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Biochemical Society Transactions ( 1 992) 20
Immunological identification of five members of the human facilitative glucose transporter family. ALISON M. BRANT, E. MICHAEL GIBBS*, GWYN W. COULD and HELEN M. THOMAS.
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Department of Biochemistry, University of Glasgow, Glasgow, G12 SQQ, Scotland, and Pfizer Central Research, Groton, CT 06340, U.S.A. The transport of glucose across the plasma membranes of mammalian cells is mediated by a family of glucose transporters of the facilitative diffusion type. To date, five such glucose transporters have been cloned from human tissues; they are the products of distinct genes and exhibit distinct tissue-specific patterns of expression [l]. Thus, GLUT 1 is expressed in many tissues, with highest levels found in the brain and at blood-tissue barriers (e.g. placenta, blood-brain barrier). GLUT 2 is expressed in hepatocytes, pancreatic beta-cells, kidney and at the basolateral surface of the adsorptive cells of the small intestine. GLUT 3 is expressed at high levels in the brain but its mRNA is also found in muscle, liver and adipose tissue. GLUT 4 is found only in tissues which exhibit acute insulin-stimulated glucose transport i.e. muscle, heart and fat. GLUT 5 is found at high levels in the small intestine but its mRNA is also present in adipose, muscle and liver [l-31. Each transporter has been functionally expressed in Xenoplrs luevis oocytes and the Km for 3-0-methyl-D-glucose determined [4]. In order to relate the tissue-specific pattern of glucose transporter expression to the contribution of different tissues to whole body glucose homeostasis, it is important firstly to establish the relative levels of each transporter isoform present in these tissues. As a first step towards this goal, we have generated a panel of anti-peptide antibodies against the C-terminal 14 amino acids of the human isoforms of GLUTS 1,2,3,4 and 5, the murine GLUT 3 isoform and the rat GLUT 2 isoform. Here we demonstrate that each antibody specifically recognises the correct transporter, and a synopsis of the tissue-specific expression of each isoform is presented. Peptides corresponding to the C-terminal 14 amino acids of each isoform, containing an additional N-terminal cysteine residue, were conjugatied to Keyhole limpet hemocyanin via sulpho-MBS. Rabbits were immunised at multiple intradermal sites and boosted at monthly intervals. Antibodies against each peptide were affinity purified using columns of immobilised peptide. Eluted antibodies were dialysed overnight against phosphate buffered saline and stored at -80' prior to use. All experiments herein were performed with affinity purified antibodies. In order to demonstrate the specificity of the antibodies obtained, we have used two independant approaches: immunoblotting of expressed proteins in oocytes [4] or the specific immunoprecipitation of D-glucose displaceable [3H]cytochalasin B (CB) binding [5]. Fig.1 shows a series of immunoblots using either anti-GLUT 2 or anti-GLUT 4 antibodies. In both cases, the antibodies produced signals only in the expected lanes and did not cross react with any of the other transporter isoforms. This approach has been used to validate antiGLUT 2, anti-human GLUT 3 (data not shown) and anti-GLUT 4. To c o n f m that the anti-human GLUT 5 and anti-murine GLUT 3 antibodies do indeed recognise a transporter, we performed a series of CB-immunoprecipitation experiments. Fig. 2 shows the data using the anti-human GLUT 5 antibody. Similar results have been obtained using the anti-murine GLUT 3 antibodies (data not shown). We have used the antibodies to examine the sites of expression of these transporters in human and murine tissues. The results of these experiments may be summarised as follows: -In murine tissues, the expression of GLUT 3 was detectable only in the brain or neurally derived cells. No immunoreactive protein was observed in murine liver, muscle, adipose tissue or 3T3-Ll adipocytes. -GLUT 2 in rat liver and the human isoform expressed in oocytes is present as several distinct bands of Mr 56.42 and 31 kDa in rat liver and 56,50 and 33 kDa for the human homologue expressed in oocytes. We have detected GLUT 2 in rat liver, islets and small intestine.
