0013.7227/92/1315-2319$03.00/0 Endocrinology Copyright (0 1992 by The Endocrine

Vol. 131, No. 5 Prrnted in U S.A.

Society

Potential Involvement of the Carboxy-Terminus Glut 1 Transporter in Glucose Transport* JEAN-FRANCOIS TANTI, NADINE EMMANUEL VAN OBBERGHEN,

AND

INSERM

06107 Nice Cedex 02, France

U-145, Facultk de Mkdecine,

GAUTIER, MIREILLE CORMONT, VRRONIQUE YANNICK LE MARCHAND-BRUSTEL

of the

BARON,

ABSTRACT The role of the carboxy-terminal domain of the Glut 1 glucose transporter was investigated using an antipeptide antibody to the Cterminal part of the molecule. The study was performed in fibroblasts transfected with the cDNA coding for the human insulin receptor. These cells acutely respond to insulin for glucose transport. Using antipeptide antibodies to Glut 1 and Glut 4, we first established that these cells expressed only Glut 1. Then, to define the role of the Cterminal part of Glut 1 in glucose transport, the antibodies were loaded into the cells by electroporation. When anti-Glut 1 immunoglobulins were introduced into the cells, a 60% increase in basal deoxyglucose

and 3-0-methylglucose transport was observed compared to that in cells electroporated with nonimmune immunoglobulins. The stimulatory action of the antipeptide was not due to an increase in the total amount of transporters. It was found only at low glucose concentrations, suggesting that the affinity of the transporter, rather than its maximal capacity, was changed. Finally, the effect of antibody was additive to that of insulin. The interaction between the anti-Glut 1 antibody and the carboxy-tail of the transporter seems to lead to an increase in the intrinsic activity of the transporter, suggesting that this part of the molecule could be implicated in the regulation of glucose uptake. (Endocrinology 131: 2319-2324, 1992)

F

be translocated acrossthe plasma membrane after a conformational change in the transporter from the outward to the inward facing form (14-16). However, the domainsinvolved in this conformational change are not known. We have identified the glucose transporter isoform in NHIR cells, which express high levels of insulin receptors and exhibit a rapid insulin stimulation of glucose transport (17). Then, we have taken advantage of antipeptide antibodies to the Cterminal part of the Glut 1 transporter, one of the regions that presentsthe greatest divergence among the transporters (13), to study a potential role for this region in glucose transport.

ACILITATED glucose transport is mediated by a family of proteins, which are differently expressed among tissues.The Glut 1 glucosetransporter cloned from the HepG2 cell line and rat brain (1, 2) is expressedat varying levels in most tissues; it is prevalent in erythrocytes, brain, and cultured cells (3). Four other transporters have been recently cloned: Glut 2, expressedin liver and pancreatic B-cells (46); Glut 3, the isoform found in human fetal muscle, brain, placenta, and kidney (7); Glut 4, found in tissues that are insulin responsive for glucose transport (8-12); and Glut 5, present in small intestine (13). All have 12 membrane-spanning domains, a relatively large middle cytoplasmic loop, a long extracellular loop with 1 glycosylation site, and the amino- and carboxy-ends oriented toward the cytoplasm (2, 13). The sequence identity among the different isotypes ranges from 50-60%, the greatest divergences being at the level of the extracellular loop and in the amino- and carboxytails (13). Despite the identification of the glucosetransporter sequence, the precise mechanism underlying glucose transport acrossthe plasma membrane is not completely understood. It has been suggestedthat the glucose-binding site of Glut 1 is formed by the membrane-spanning segments9, 10, and 11 (14). Several lines of evidence pointed to the asymmetry of Glut 1 and described it as a gated channel. In this model the glucose-binding site could exist alternatively on the outer or the inner side of the channel, but not simultaneously at both. The glucose bound to the outer side would Received April 29, 1992. Address all correspondence and requests for reprints to: Dr. J.-F. Tanti, INSERM U-145, FacultC de Mkdecine, avenue de Valombrose, 06107 Nice Cedex 02, France. *This work was supported by grants from INSERM, the Rigion Provence-Alpes-C&e d’Azur, the University of Nice, and the French Association for the Study of Myopathy.

