THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 25, Issue of September 5, pp. 17502-17507,1992 Printed in U.S.A.

Site-directed Mutagenesis of GLUT1 in Helix 7 Residue 282 Results in Perturbation of Exofacial Ligand Binding*

(Received for publication, January 9, 1992)

Mitsuru HashiramotoS, Takashi KadowakiQ11, Avril E. Clark11 , Akihiro Muraoka85, Kaoru MomomuraQ, Hiroshi SakuraS, Kazuyuki TobeS, Yasuo Akanuma11, Yoshio YazakiQ, Geoffrey D. Holman[[, and Masato KasugaS From the $Second Department of Internal Medicine, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo- ku, Kobe 650, the §Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo- ku, Tokyo 113, the Tlnstitute for Diabetes Care and Research, Asahi Life Foundation, 1-6-1 Marunouchi, Chiyoda-ku, Tokyo 100, Japan, and the 11 Department of Biochemistry, University of Bath, Claverton Down, Bath BA2 7A Y, United Kingdom

The structure-function relationship of the HepG2/ erythrocyte-type glucose transporter (GLUTl) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose trans- porters were transfected were shown by Western blot- ting with a GLUTl anti-COOH-terminal peptide anti- body to have expression levels of GlnZs2 + Leu, AsnZ8’ + Ile, and Asn317 + Ile mutations that were comparable with the wild type. All three mutant GLUTl clones had high 2-deoxy-~-glucose transport activity com- pared with a nontransfected clone, suggesting that these residues are not absolutely required for the trans- port function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific li- gand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-l,3- bis(~-mannos-4-y~oxy)-2-propy~amine (ATB-BMPA). All three mutant GLUTl clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 GlnZs2 + Leu mu- tant was labeled by ATB-BMPA to a level that was only 6% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-~-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-~-glucose. 4,6-0- Ethylidene-D-glucose inhibited wild-type transport with a Ki of =12 mM, but this was increased to >120 mM in the GlnZs2 + Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-0- ethylidene-D-glucose.

The facilitated uptake of glucose into mammalian cells is mediated by at least five homologous glucose transporter

* This work was supported by grants from Ohtsuka Pharmaceutical Co., Ltd. for diabetes research (to M. K.), the Juvenile Diabetes Foundation International (to T. K.), and the Membrane Initiative of the Science and Engineering Research Council (United Kingdom) (to <:. D. H. and A. E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

isoforms (1). All five are predicted to possess nearly identical two-dimensional membrane topography and are likely to have similar tertiary structure. The glucose transporters are highly conserved proteins, and the mammalian transporters possess high levels of sequence identity to transporters found in many species including cyanobacteria (2), bacteria Escherichia coli (3, 4) and Zymomonm mobilis (5), yeast (6-8), algae (9), protozoa (lo), and plants (11). Given this high level of se- quence similarity, a common mechanism for facilitation of transport is likely. The transport of hexoses is generally thought to occur via a conformational change mechanism in which binding sites are alternately exposed to the outside and inside solutions (12-14). This model was partly based on studies involving site-specific hexose analogues such as 4,6- 0-ethylidene-D-glucose, an outside site-specific ligand, and n‘-propyl-P-D-glucoside, an inside site-specific ligand (12,15). These studies suggested that glucose is transported with a distinct polarity. Thus, there may be a structural link between outside and inside sites that forces the sugar to enter the transporter from outside with C-1 first and with C-4 and C-6 trailing. This relationship between sites in the transport pathway and the alternating conformational change model for transport can be usefully examined by site-directed mu- tagenesis.