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Figure 1. Immunoblot analysis of a-GLUT 2 and a-GLUT 4. Membranes were prepared from oocytes ex essin the human isoforms of either GLUT 4 or GLUT 2. In the left han&anel.%nes 1 and 2 contain the equivalent of 6 or 3 oocytes expressm G L m 4 lanes 3 and 4 e uivalenl amounts of oocyles ex ressing G L d 2 . In the ht hand panelLne 1 conauns 50 pg of 3T3-%1 a d i r p membmes, fanes 2 and 3 GLUT 4 iniected oocytes, lanes 4 and G UT 2 iniccted oocytes and lanes 6 and 7 water in'ected oocytes. The left panel shows an anti-GLUT 2 blot, the right an anti-GLbT 4 blot.
f 1 Z J P 5 6 1 8 9 l 0 1 1
Fi ure 2 Cytochalasin B 1a8e11in 'of GLUT 5. Human Ckuodena! membranes were photolabelled with cytochalasin B 18.7 nM 3H CB m the resence of eother 6.52M ( ) glucose ( ). Membranes were solubilised in 2% Triton X-100and GLUT 5 immunopreci itated. The antlmv-antieen comoPex was recove& usi; Protein A agarose, eluted in SDS-!AGE buffer, and proteins separated on,a 10% aemmli el The radioactlvity associate8 with each section of the
d- art-
posiuon of molecular weight stan&&.
-GLUT 5 is expressed at high levels in the human small intestine. Immunolocalisation has demonstrated that this transporter is localised to the apical membrane of the adsorptive epithelia [5]. Moreover, the protein is expressed in isolated human adipocytes, human muscle and human brain (Peter R.Shepherd, G.W.G., E.M.G. and Barbara B.Kahn, submitted). This work was supported by grants from the MRC and the British Diabetic Association (to G.W.G.) [ l ] Could CAW.8t Bell, G I . (1990) Trends Biochem. Sci. 15, IU-LJ.
[2] Bell G I Ka ano T Buse J.B Burrant C.F., Takeda, 1. e t a . (1690 Dlibetes kare;'l3, 198:208. 131 Meuckler, M (199b;Diabetes, 39,6-11. 4 Could G.W Thomas, H.M. Jess, T.J. and Bell, G.I. (1391 B k h e m i s 30,5139-5143. [5] Wesss;g&A'&-tiochy.F. (1984) Biochim. Biophys. Acta. I I I , ILJ-IJL.
[6] Davidson N.O., Hausmann, A.M.L., Ifkovits C.A., Buse J.B., bould,.G.W., Burrant, C.F. and Bell, (3.1. (1992) Am. J. Physiol. in press.
Biochemical Society Transactions ( 1 992) 20
Immunological identification of five members of the human facilitative glucose transporter family. ALISON M. BRANT, E. MICHAEL GIBBS*, GWYN W. COULD and HELEN M. THOMAS.
-116.5
-80
Department of Biochemistry, University of Glasgow, Glasgow, G12 SQQ, Scotland, and Pfizer Central Research, Groton, CT 06340, U.S.A. The transport of glucose across the plasma membranes of mammalian cells is mediated by a family of glucose transporters of the facilitative diffusion type. To date, five such glucose transporters have been cloned from human tissues; they are the products of distinct genes and exhibit distinct tissue-specific patterns of expression [l]. Thus, GLUT 1 is expressed in many tissues, with highest levels found in the brain and at blood-tissue barriers (e.g. placenta, blood-brain barrier). GLUT 2 is expressed in hepatocytes, pancreatic beta-cells, kidney and at the basolateral surface of the adsorptive cells of the small intestine. GLUT 3 is expressed at high levels in the brain but its mRNA is also found in muscle, liver and adipose tissue. GLUT 4 is found only in tissues which exhibit acute insulin-stimulated glucose transport i.e. muscle, heart and fat. GLUT 5 is found at high levels in the small intestine but its mRNA is also present in adipose, muscle and liver [l-31. Each transporter has been functionally expressed in Xenoplrs luevis oocytes and the Km for 3-0-methyl-D-glucose determined [4]. In order to relate the tissue-specific pattern of glucose transporter expression to the contribution of different tissues to whole body glucose homeostasis, it is important firstly to establish the relative levels of each transporter isoform present in these tissues. As a first step towards this goal, we have generated a panel of anti-peptide antibodies against the C-terminal 14 amino acids of the human isoforms of GLUTS 1,2,3,4 and 5, the murine GLUT 3 isoform and the rat GLUT 2 isoform. Here we demonstrate that each antibody specifically recognises the correct transporter, and a synopsis of the tissue-specific expression of each isoform is presented. Peptides