Materials

and Methods

Cell culture NHIR cells, mouse embryo fibroblasts transfected with an expression plasmid encoding for the human insulin receptor, were a gift from Dr. J. Whittaker (Stony Brook, NY). They were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (17).

Antibodies Antipeptide antibodies to the last 14 amino acid residues of the carboxy-terminus of Glut 1 (serum 4C) or Glut 4 (serum 5A) were obtained as previously described (18). Antipeptide antibodies were affinity purifieh as previously detailed (19). Briefly, 15 mg of each peptide were coupled to 5 ml Affi-Gel 10 (Bio-Rad, Richmond, CA) in 50 rnM HEPES b;ffer, pH 7.4, at 4 C for i6 h before the addition’of 100 mM Tris-HCl, pH 8.0. The Affi-Gel column was equilibrated with PBS, pH 7.4. Each antiserum (15 ml) was passed three times over the corresponding column. The columns were washed successively with 10 ml PBS, PBS supplemented with 2 M NaCl, and PBS. The antibodies were eluted with 3.5 M NaSCN in PBS, pH 6.6, dialyzed overnight against PBS, and concentrated to 0.5-l mg/ml. Control immunoglobulins (Igs) were similarly treated, with the exception of the column step.

2319

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2320

GLUT

Membrane

preparation

l-CARBOXY-TAIL

and immunoblotting

NHIR cells from 35-mm plates were scraped and lysed for 15 min at 4 C in Tris-HCl (1 mM), pH 7.4. Cells were homogenized with 10 hand strokes in a Dounce homogeneizer (Kontes, Vineland, NJ). The buffer was adjusted to 250 mM sucrose, and the homogenate was centrifuged at 1,000 x g for 10 min. The supernatant was then centrifuged at 200,000 X g for 30 min. The resulting pellets con-responding to total crude membranes were resuspended in Tris-sucrose buffer and stored at -80 C. Membranes (80 pg) were diluted in solubilizing buffer [3% sodium dodecyl sulfate (SDS), 70 rnM Tris, 10% glycerol, and 100 rnM P-mercaptoethanol] and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with a 10% acrylamide resolving gel and a 3% stacking gel (20). After transfer to nitrocellulose, the blots were incubated with blocking buffer (5% fat skimmed dry milk in PBS, pH 7.4) for 2 h at room temperature. The sheets were incubated overnight at 4 C with affinity-purified antibodies to the glucose transporter Glut 1 or Glut 4 or nonimmune Ig (10 Kg/ml). After three washes (10 min) in PBS containing 1% Triton X-100, sheets were incubated for 1 h at room temperature with [iZ51]protein-A (10’ cpm/ml) and washed as described above. The sheets were autoradiographed with Trimax film (3M Co., Italy) for 12-24 h at -80 C.

Injection

of antibodies

into cetls

Injection was performed by electroporation using the Electropulsing Unit (Apelex, Massy, France) (21, 22). Briefly, confluent NHIR cells were trypsinized, washed once with DMEM-10% FCS and once with PBS, and resuspended at 2.5 x lob/ml in PBS containing 300-400 fig/ml affinity-purified anti-Glut 1 or anti-Glut 4 Ig or nonimmune Ig. Cells were transferred to the sterile electroporation chamber and exposed to a single electric pulse of 750 V/cm for 0.93 msec at 23 C. Thev were mainTained for lb min at 4 C. Then, they were diluted into DMEM-10% FCS and ulated at high densitv (2 X lo5 cells/well) in 12.well dishes. Usmg this’experimentll condition: approximately 6 ;g Ig/ml cell volume were transferred into cells (21), and most of the cells were electroporated, as shown by the entry of lucifer yellow (Baron, V., personal communiCatIon)