Sequence alignment of the “glucose” transporter sequences (1-11, 16) shows that asparagine and glutamine residues are highly conserved and would therefore seem to be good candi- dates for substitution by site-directed mutagenesis. Since residues of this type can participate in hydrogen bonding to sugars (17), they may form part of a channel through the protein (18). Interestingly, we have found that these residues can be divided into those that are important for binding the inside site-specific ligand cytochalasin B and those that are important for binding the outside site-specific ligands ATB- BMPA’ and 4,6-O-ethylidene-~-glucose. We report here the effects of substitution at glutamine 282 and asparagine 288, which is predicted to occur in transmembrane helix 7 (TM7), and of asparagine 317, which is predicted to occur in trans- membrane helix 8 (TM8), and have examined the relative importance of these residues for transport and inner and outer binding site functions. This is the first demonstration by mutagenesis that helix 7 constitutes part of the outside ligand-binding site.

The abbreviations used are: ATB-BMPA, 2-N-4-(1-azi-2,2,2-tri- fluoroethyl)benzoyl-l,3-bis(o-mannos-4-yloxy)-2-propy~amine; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; TM, trans- membrane helix.

17502

Mutagenesis of GLUTl

EXPERIMENTAL PROCEDURES

Materials-Phloretin, cytochalasin E, galactose oxidase, neuramin- idase, protein A-Sepharose, keyhole limpet hemocyanin, and molec- ular weight markers were from Sigma. [4-3H]Cytochalasin B and lZ51- protein A were from Amersham International. 2-Deo~y-D-[2,6-~HI glucose was obtained either from Amersham International or Sigma. The detergent nonaethylene glycol dodecyl ether (CIZEg) was from Boehringer Mannheim. ATB-BMPA was synthesized as described by Clark and Holman (19).

Construction of Expression Vector-The vector pRC/CMV, con- structed for high level and stable expression in eukaryotic cells, is 5446 base pairs long. This pUC19 vector contains the plasmid origin for DNA replication and the p-lactamase gene. This vector also has the bacterial neomycin resistance gene fused to the SV40 promoter. The remaining region consists of the human cytomegalovirus IE1 promoter and the potylinker site fused to both the T7 and SP6 promoters. A full-length human glucose transporter (GLUT1) cDNA, constructed in the pGEM4z vector and called pGEM4z-GT1, was kindly provided by Dr. G. I. Bell (University of Chicago, Chicago). The Sal1 fragment of pGEM4z-GT1, with nucleotides 52-1803, which encompass the whole coding region, was filled in with the Klenow fragment in the presence of deoxynucleotide triphosphates and ligated into the HindIII linker. This GLUTl cDNA insert was subcloned into the HindIII site of the cytomegalovirus promoter of pRC/CMV, yielding an expression vector designated pRC/CMV-GTl(WT).

Site-directed Mutagenesis of Human GLUTl cDNA and Construc- tion of Mutant Expression Plasmids-Point mutations were intro- duced into the cDNA of human GLUTl by oligonucleotide-directed mutagenesis, which replaced glutamine 282 with leucine, asparagine 288 with isoleucine, and asparagine 317 with isoleucine. The template for mutagenesis was prepared by cloning a 1752-base HindIII-Hind111 fragment from pGEM4z-GT1 into M13mp19. A uracil-rich template was prepared in E. coli RZ1032, and mutagenesis was carried out by the method of Kunkel (20) using the primer 5"CAGCTGTCCCTG- CAGCTGTCT-3' for Q282L, 5'-TCTGGCATCATCGCTGTCTTC- 3' for N2881, and 5'-GGTATCGTCATCACGGCCTTC-3' for N3171, respectively, with each containing one base pair change from the sequence determined by Mueckler et al. (18). Suitable mutant clones were selected, and their sequences were confirmed by dideoxynucle- otide sequencing in M13. The final construct encoded amino acid sequences Val-Leu-Gln-Leu-Ser-Leu-Gln-Leu-Ser-Gly-Ile (Q288L), Gln-Leu-Ser-Gly-Ile-Ile-Ala-Val-Phe-Tyr-Tyr (N2881), and Gly-Ser- Gly-Ile-Val-Ile-Thr-Ala-Phe-Thr-Val (N317I). These sequences span residues 277-287 (Q282L), 283-293 (N288I), and 312-322 (N317I). The mutant fragments were reinserted into the BstEII-NheI site of pRC/CMV-GTl(WT).

