GLUT8, a Novel Member of the Sugar Transport Facilitator Family with Glucose Transport Activity

GLUT8 is a novel glucose transporter-like protein that exhibits significant sequence similarity with the members of the sugar transport facilitator family (29.4% of amino acids identical with GLUT1). Human and mouse sequence (86.2% identical amino acids) comprise 12 putative membrane-spanning helices and several conserved motifs (sugar transporter signatures), which have previously been shown to be essential for transport activity, e.g. GRK in loop 2, PETPR in loop 6, QQLSGVN in helix 7, DRAGRR in loop 8, GWGPIPW in helix 10, and PETKG in the C-terminal tail. An expressed sequence tag (STS A005N15) corresponding with the 3′-untranslated region of GLUT8 has previously been mapped to human chromosome 9. COS-7 cells transfected with GLUT8 cDNA expressed a 42-kDa protein exhibiting specific, glucose-inhibitable cytochalasin B binding (KD = 56.6 ± 18 nm) and reconstitutable glucose transport activity (8.1 ± 1.4 nmol/(mg protein × 10 s)versus 1.1 ± 0.1 in control transfections). In human tissues, a 2.4-kilobase pair transcript was predominantly found in testis, but not in testicular carcinoma. Lower amounts of the mRNA were detected in most other tissues including skeletal muscle, heart, small intestine, and brain. GLUT8 mRNA was found in testis from adult, but not from prepubertal rats; its expression in human testis was suppressed by estrogen treatment. It is concluded that GLUT8 is a sugar transport facilitator with glucose transport activity and a hormonally regulated testicular function. GLUT8 is a novel glucose transporter-like protein that exhibits significant sequence similarity with the members of the sugar transport facilitator family (29.4% of amino acids identical with GLUT1). Human and mouse sequence (86.2% identical amino acids) comprise 12 putative membrane-spanning helices and several conserved motifs (sugar transporter signatures), which have previously been shown to be essential for transport activity, e.g. GRK in loop 2, PETPR in loop 6, QQLSGVN in helix 7, DRAGRR in loop 8, GWGPIPW in helix 10, and PETKG in the C-terminal tail. An expressed sequence tag (STS A005N15) corresponding with the 3′-untranslated region of GLUT8 has previously been mapped to human chromosome 9. COS-7 cells transfected with GLUT8 cDNA expressed a 42-kDa protein exhibiting specific, glucose-inhibitable cytochalasin B binding (KD = 56.6 ± 18 nm) and reconstitutable glucose transport activity (8.1 ± 1.4 nmol/(mg protein × 10 s)versus 1.1 ± 0.1 in control transfections). In human tissues, a 2.4-kilobase pair transcript was predominantly found in testis, but not in testicular carcinoma. Lower amounts of the mRNA were detected in most other tissues including skeletal muscle, heart, small intestine, and brain. GLUT8 mRNA was found in testis from adult, but not from prepubertal rats; its expression in human testis was suppressed by estrogen treatment. It is concluded that GLUT8 is a sugar transport facilitator with glucose transport activity and a hormonally regulated testicular function. glucose transporter expressed sequence tag rapid amplification of cDNA ends polymerase chain reaction Hexose transport into mammalian cells is catalyzed by the members of a small family of 45–55-kDa membrane proteins, GLUT1–GLUT5 (1.Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1136) Google Scholar, 2.Mueckler M. Diabetes. 