Measurement

of deoxyglucose uptake

Unless otherwise indicated, 24 h after electroporation, cells were washed twice with DMEM-0.2% BSA and incubated for 3 h in this medium. They were then washed twice with Krebs-Ringer phosphate buffer and incubated without or with insulin (10e7 M) for 30 min in the same buffer supplemented with 0.2% BSA. Thereafter, [2-3H]deoxyglucase was added (0.1 &i/well; 0.1 mM) for 5 min. After three washes with ice-cold PBS, cells were solubilized in 500 @cl0.1 N NaOH, and radioactivity was counted. To determine the half-maximal inhibition constant (K,) for cytochalasin-B, the cells were electroporated as described above. Deoxyglucose transport (50 PM; 0.1 pCi/well) was then measured in the presence of various concentrations of cytochalasin-B (0.1-l PM). To ensure that the effects observed were specific for anti-Glut 1 Ig, the antibodies were incubated overnight at 4 C with the corresponding 14.amino acid peptide immobilized on Affi-Gel 10. The remaining solution was microinjected 24 h before deoxyglucose uptake measurement, as described above.

3-O-o-Methylglucose

transport

assays

Measurements of zero trans influx were performed as previously described (23). Twenty-four hours after electroporation, cells were washed twice and incubated for 3 h in DMEM-0.2% BSA. They were then washed twice with Krebs-Ringer phosphate buffer and incubated in 0.5 ml of the same buffer supplemented with 0.2% BSA. The multiwell plates were placed at room temperature for 10 min. Glucose transport assays were initiated by the addition of 3-O-methyl-[‘4C]glucose (l-2.5 &i/assay) at the concentration indicated in Table 3, and initial rates of transport were determined at room temperature. Transport was termi nated by the addition of PBS containing 500 FM phloretin, and the

AND

GLUCOSE

TRANSPORT

Endo. Voll31.

1992 No 5

content of each well was immediately aspirated. Four rapid washes were performed with the same buffer, the cells were solubilized in 500 11 0.1 N NaOH, and radioactivity was counted. Nonspecific glucose transport was measured in the presence of 40 PM cytochalasin-B.

Results Characterization NHIR cells

of glucose transporter

isoforms expressed in

The studies were performed in NHIR fibroblasts transfected with the human insulin receptor cDNA and expressing a high level of these receptors (17). When cells were incubated with the hormone (10m7 M) for 30 min, and deoxyglucase was added for 5 min, insulin induced a 2-fold increase in glucose transport (76 + 7 and 164 f 8 pmol/min . assay in basal and insulin-stimulated conditions, respectively). To look for the presence of Glut 1 and Glut 4 isotypes, cell membranes were subjected to SDS-PAGE, the proteins were transferred to nitrocellulose, and sheets were immunoblotted using specific antibodies to Glut 1 or Glut 4 (18). Anti-Glut 1 antibodies revealed one protein that had an electrophoretic mobility around 45-50 kilodaltons, compatible with the mol wt of Glut 1, while no bands were detected with the antiGlut 4 antibodies (Fig. 1). The same result was obtained with the nontransfected cells (data not shown). Effect of anti-Glut