Overexpression oi Human GLUTl in Chinese Hamster Ouary Cells-CHO-K1 cells, which have low endogenous glucose transport activity (Zl), were maintained in Ham's F-12 medium containing 10% fetal calf serum, transfected with pRC/CMV-GTl(WT), Q282L, N2881, and N317I by the calcium phosphate method, and selected by their resistance tc a 600 pg/ml concentration of the neomycin deriv- ative G418 (GIBCO). The cells expressing these glucose transporters were identified by Western blot analysis of the cell lysate using an anti-peptide antibody against the COOH-terminal domain of human GLUTl as described below. Mutant clones expressing amounts of protein similar to those of the wild type were selected for further study.

Peptide Synthesis and Production of COOH-terminal Peptide An- tibody-Peptide TPEELFHPLGADSQV, corresponding in sequence t o residues 478-492 of human GLUT1, was coupled to keyhole limpet hemocyanin. The conjugate (1.5 mg) was emulsified with Freund's complete adjuvant and injected into New Zealand White rabbits. Rabbit antibody production was boosted by injection of the antigen emulsified in Freund's complete adjuvant. The antibody was purified using peptide coupled to an Affi-Gel 15 column (Bio-Rad) (22).

Western Blot Analysis-Control CHO-K1 and GLUT1-overex- pressing CHO-K1 cell lines were grown in 6-well (35-mm) culture plates in Ham's F-12 medium containing 10% fetal calf serum with or without 600 pg/ml G418. Near-confluent cells were homogenized in 120 p1 of TES (10 mM Tris-HCI, 1 mM EDTA, and 250 mM sucrose, pH 7.4) with 1 mM phenylmethylsulfonyl fluoride at 4 "C. The ho- mogenates were centrifuged at 900 X g,,, for 10 min to sediment the fraction containing mainly nuclei, and the resulting supernatant was centrifuged at 17,000 X gmax for 75 min at 4 "C. 100 pg of the pellets were resuspended in 2% lauryl sodium dodecyl sulfate (SDS) (Pierce Chemical Co.), 75 mM dithiothreitol, 10% glycerol, 0.05% bromphenol

Exofacial Binding Site 17503

blue, and 50 mM Tris, pH 6.8. SDS-polyacrylamide gel electrophoresis was in a Laemmli (23) discontinuous buffer system with a 4% stacking gel and a 10% resolving gel. Proteins in the gels were electrophoreti- cally transferred to nitrocellulose filters (24) at 4 "C for 3 h. The filters were blocked in 25 mM Tris, 150 mM NaC1, and 0.1% Tween 20, pH 7.5, with 3% bovine serum albumin and then incubated overnight with an anti-COOH-terminal peptide antibody at a 1:20 dilution. The filters were then incubated with 0.5-1.0 pci of Iz5I-

protein A at room temperature for 1 h. The dried filters were auto- radiographed using Kodak AR x-ray films and intensifying screens at -70 "C. To quantify the Western blot analysis, the locations of immunoreactive proteins on the nitrocellulose were identified from autoradiographs. The glucose transporter proteins were then excised from the filters, and bound '''I was estimated by y-counting. Nitro- cellulose sections with surface areas equal to those of the excised bands were cut from the same filters immediately above and below each glucose transporter band and used as an estimate of background (or nonspecifically bound) radioactivity.

Labeling of Transporter Cell-surface Carbohydrate with Tritiated Sodium Borohydride-The CHO-K1 clones transfected with either wild-type or mutant GLUTl were grown to confluence in 35-mm culture dishes. The cells were then simultaneously treated with 10 units of galactose oxidase and 1 unit of neuraminidase in 1 ml of PBS for 30 min at 18 "C. The dishes were washed three times with 2 ml of PBS, and then 2 mCi of sodium borohydride (specific activity of 13.7 Ci/mmol) in 40 p1 of 50 mM NaOH were rapidly mixed with 20 p1 of 45 mM HC1 and added immediately to the cells in 1 ml of PBS (154 mM NaCl, 12.5 mM Na2P04, pH 7.4) (25). After maintaining the cells at 18 "C for 10 min, the dishes were washed four times in PBS, and then the cells were solubilized in 1 ml of detergent buffer containing 2% CIZEg, 5 mM sodium phosphate, and 5 mM EDTA, pH 7.2, with the proteinase inhibitors antipain, aprotinin, pepstatin, and leupep- tin, each at 1 pg/ml. Following centrifugation at 20,000 X g,,, for 20 min, the supernatant was subjected to immunoprecipitation with 20 p1 of protein A-Sepharose coupled to 100 pl of anti-GLUT1 antiserum. The immunoprecipitates were then subjected to electrophoresis for analysis of labeled transporters as described below for the ATB- BMPA labeling procedure.