1990; 39: 6-11Crossref PubMed Google Scholar, 3.Bell G. Kayano T. Buse J. Burant C. Takeda J. Lin D. Fukumoto H. Seino S. Diabetes Care. 1990; 13: 198-206Crossref PubMed Scopus (670) Google Scholar, 4.Gould G.W. Holman G.D. Biochem. J. 1993; 295: 329-341Crossref PubMed Scopus (652) Google Scholar). These hexose transporters belong to the larger family of transport facilitators, which comprises yeast hexose transporters, plant hexose-proton symporters, bacterial sugar-proton symporters (5.Marger M.D. Saier M.H. Trends Biochem. Sci. 1993; 18: 13-20Abstract Full Text PDF PubMed Scopus (747) Google Scholar, 6.Sauer N. Tanner W. Botanica Acta. 1993; 106: 277-286Crossref Scopus (39) Google Scholar), and organic anion as well as organic cation transporters (7.Jacquemin E. Hagenbuch B. Stieger B. Wolkoff A.W. Meier P.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 133-137Crossref PubMed Scopus (545) Google Scholar, 8.Grundemann D. Gorboulev V. Gambaryan S. Veyhl M. Koepsell H. Nature. 1994; 372: 549-552Crossref PubMed Scopus (604) Google Scholar). Defining characteristics in the family of hexose transporters are the presence of 12 membrane-spanning helices and a number of conserved residues and motifs (see Fig. 3). These sugar transporter signatures have been characterized by sequence comparisons as well as by mutagenesis. Substitutions, e.g. of the conserved arginine and glutamate residues on the cytoplasmic surface (9.Schürmann A. Doege H. Ohnimus H. Monser V. Buchs A. Joost H.-G. Biochemistry. 1997; 36: 12897-12902Crossref PubMed Scopus (63) Google Scholar), of tryptophan residues 388 and 412 in helix 10 and 11 (10.Garcia J.C. Strube M. Leingang K. Keller K. Mueckler M.M. J. Biol. Chem. 1992; 267: 7770-7776Abstract Full Text PDF PubMed Google Scholar, 11.Schürmann A. Keller K. Monden I. Brown F.M. Wandel S. Shanahan M.F. Joost H.-G. Biochem. J. 1993; 290: 497-501Crossref PubMed Scopus (38) Google Scholar), tyrosines 146 and 292/293 in helix 4 and 7 (12.Mori H. Hashiramoto M. Clark A.E. Yang J. Muraoka A. Tamori Y. Kasuga M. Holman G.D. J. Biol. Chem. 1994; 269: 11578-11583Abstract Full Text PDF PubMed Google Scholar, 13.Wandel S. Schürmann A. Becker W. Summers S.A. Shanahan M.F. Joost H.-G. FEBS Lett. 1994; 348: 114-118Crossref PubMed Scopus (23) Google Scholar), glutamine 161 in helix 5 (14.Mueckler M. Weng W. Kruse M. J. Biol. Chem. 1994; 269: 20533-20538Abstract Full Text PDF PubMed Google Scholar), and glutamine 282 (15.Hashiramoto M. Kadowaki T. Clark A.E. Muraoka A. Momomura K. Sakura H. Tobe K. Akanuma Y. Yazaki Y. Holman G.D. Kasuga M. J. Biol. Chem. 1992; 267: 17502-17507Abstract Full Text PDF PubMed Google Scholar), have been shown to markedly affect transporter function. In addition, mutagenesis experiments have implicated a motif (QLS) in helix 7 in determining the sugar recognition of GLUT1–GLUT5 (16.Seatter M.J. De la Rue S.A. Porter L.M. Gould G.W. Biochemistry. 1998; 37: 1322-1326Crossref PubMed Scopus (93) Google Scholar). The known glucose transporter (GLUT)1 isoforms differ in their expression in different tissues, in their kinetic characteristics, i.e. K m values (2.Mueckler M. Diabetes. 1990; 39: 6-11Crossref PubMed Google Scholar), and in their substrate specificity. GLUT1 mediates glucose transport into erythrocytes and through the blood-brain barrier, and appears to provide a basal supply of glucose for most cells. GLUT2 catalyzes glucose uptake into the liver (17.Thorens B. Sarkar H.K. Kaback H.R. Lodish H.F. Cell. 1988; 55: 281-290Abstract Full Text PDF PubMed Scopus (659) Google Scholar), and is an essential component of the glucose sensing mechanism of the pancreatic β cell (18.Guillam M.T. Hummler E. Schaerer E. Wu J.Y. Birnbaum M.J. Beermann F. Schmidt A. Deriaz N. Thorens B. Nat. Genet. 1997; 17: 327-330Crossref PubMed Scopus (333) Google Scholar). GLUT3 is predominantly expressed in neuronal cells (19.Gould G.W. Brant A.M. Kahn B.B. Shepherd P.R. McCoid S.C. Gibbs E.M. Diabetologia. 