1 antibodies

on deoxyglucose uptake

To investigate the role of the carboxy-terminus of Glut 1 in glucose transport, antipeptide antibodies directed to the C-terminal part of Glut 1 were introduced into cells by electroporation. Anti-Glut 1 antibodies induced a 60% increase in deoxyglucose uptake, an effect comparable to the insulin stimulation of glucose uptake in cells electroporated with nonimmune lg (Fig. 2). Further, the effect of anti-Glut 1 was additive to that of insulin stimulation. Anti-Glut 4 antibodies did not modify glucose uptake, an expected result, since those cells did not express this transporter isoform (Fig. 2). Moreover, when anti-Glut 1 antibodies were preblocked with the peptide before electroporation, glucose transport was unchanged compared to that in nonimmune Ig-loaded cells (85 + 1.8 and 83 f 3 pmol/min.assay, respectively). Furthermore, glucose transport was not increased in cells electroporated with antipeptide antibodies directed to the Cterminal part of the insulin receptor (21). The effect of antiGlut 1 Igs was present 9 h after electroporation (Table l), which is the earliest tested time due to the fact that cells have to recover from the trypsin treatment and the electroporation itself. The effect was maximal at 28 h, the time period chosen for the following experiments, and it disappeared after 2 days, in accordance with the half-life of electroporated Ig (data not shown). In control cells and cells electroporated with anti-Glut 1 antibodies, deoxyglucose uptake was inhibited in the presence of 30 PM cytochalasin-B (Table 2). To investigate whether the anti-Glut 1 antibodies modified the sensitivity of glucose transport to cytochalasin-B, deoxyglucose uptake was measured at a low concentration (50 PM) in the presence of increasing concentrations of inhibitor (Fig. 3). Under these

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GLUT

l-CARBOXY-TAIL

B

AND GLUCOSE TABLE cells

C

TRANSPORT

1. Effect of anti-Glut

2321 1 Igs on glucose uptake in NHIR Deoxyglucose

Hours after

uptake

electroporation

Nonimmune Ig

Anti-Glut 1 Ig

9 28 48

100 100 100

143 z!z 4” 155 & 3” 109 f 12

NHIR cells were loaded by electroporation with nonimmune Ig or anti-Glut 1 antibodies (400 pg/ml) as described in Materials and Methods. Cells were depleted in DMEM-0.2% BSA for 3 h before glucose uptake determination. Deoxyglucose uptake was measured for 5 min in Krebs-Ringer phosphate-0.2% BSA supplemented with 2-[3H] deoxyglucose (0.1 &i/well; 0.1 mM). Results are expressed as a percentage of values obtained for cells electroporated with nonimmune Ig and are the mean f SEM of 6-12 wells. n The effect of anti-Glut 1 Ig is statistically significant (P < 0.005). TABLE 2. Cytochalasin-B inhibition glucose transport in NHIR cells

45-

l-stimulated

Deoxyglucoseuptake (pmol/min assay) Nonimmune Ig Anti-Glut 1 Ig conditions -Cyto B

Mr X IO-3

55.6 -c 1.7 85.2 c!z 1.1 4.6 f 0.4 3.5 + 0.6 NHIR cells were loaded by electroporation with nonimmune Ig or anti-Glut 1 antibodies (400 pg/ml) as described in Materials and Methods. Cells were depleted in DMEM-0.2% BSA for 3 h before glucose uptake determination. Deoxyglucose uptake was measured as described in Materials and Methods without or with 30 jtM cytochalasinB (Cyto B). Results are expressed as the mean f SEM of six determinations. +Cyto

Non Immune

anti Glut 1

anti Glut 4

FIG. 1. Characterization of glucose transporter isoforms in NHIR cells. Total cellular membranes from NHIR cells were prepared as described in Materials and Methods. Proteins (80 rg) were separated by SDSPAGE with a 7.5% resolving gel, transferred to nitrocellulose, and blotted with nonimmune Ig (lane A), anti-Glut 1 antibodies (lane B), or anti-Glut 4 antibodies (lane C). The antipeptide antibodies were affinity purified as described in Materials and Methods and used at 10 pg/ml. Autoradiograms were exposed for 16 h at -80 C. Mr, Mol wt.