2-Deoxy-D-g~ucose Uptake in CHO-KI Cells-The measurement of 2-deoxy-~-glucose uptake was performed essentially as described by Harrison et al. (26). Cells were cultured in 24-well (16-mm) plates and grown to confluence for 2-3 days. Cells were then washed three times with PBS. Next, the cells were washed once with 0.5 ml of Krebs-Ringer phosphate buffer containing 130 mM NaCI, 5 mM KC1, 1.3 mM CaClZ, 1.3 mM MgSO,, and 10 mM Na2HP04, pH 7.4, and incubated with an additional 0.5 ml for 20 min at 37 "C. P -Deoxy-~ [2,6-3H]glucose (0.3-1 pCi) was then added to give a final assay concentration of 0.1 m ~ , and cells were maintained at 37 'C fur 5-10 min. Uptake was terminated by addition of 1 ml/well ice-cold PBS containing 0.3 mM phloretin and three rapid washes with ice-cold PBS/phloretin. In experiments in which the inhibition of 2-deoxy-D- glucose transport by 4,6-0-ethylidene-~-glucose was studied, the transport assays were carried out in 35-mm culture dishes. The cells were incubated with the inhibitor at the indicated final concentrations for 10 min. Following the arrest of transport, cells were solubilized in 0.4 ml of 0.1% SDS or 1 ml of 0.1 M NaOH, and the extract was added to scintillant for estimation of radioactivity. Nonspecific 2- deoxy-D-glucose uptake was measured in the presence of 20 or 50 p~ cytochalasin B and 0.3 mM phloretin and was subtracted from each determination to obtain specific uptake.

ATB-BMPA Photolabeling of GLUTl-transfected CHO-Kl Cells- Confluent cells in 35-mm culture dishes were washed with PBS, incubated at -18 "C with 100 pCi of ATB-[2-3H]BMPA (specific activity of 10 Ci/mmol) in 200 pl of PBS for 2 min, and then irradiated for 1 min in a Rayonet RPR-100 photochemical reactor with 300-nm lamps (19). Following irradiation, the dishes were washed five times in PBS and dissolved in 250 p1 of 2% C,,E9 detergent buffer. 1 ml of methanol and 0.25 ml of chlorform were then added to the cell lysate, which was centrifuged at 9000 X gmaX for ~ 1 0 s . Then 750 p1 of water were added, and the sample was respun at 9000 X gmax for 1 min. The upper phase was removed, an additional 750 pl of methanol was added, and the sample was respun at 9000 x gmax for 2 min. The precipitated pellet was washed once in TES and then resuspended in 200 pl of TES. After adjusting the protein concentration to compare the mutant and wild-type transporter clones, the pellets were solu- bilized in electrophoresis sample buffer containing 10% SDS, 6 M urea, and 10% mercaptoethanol and subjected to electrophoresis using 10% acrylamide resolving gels and the Laemmli (23) discontinuous

17504 Mutagenesis of GLUTl Exofacial Binding Site buffer system. Gel slices, in scintillation vials, were dried at 80 “C for 2 h and then dissolved in 0.5 ml of alkaline hydrogen peroxide (2% (v/v) ammonium hydroxide in 30% hydrogen peroxide) at 80 “C for an additional 2 h. Scintillant was added, and the radioactivity was counted. The positions of the photolabeled peaks were compared with the positions of molecular weight markers in adjacent lanes. The levels of radioactivity associated with each peak were obtained by summing the radioactivity in all the slices under the peak and subtracting a background based on the average radioactivity of the slices on either side of the peak.