1992; 35: 304-309Crossref PubMed Scopus (46) Google Scholar), whereas GLUT4 is exclusively found in muscle and adipose tissue (20.James D.E. Strube M. Mueckler M. Nature. 1989; 333: 83-87Crossref Scopus (671) Google Scholar, 21.Birnbaum M.J. Cell. 1989; 57: 305-315Abstract Full Text PDF PubMed Scopus (462) Google Scholar); its subcellular localization is controlled by insulin (22.Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, 23.Holman G.D. Cushman S.W. BioEssays. 1994; 16: 753-759Crossref PubMed Scopus (134) Google Scholar). GLUT5 mediates transport of fructose, but probably not glucose, in intestine and spermatozoa (24.Burant C.F. Takeda J. Brot-Laroche E. Bell G.I. Davidson N.O. J. Biol. Chem. 1992; 267: 14523-14526Abstract Full Text PDF PubMed Google Scholar). The diverse tissue distribution and the specific functions of GLUT1–GLUT5 appear to indicate that these genes are sufficient to control glucose uptake in all mammalian tissues. However, two arguments may be raised that suggest the possibility that additional sugar transport facilitators exist. First, in some tissues, only low levels of mRNA of the known isoforms were detected (25.Kayano 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). Second, GLUT4 knockout mice exhibited an almost normal glucose transport in muscle, although no compensatory increase of the GLUT1 or GLUT3 gene expression was detected (26.Katz E.B. Stenbit A.E. Hatton K. DePinho R. Charron M.J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (395) Google Scholar). Therefore, in order to identify additional hexose transporters, we conducted a search of the EST data bases taking advantage of the conserved “sugar transporter signatures.” This search led to the identification of several novel GLUT-like genes. Here we describe the identification and characterization of GLUT8, a novel sugar transporter with unusual structural features and tissue-specific gene expression. Tissues were homogenized in 4 m guanidine thiocyanate, and total RNA was isolated by centrifugation on a cesium chloride cushion (5.88 m) at 33,000 rpm (rotor SW40) for 22 h. 5′-RACE (rapid amplification of cDNA ends) was performed with a kit from Life Technologies, Inc., Eggenstein, Germany, according to the instructions of the manufacturer. Primers for cDNA synthesis and the first amplifications were derived from the sequence of the IMAGE clone 46121. DNA fragments were isolated and subcloned into pUC18 with the SureCloneR kit (Amersham Pharmacia Biotech, Freiburg, Germany). Since the first RACE amplification yielded fragments still lacking the 5′ end of the cDNA, a second and third amplification was performed on the basis of the sequence information obtained in the previous RACE procedures. All cDNA clones and PCR products were sequenced in both directions by the method of Sanger (ThermoSequenase fluorescent labeled primer cycle sequencing kit; Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) with the aid of an automated sequencer (LI-COR, Lincoln, NE). Samples of total RNA (10 μg) were separated by electrophoresis on 1% agarose gels containing 1% formaldehyde and transferred onto nylon membranes (Hybond N+, Amersham Pharmacia Biotech, Braunschweig, Germany). Blots generated with RNA from different human tissues were purchased from CLONTECH (Palo Alto, CA). Probes were generated with the Klenow fragment of DNA polymerase I and α-32PdCTP by random oligonucleotide priming (27.Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16646) Google Scholar). The nylon membranes were hybridized at 42 °C and washed two times at 55 °C with 0.12 m NaCl, 0.012 m sodium citrate, 0.1% SDS. A fragment of the GLUT8 cDNA comprising the 5′-untranslated region and the full reading frame was amplified by PCR and was subcloned into the mammalian expression vector pCMV, which harbors an SV40 origin, a cytomegalovirus promoter, and a polyadenylation site. GLUT4 cDNA (21.Birnbaum M.J. Cell. 1989; 57: 305-315Abstract Full Text PDF PubMed Scopus (462) Google Scholar) was subcloned into the same expression vector as described (28.Schürmann A. Monden I. Joost H.