B

not different from the K, (611 + 30 nM) in cells loaded with anti-Glut 1 antibodies. Effect of anti-Glut

0

Basal

0 Non Immune

Anti Glut 1

Anti Glut 4

FIG. 2. Effect of anti-Glut Igs on glucose uptake in NHIR cells. NHIR cells were loaded by electroporation with nonimmune Ig, anti-Glut 1, or anti-Glut 4 antibodies (300 rg/ml) as described in Materials and Methods. Deoxyglucose uptake was mesured in the absence (0) or presence of 10e7 M insulin (m). The results are the mean zk SEM of 410 different electroporation experiments.

the Ki can be calculated from the equation vo/v = 1 + I/Kit where vo/v was the fractional inhibition, and I was the inhibitor concentration (24). The calculated Ki was 688 + 50 nM in cells loaded with nonimmune Ig, which was

conditions,

of anti-Glut

1 antibodies

on 3-0-methylglucose

uptake

To determine whether the effects of anti-Glut 1 antibodies on 2-deoxyglucose uptake represent increased glucosetransport rates rather than changesin the rates of glucose phosphorylation, cells were also assayed for 3-O-methyl-[‘4C] glucose transport under zero trans influx conditions (Table 3). At low concentrations (25-50 PM), 3-0-methylglucose uptake was increased by 60-70s in cells loaded with antiGlut 1 antibodies and was inhibited in the presence of cytochalasin-B (data not shown). This uptake was saturable in cells electroporated with nonimmune Ig or anti-Glut 1 antibodies (Table 3). Maximum rates of transport were reached between 3-8 mM 3-0-methylglucose. However, at high sugar concentrations, the effect of anti-Glut 1 antibodies disappeared, suggesting that the antibodies increased the affinity of the transporter without changing its maximal velocity. Effect of anti-Glut

1 antibodies

on total Glut 1 protein

level

To determine whether the effects of anti-Glut 1 antibodies were related to an increase in glucose transporter protein levels, Glut 1 protein was quantified by immunoblot analysis. Total cellular membranes were prepared from NHIR cells

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GLUT

2322

l-CARBOXY-TAIL

AND

GLUCOSE

TRANSPORT

AB

Endo Voll31.

l

1992 No 5

CD

2.5

()

Cytochalasin

Non Immune

B (nM)

FIG. 3. Effect of anti-Glut 1 Igs on glucose transport inhibition by cytochalasin-B. NHIR cells were loaded by electroporation with nonimmune Ig or anti-Glut 1 antibodies (400 pg/ml) as described in Materials and Methods. Deoxyglucose uptake was measured in the presence of different cytochalasin-B concentrations, as indicated on the abscissa. The ratio of the rate constant for uptake of 50 pM deoxyglucose in the absence and presence of cytochalasin-B (vo/v) was plotted against the cytochalasin-B concentration (I). The results are from two different electroporation experiments, with duplicate determinations of the rate constant. The line was derived by fitting the equation vo/v= 1 + I/Ki. This gave Ki values of 688 + 50 and 611+ 30 nM in cells electroporated with nonimmune and anti-Glut 1 Ig, respectively. TABLE 3. Effect of anti-Glut 1 antibodies on 3-0-methylglucose uptake in NHIR cells 3-0-Methylglucose (PM) 25 50 100 1000 3000 8000

3-O-Methylglucose uptake (pmol/20 sec. assay) Nonimmune 2.3 4.1 12.5 115 153 172

+ + + & + +

Ig 0.5 0.1 0.7 2 14 28

Anti-Glut 3.7 7.1 15.8 145 146 160

f -e If: + f +

1 Ig 0.2” 0.4” 0.7” 18” 18 29

Stimulation (%) 60 73 26 26 0 0

NHIR cells were electroporated with nonimmune Ig or anti-Glut 1 antibodies (400 @g/ml) as described in Materials and Methods. Cells were depleted in DMEM-0.2% BSA for 3 h before glucose uptake determination. 3-0-methylglucose uptake was measured for 20 set in Krebs-Ringer phosphate buffer-0.2% BSA supplemented with 3-Omethyl-[“Clglucose (l-2.5 &i) at the concentration indicated in the table. Results are expressed as the mean + SEM of six determinations, performed in one experiment. The experiment was repeated twice with similar results. Stimulation is calculated as a percentage of the antiGlut 1 antibody effect over the control condition (nonimmune Ig). ” The effect of anti-Glut 1 Ig is statistically significant (P < 0.005).