corresponding to the C-terminal 14 amino acids of each isoform, containing an additional N-terminal cysteine residue, were conjugatied to Keyhole limpet hemocyanin via sulpho-MBS. Rabbits were immunised at multiple intradermal sites and boosted at monthly intervals. Antibodies against each peptide were affinity purified using columns of immobilised peptide. Eluted antibodies were dialysed overnight against phosphate buffered saline and stored at -80' prior to use. All experiments herein were performed with affinity purified antibodies. In order to demonstrate the specificity of the antibodies obtained, we have used two independant approaches: immunoblotting of expressed proteins in oocytes [4] or the specific immunoprecipitation of D-glucose displaceable [3H]cytochalasin B (CB) binding [5]. Fig.1 shows a series of immunoblots using either anti-GLUT 2 or anti-GLUT 4 antibodies. In both cases, the antibodies produced signals only in the expected lanes and did not cross react with any of the other transporter isoforms. This approach has been used to validate antiGLUT 2, anti-human GLUT 3 (data not shown) and anti-GLUT 4. To c o n f m that the anti-human GLUT 5 and anti-murine GLUT 3 antibodies do indeed recognise a transporter, we performed a series of CB-immunoprecipitation experiments. Fig. 2 shows the data using the anti-human GLUT 5 antibody. Similar results have been obtained using the anti-murine GLUT 3 antibodies (data not shown). We have used the antibodies to examine the sites of expression of these transporters in human and murine tissues. The results of these experiments may be summarised as follows: -In murine tissues, the expression of GLUT 3 was detectable only in the brain or neurally derived cells. No immunoreactive protein was observed in murine liver, muscle, adipose tissue or 3T3-Ll adipocytes. -GLUT 2 in rat liver and the human isoform expressed in oocytes is present as several distinct bands of Mr 56.42 and 31 kDa in rat liver and 56,50 and 33 kDa for the human homologue expressed in oocytes. We have detected GLUT 2 in rat liver, islets and small intestine.
-49.5
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Figure 1. Immunoblot analysis of a-GLUT 2 and a-GLUT 4. Membranes were prepared from oocytes ex essin the human isoforms of either GLUT 4 or GLUT 2. In the left han&anel.%nes 1 and 2 contain the equivalent of 6 or 3 oocytes expressm G L m 4 lanes 3 and 4 e uivalenl amounts of oocyles ex ressing G L d 2 . In the ht hand panelLne 1 conauns 50 pg of 3T3-%1 a d i r p membmes, fanes 2 and 3 GLUT 4 iniected oocytes, lanes 4 and G UT 2 iniccted oocytes and lanes 6 and 7 water in'ected oocytes. The left panel shows an anti-GLUT 2 blot, the right an anti-GLbT 4 blot.
f 1 Z J P 5 6 1 8 9 l 0 1 1
Fi ure 2 Cytochalasin B 1a8e11in 'of GLUT 5. Human Ckuodena! membranes were photolabelled with cytochalasin B 18.7 nM 3H CB m the resence of eother 6.52M ( ) glucose ( ). Membranes were solubilised in 2% Triton X-100and GLUT 5 immunopreci itated. The antlmv-antieen comoPex was recove& usi; Protein A agarose, eluted in SDS-!AGE buffer, and proteins separated on,a 10% aemmli el The radioactlvity associate8 with each section of the
d- art-
posiuon of molecular weight stan&&.
-GLUT 5 is expressed at high levels in the human small intestine. Immunolocalisation has demonstrated that this transporter is localised to the apical membrane of the adsorptive epithelia [5]. Moreover, the protein is expressed in isolated human adipocytes, human muscle and human brain (Peter R.Shepherd, G.W.G., E.M.G. and Barbara B.Kahn, submitted). This work was supported by grants from the MRC and the British Diabetic Association (to G.W.G.) [ l ] Could CAW.8t Bell, G I . (1990) Trends Biochem. Sci. 15, IU-LJ.
[2] Bell G I Ka ano T Buse J.B Burrant C.F., Takeda, 1. e t a . (1690 Dlibetes kare;'l3, 198:208. 131 Meuckler, M (199b;Diabetes, 39,6-11. 4 Could G.W Thomas, H.M. Jess, T.J. and Bell, G.I. (1391 B k h e m i s 30,5139-5143. [5] Wesss;g&A'&-tiochy.F. (1984) Biochim. Biophys. Acta. I I I , ILJ-IJL.
[6] Davidson N.O., Hausmann, A.M.L., Ifkovits C.A., Buse J.B., bould,.G.W., Burrant, C.F. and Bell, (3.1. (1992) Am. J. Physiol. in press.