-G. Keller K. Biochim. Biophys. Acta. 1992; 1131: 245-252Crossref PubMed Scopus (29) Google Scholar). COS-7 cells were transfected with calcium phosphate/DNA co-precipitates as described in detail previously (29.Doege H. Schürmann A. Ohnimus H. Monser V. Holman G.D. Joost H.-G. Biochem. J. 1998; 329: 289-293Crossref PubMed Scopus (36) Google Scholar), and were harvested 64 h after transfection. Cells transfected with glucose transporter cDNA were homogenized and fractionated as described previously (28.Schürmann A. Monden I. Joost H.-G. Keller K. Biochim. Biophys. Acta. 1992; 1131: 245-252Crossref PubMed Scopus (29) Google Scholar) with a modification of a protocol employed in 3T3-L1 cells (30.Weiland M. Schürmann A. Schmidt W.E. Joost H.-G. Biochem. J. 1990; 270: 331-336Crossref PubMed Scopus (71) Google Scholar). For detection of GLUT8 in the membrane fractions (plasma membranes, 13,000 ×g; high density microsomes, 45,000 × g; low density microsomes, 200,000 × g), antiserum against a C-terminal peptide (sequence in single-letter code: KGRTLEQVTAHFEGR) was used. Equilibrium cytochalasin B binding in plasma membranes from transfected cells was assayed by a method established with fat cell membranes (31.Weber T.M. Joost H.-G. Simpson I.A. Cushman S.W. Kahn C.R. Harrison L. Receptor Biochemistry and Methodology. Vol. 12B : Insulin Receptors. Biological Responses, and Comparison to the IGF-I Receptor. Alan R. Liss Inc., New York1988: 171-187Google Scholar) with modifications described in detail elsewhere (9.Schürmann A. Doege H. Ohnimus H. Monser V. Buchs A. Joost H.-G. Biochemistry. 1997; 36: 12897-12902Crossref PubMed Scopus (63) Google Scholar). Scatchard plots were evaluated graphically as described previously (32.Rosenthal H. Anal. Biochem. 1967; 20: 525-532Crossref PubMed Scopus (1347) Google Scholar, 33.Joost H.-G. Steinfelder H.J. Mol. Pharmacol. 1987; 31: 279-283PubMed Google Scholar). Glucose transporter protein in plasma membranes was solubilized and reconstituted into lecithin liposomes as described previously (34.Robinson F.W. Blevins T.L. Suzuki K. Kono T. Anal. Biochem. 1982; 122: 10-19Crossref PubMed Scopus (34) Google Scholar, 35.Schürmann A. Rosenthal W. Hinsch K.D. Joost H.-G. FEBS Lett. 1989; 255: 259-264Crossref PubMed Scopus (26) Google Scholar). Initial uptake rates ofd-U-14Cglucose were assayed after 10 s at a substrate concentration of 1 mm. The data were corrected for non-carrier-mediated uptake with tracerl-3Hglucose. In order to identify unknown glucose transporter-like sequences, we performed a search of the EST data bases with the protein sequences of the known GLUT isoforms (tblastx program). A total of approximately 200 EST sequences found in this search were further analyzed by individual comparisons. Among these, several human and murine EST sequences exhibited significant similarity with the GLUT family but differed from the known GLUT-isoforms. By sequencing a clone obtained from the IMAGE consortium (clone no. 46121, EST HS414155) we generated a partial cDNA sequence of a glucose transporter-like protein; this sequence exhibited significant similarity with a portion of the GLUT1 comprising membrane-spanning helices 5–12. Screening of several cDNA libraries failed to isolate longer clones. the sequence information of the 5′ portion was obtained by 5′-RACE amplifications with cDNA from human the mouse cDNA sequence was obtained by sequencing of a partial IMAGE clone (clone EST and