loaded with either anti-Glut 1 antibodies or nonimmune Ig. As shown in Fig. 4, no differences were observed in the amount of Glut 1 in the two preparations. This excludes the possibility that a change in total glucose transporter content could be responsible for the increase in glucose transport in anti Glut l-loaded cells. Discussion We have used NHIR cells becausethesefibroblasts express high levels of insulin receptors (17) and display a rapid

29 -

Mr X 10m3 Non immune

electroporated

anti Glut 1

Ig

FIG. 4. Lack of effect of anti-Glut 1 antibodies on the amount of Glut 1 in NHIR cells. NHIR cells were loaded with nonimmune Ig (lanes A and B) or anti-Glut 1 antibodies (lanes C and D; 300 pg/ml). Twenty-four hours after the electroporation, crude membranes were prepared as described in Materials and Methods. The proteins [5 rg (lanes A and C) or 30 pg (lanes B and D)] were separated by SDSPAGE with a 10% resolving gel and transferred to nitrocellulose, and the sheets were blotted with anti-Glut 1 antibodies as described in Materials and Methods. Autoradiograms were exposed for 16 h at -80 C. Mr, Mol wt.

insulin-induced stimulation of glucose transport. We show that these cells expressed only Glut 1, as do most cell lines (2), and that overexpression of the insulin receptor did not induce the expression of Glut 4. A rapid insulin effect on glucoseuptake is not commonly observed in Glut l-containing cells, such as erythrocytes, brain, and HepG2 (l-3), in which no intracellular pool of Glut 1 molecules exists (25). By contrast, in Chinese hamster ovary cells, undifferentiated or differentiated 3T3-Ll, and rat adipocytes, Glut 1 is present both in an intracellular compartment and at the plasma membrane and is translocated in responseto insulin (11, 23, 26-29). Likewise, in NHIR cells, Glut 1 is located at similar levels in both the intracellular compartment and the plasma membranes, and the insulin stimulation of glucose uptake results from translocation of Glut 1 molecules to the plasma membranes(data not shown). Despite the identification of the glucose transporters sequences, the different domains of these molecules involved in glucosetransport regulation are not precisely known. Here, we have approached the role of the carboxy-tail of this transporter in glucosetransport, using antipeptide antibodies to the C-terminus of Glut 1. Since this part of the molecule

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GLUT

l-CARBOXY-TAIL

AND

is intracellular (2), we have used an electroporation procedure to allow the entry of Igs into cells, while preserving a high viability. This approach has been used to study the structurefunction relationship of some protein domains (21, 22). The electroporation of NHIR cells did not modify the insulin response for glucose transport. Furthermore, it does not impair insulin-induced amino acid uptake or cellular growth (21). Our results indicate that antibodies to Glut 1 specifically increased deoxyglucose uptake to a level similar to the insulin effect. This uptake was inhibited by cytochalasin-B, the sensitivity to this drug being similar in cells electroporated with control or anti-Glut 1 antibodies. Since 3-O-methylglucase uptake was stimulated to a similar extent as 2-deoxyglucase uptake, this suggests that glucose uptake per se was stimulated rather than glucose phosphorylation. The stimulatory effect of anti-Glut 1 antipeptides on glucose transport could be due to several mechanisms. A first possibility would be that anti-Glut 1 antibodies induced an increase in the total cellular Glut 1 level. This can be ruled out, since the amount of Glut 1 was similar in cells loaded with anti-Glut 1 antibodies and those loaded with nonimmune Ig. A second possibility would be that anti-Glut 1 antibodies induced glucose transporter recruitment to the plasma membrane. Due to the high number of cells required to perform subcellular fractionation, it was not feasible to test this hypothesis after electroporation. However, the additivity of the effects induced by anti-Glut 1 antipeptides and insulin was not in favor of such a hypothesis. Further, the effect of the antibodies is observed only at low sugar concentrations, suggesting that the affinity of the transporter was increased without any change in its maximal velocity. Hence, the biological effects of anti-Glut 1 antipeptides could be explained by a third possibility, which is that a direct interaction between the C-terminal part of the glucose transporters and the antipeptide enhanced the intrinsic activity of the transporters present at the cell surface and, thus, increased glucose transport across the membranes. Along this line, it has been recently reported that the intrinsic activity of Glut 1 can be modulated. Protein synthesis inhibitors or cadmium increase Glut 1 affinity for glucose in 3T3 Ll cells (23, 30, 31), while differentiation is accompanied by a decrease in Glut 1 activity (32). The Glut 1 carboxy-tail is not directly involved in glucose binding (33), but it could play a role in the regulation of the activity of the transporter. As it has been suggested that the amino-terminus of the transporters would be implicated in their cellular targeting (34), the COOH-terminus would be more closely involved in transporter activity. Indeed, a deletion of the C-terminal part appears to lock the glucose-binding site into the inward facing form, since it blocks glucose transport (35). The carboxy-tail of the transporter could, thus, play a role in the reorientation of the transporter from the inward to the outward conformational state. It has been proposed that putative regulator proteins could interact with glucose transporters, inhibiting their activity (36). Covalent modifications of the transporters, such as phosphorylation, could also change transport activity without modification of the transporter number at the cell surface. It is conceivable that the interac-

