasma membrane the in GLUT9 be for basolateral expression in cells, as for proteins this T. P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. P. PubMed Scopus Google Scholar, EMBO J. PubMed Scopus Google Scholar). However, residues the be in basolateral of glucose transporters in polarized cells was shown to be in an K. James D.E. Am. J. Physiol. PubMed Google Scholar). Here, we have described for the first that alternative splicing of a glucose transporter isoform in differential targeting, a that been described for membrane proteins B. K. S. J. PubMed Google Scholar) and transporters J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Based on the characteristics of the glucose transporter family members with to their and expression as well as their functional properties, these isoforms be in transport of hexoses, in polarized cells and in cells. data a of regulation of hexose transport in cells by alternative resulting in differential Doege for the for the cytochalasin B binding assay, of and Washington University) for the kidney and Washington University) for the staining of GLUT9 in human the J. at Washington of and the for the of the that human RNA and tissue and for and and Mueckler for of the