data with the GLUT8 cDNA led to the identification of a human EST (STS for which localization has been by This sequence tag is identical with the end of 12 GLUT8 cDNA clones. be concluded that the sequence tag the localization of GLUT8 to chromosome 9. The of both mouse and human GLUT8 reading a sequence of amino acids the of the and of the amino acids are The amino sequence of human GLUT8 is identical with that of the GLUT1 of the residues that are identical in all mammalian GLUT isoforms by in Fig. are conserved in of the sequence with the the presence of 12 putative membrane-spanning with the of a transport facilitator The sequence all motifs (sugar transporter that are for the family of sugar transporters, in two motifs to the motifs helices and 12 motifs corresponding with the motifs in and 8, and glutamate and arginine residues in the 4 and 10 are in tryptophan residues corresponding with and in GLUT1 are has been implicated in the binding of the transport cytochalasin B. are from 1 is that of GLUT1 and a the appears to be in the larger loop 9. the conserved motif in loop 7 is by membrane of GLUT8 and sequences of its sugar transporter The is on structural obtained with the and is according to that by Mueckler (1.Mueckler M. Caruso C. Baldwin S.A. Panico M. Blench I. Morris H.R. Allard W.J. Lienhard G.E. Lodish H.F. Science. 1985; 229: 941-945Crossref PubMed Scopus (1136) Google Scholar). residues of GLUT8 that with the sugar transporter signatures by sequence comparisons of are of these residues the GLUT8 The a in loop Fig. a of an of GLUT8 with its The protein with the similarity is novel transport facilitator identical amino which was by Doege and H.-G. The are the mammalian glucose transporters identical the transporter identical and the and transporters and identical amino of these sequences that the similarity of GLUT8 with the transporter identical amino acids) is that of the transporter with the GLUT1 amino GLUT8 may be with a of the sugar transporter family the GLUT The mouse GLUT8 cDNA was subcloned into an expression vector by the cytomegalovirus promoter, and COS-7 cells were transfected with this membranes from transfected cells were isolated by centrifugation and were with B and different of is in of GLUT8 protein a increase in specific binding of cytochalasin B. The derived from Scatchard plots of the binding not was 56.6 ± 18 and is well the values assayed for binding of cytochalasin B to members of the GLUT family (22.Cushman S.W. Wardzala L.J. J. Biol. Chem. 1980; 255: 4758-4762Abstract Full Text PDF PubMed Google Scholar, B. Joost H.-G. Mol. Pharmacol. Google Scholar). B binding to GLUT8 is by glucose with an of approximately mm. membranes from cells transfected with GLUT8 cDNA were and were reconstituted into lecithin liposomes for of their glucose transport is in with GLUT8 cDNA an increase transport activity as with membranes from cells transfected with with GLUT4 cDNA a that this to a of GLUT4 in the reconstituted membranes, of transport rates for cytochalasin B a activity of GLUT8 ± of of cytochalasin ± in membranes with In order to an additional of the glucose transport of GLUT4 and GLUT8, membranes were from transfected cells on a larger a full Scatchard of the number of cytochalasin B binding In this GLUT8 of of cytochalasin as with by In order to the expression of GLUT8 with a of the membrane fractions were analyzed with antiserum against a C-terminal is in the of Fig. cells transfected with the GLUT8 cDNA expressed a protein with a that of the GLUT4 no was found in cells transfected with A second specific was detected at approximately Since glucose transporters to this a of By a 2.4-kilobase pair transcript corresponding with GLUT8 mRNA from the was predominantly