GLUCOSE

TRANSPORT

tion of anti-Glut 1 antibodies with the C-terminus of Glut 1 could prevent such interaction or modification. Facilitated glucose transporter isoforms share structural homologies, but the greatest sequence divergence is located in the C-terminal part of the transporter (13). Further experiments are required to know whether the C-terminal part of other isoforms plays a role in the conformational changes in the transporter. Acknowledgments We thank A. Grima, G. Visciano, and C. Minghelli for illustration work. We thank Dr. S. Heydrick for fruitful discussions. The gift of NHIR cells by Dr. Whittaker is greatly acknowledged.

References 1. Birnbaum MJ, Haspel HC, Rosen OM 1986 Cloning and characterization of a cDNA encoding the rat brain gluco&transporter protein. Proc Nat1 Acad Sci USA 83:5784-5788 2. Mueckler M, Caruso C, Baldwin SA, Panic0 M, Blench I, Morris HR, Allard WJ, Lienhard GE, Lodish HF 1985 Sequence and structure of a human glucose transporter. Science 229:941-945 3. Flier JS, Mueckler M, McCall AL, Lodish HF 1987 Distribution of glucose transporter messenger RNA transcripts in tissues of rat and man. J Clin Invest 79:657-661 4. Fukumuto H, Seino S, Imura H, Seino Y, Eddy RL, Fukushima Y, Byers MG, Shows TB, Bell GI 1988 Sequence, tissue distribution, and chromosomal localization of mRNA encoding a human glucose transporter-like protein. Proc Nat1 Acad Sci USA 85:5434-5438 5. Orci L, Thorens B, Ravazzola M, Lodish HF 1989 Localization of the pancreatic beta cell glucose transporter to specific plasma membrane domains. Science 245:295-297 6. Thorens B, Sarkhar HK, Kaback HR, Lodish HF 1988 Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and P-pancreatic islet cells. Cell 55:281-291 7. Kayano T, Fukumoto H, Eddy RL, Fan Y-S, Byers MG, Shows TB, Bell GI 1988 Evidence for a family of human glucose transporter-like proteins. Sequence and gene localization of a protein expressed in fetal skeletal muscle and other tissues. J Biol Chem 263:15245-15248 8. Birnbaum MJ 1989 Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell 57:305-315 9. Charron MJ, Brosius III FC, Alper SL, Lodish HF 1989 A glucose transport protein expressed predominantelv in insulin-responsive tissues. Prbc Nat1 Acad Sci USA 86:2535-2539 10. Fukumoto H, Kavano T. Buse IB. Edwards Y. Filch PF. Bell GL Seino S 1989 Cloning and characterization of the major insulin: responsive glucose transporter expressed in human skeletal muscle and other insulin-responsive tissues. J Biol Chem 264:7776-7779 11. James DE, Strube M, Mueckler M 1989 Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 338:83-87 12. Kaestner KH, Christy RJ, McLenithan JC, Braiterman LT, Cornelius P, Pekala PH, Lane MD 1989 Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-Ll adipocytes. Proc Nat1 Acad Sci USA 86:3150-3154 13. Gould GW, Bell GI 1990 Facilitative glucose transporters: an expanding family. Trends Biochem Sci 15:18-23 14. Walmsley AR 1988 The dynamics of the glucose transporter. Trends Biochem Sci 13:226-231 15. Gorga FR, Lienhard GE 1981 Equilibria and kinetics of ligand binding to the human erythrocyte glucose transporter. Evidence for an alternating conformation model for transport. Biochemistry 20:5108-5133 16. Holman GD, Rees WD 1987 Photolabelling of the hexose transporter at external and internal sites fragmentation patterns and

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GLUT evidence for 897:395-405

a conformational

change.

l-CARBOXY-TAIL Biochim

Biophys

AND Acta

17.

Whittaker J, Okamoto AK, Thys R, Bell GI, Steiner DF, Hofmann CA 1987 High-level expressionof human insulin receptor cDNA in

18.

Le Marchand-Brustel Y, Olichon-Berthe C, Grbmeaux T, Tanti JF, Rochet N, Van Obberghen E 1990 Glucose transporter in insulin

mouse

NIH

3T3

cells. Proc

Nat1 Acad

sensitive tissues of lean and obese agent BRL 26830A. Endocrinology 19.

Sci USA

GLUCOSE 27.

28.

Calderhead DM, Kitagawa K, Tanner LI, Holman GD, Lienhard GE 1990 Insulin regulation of the two glucose transporters in 3T3-

29.

Zorzano A, Wilkinson W, Kotliar N, Thoidis G, Wadzinkski BE, Ruoho AE, Pilch PF 1989 Insulin-regulated glucose uptake in rat

Ll adipocytes.

Oka Y, Asano T, Shibasaki Y, Kasuga M, Kanazawa Y, Takaku F

1988 Studies with antipeptide antibody suggest the presence of at least two types of glucose transporter in rat brain and adipocyte. J Biol Chem 263:13432-13439 20. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 21. Baron V, Gautier N, Kaliman P, Dolais-Kitabgi J, Van Obberghen E 1991 The carboxyl-terminal domain of the insulin receptor: its potential role in growth promoting effects. Biochemistry 30:93659370 22. Chakrabarti R, Wylie DE, Schuster SM 1989 Transfer of monoclonal antibodies into mammalian cells by electroporation. J Biol Chem 264:15494-15500 23. Clancy BM, Harrison SA, Buxton JM, Czech MP 1991 Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3-Ll adipocytes. J Biol Chem 266:10122-10130 24. Palfreyman RW, Clark AE, Denton RM, Holman GD, Kozka IJ 1992 Kinetic resolution of the separate Glut1 and Glut4 glucose transport activities in 3T3-Ll cells. Biochem J 284:275-281 25 Haney PM, Slot JW, Piper RC, James DE, Mueckler M 1991 Intracellular targeting of the insulin-regulatable glucose transporter (GLUT4) is isoform specific and independent of cell type. J Cell Biol 114:689-699 26 Harrison SA, Buxton JM, Helgerson AL, MacDonald RG, Chlapowski FJ, Carruthers A, Czech MP 1990 Insulin action on activity and cell surface disposition of human HepG2 Glucose transporters expressed in Chinese hamster ovary cells. J Biol Chem 265:57935801

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Potential involvement of the carboxy-terminus of the Glut 1 transporter in glucose transport.

The role of the carboxy-terminal domain of the Glut 1 glucose transporter was investigated using an antipeptide antibody to the C-terminal part of the...
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