found in amounts were detected in most other tissues small intestine, heart, and skeletal of the expression of the GLUT8 in testis, we its expression in testicular and in testicular tissue from with is in GLUT8 mRNA was not in from two with testicular carcinoma. estrogen suppressed the expression of GLUT8 in of GLUT8 expression in mRNA isolated from the of human testis testis, was and hybridized with human GLUT8 mRNA isolated from testis of prepubertal and was and hybridized with mouse GLUT8 of RNA was controlled by The of the that GLUT8 is with and that its expression is controlled by Therefore, we the mRNA levels in testis from of different is in Fig. 7 the 2.4-kilobase pair transcript was found in testis from and but not in prepubertal The novel transporter protein is a of the glucose transport facilitators and their to the of glucose in a of reconstituted membranes, and their to the specific cytochalasin B in a glucose-inhibitable sequence all (sugar transporter that are for the GLUT family and are for their as hexose transporters (9.Schürmann A. Doege H. Ohnimus H. Monser V. Buchs A. Joost H.-G. Biochemistry. 1997; 36: 12897-12902Crossref PubMed Scopus (63) Google H. Schürmann A. Ohnimus H. Monser V. Holman G.D. Joost H.-G. Biochem. J. 1998; 329: 289-293Crossref PubMed Scopus (36) Google Scholar). the protein is a novel of the family of sugar transport facilitators and was However, its similarity with the GLUT isoforms is not that with and transporter and with that of the S. with a second novel transport is on a the family of hexose the basis of the sequence GLUT8 and the GLUT we the protein to cytochalasin B with high in a glucose-inhibitable The this and suggest that binding of this the presence of of the sugar transporter tryptophan In to glucose-inhibitable cytochalasin B GLUT8 exhibited a reconstitutable glucose transport activity to that of the This was we that the substrate recognition a similarity with the GLUT1–GLUT5 In GLUT8 harbors a motif in the loop 7 that markedly from that of the glucose transporters mutagenesis from that these residues are for the the transport (29.Doege H. Schürmann A. Ohnimus H. Monser V. Holman G.D. Joost H.-G. Biochem. J. 1998; 329: 289-293Crossref PubMed Scopus (36) Google Scholar). the of this motif that the residues in this loop the glucose of these However, the data against a of the motif in determining the sugar of the GLUT the data are with the that is the motif in helix 7 in that of a GLUT (19.Gould G.W. Brant A.M. Kahn B.B. Shepherd P.R. McCoid S.C. Gibbs E.M. Diabetologia. 1992; 35: 304-309Crossref PubMed Scopus (46) Google Scholar). However, the possibility be that GLUT8 other and are to the substrate of this sugar transport The tissue distribution of mRNA of GLUT8 that is expressed in different glucose tissues as testis, muscle, and is that GLUT8 is the unknown glucose transporter that has been to the of GLUT4 in GLUT4 knockout mice (26.Katz E.B. Stenbit A.E. Hatton K. DePinho R. Charron M.J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (395) Google Scholar). However, mRNA levels were detected in testis, and appears to that this expression a function. GLUT8 was expressed in testis from adult, but not from we that the human testicular expression is markedly by estrogen which is known to L. J. PubMed Scopus Google Scholar). the are with a of the GLUT8 expression by a on GLUT8 be in the of glucose for DNA synthesis in cells. of this a glucose transporter cDNA was described and B. J. Biol. Chem. which is identical with that of The cDNA sequences of human GLUT8 and human differ in 5 of these the amino sequence The cDNA sequences of mouse GLUT8 and differ in 4 in the of amino acids S. for S. for mRNA and and W. Becker for reading the