Cytochalasin H Photolabeling of GLUTI-transfected CHO-K1 Cd.s-CHO-Kl cells were grown to confluence in 90-mm culture dishes and, after homogenization in 2 ml of TES, were centrifuged a t 900 X g,... to remove a fraction containing mainly nuclei and then a t 20,000 x g,,., for 20 min to obtain a membrane fraction. 200 pg of membrane pellet in 400 pl of TES containing 100 p~ cytochalasin E were labeled with 1.4 pCi of [4-:’H]cytochalasin B by irradiation for 46 s in the Rayonet photochemical reactor. Following irradiation, the membranes were washed twice by centrifugation a t 20,000 X gmax for 20 min and resuspension in 3 ml of TES. The final pellet was resuspended in 100 pl of TES, solubilized in electrophoresis sample buffer, and subjected to electrophoresis for analysis of labeled trans- porters as described above for the ATB-BMPA labeling procedure.

RESULTS

CHO-K1 cell lines were transfected with GLUTl cDNA containing base changes that resulted in the following amino acid substitutions: Gln2R2 + Leu (Q282L), + Ile (N288I), and Asn:”’ + Ile (N3171). Each of these clones was shown by Western blotting with an anti-peptide antibody directed against sequence 478-492 of GLUTl (18) to have expression levels that were comparable with the wild type (Fig. 1) and that were 4-&fold higher than the nontransfected level.

The CHO-K1 cell line was chosen because this line has low levels of endogenous glucose transport activity (21). 2-Deoxy- D-glucose uptake measurements in each clone showed that the wild-type clone had a large increase (5.2-fold) in hexose uptake activity compared with the nontransfected CHO-K1 cells (Table I). The Q282L mutant clone had transport activ- ity similar to that of the wild-type clone. The K,,, and V,,, values for 2-deoxy-D-glucose net influx were also similar to those of the wild type (Fig. 2). The K,,, and VmaX values calculated from these data were 1.83 f 0.2 mM and 1458 f 97 pmol/min/mg of protein for the wild type and 1.07 & 0.38 mM and 735 f 136 pmol/min/mg of protein for the Q282L cells, respectively. Thus, at low concentrations of substrate, where the transport rate constant is equal to V,,,/K,, the wild-type and Q282L values are similar (the V,,,/K,,, ratios were 0.8 and 0.69 nmol/min/mg of protein/mM for the wild type and

75 K -

49 K -

1 2 3 4 5

FIG 1. Expression of mutant GLUTl transporters in CHO- K1 cells. CHO-K1 cells were transfected with cDNA coding for wild- type (lane 2), Q282L (lane 3 ) , N288I (lane 4 ) , and N317I (lune 5) transporters and compared with nontransfected cells (lane 1 ). A crude membrane fraction was obtained from stably transfected cells and subjected to Western blotting with a GLUTl COOH-terminal peptide antibody as described under “Experimental Procedures.” 100 pg of crude membrane fraction were added to each gel lane. Nitrocellulose sections were cut and counted to obtain quantification of the expres- sion level shown in Table I.

TABLE I Characterization of helix 7 and 8 mutants

Exprcwon Lcvcl (-hid)' S.Jrl.4 5.3~1.0 4 . 2 ~ 1 2 mcan7.X ohlalncd by (6.2. 9.3)

Wcslcrn Blomng ( 9L )h 9X.Y 7 x 3 143. I (n=3)d (n=3) (n=2)

2-dcoxy-D- ( Yo )b 1 0 0 Y2.2t6.1 5l.Ot3.X 7O.Oe16.1 Glucosc

Transport (Yo). Y3.2 65.1 48.9

(n=S) (n=5) (n=5)

Cell-Surl’acc ( ‘% )* 100 Y7.0t14.X mean 57.7 1 6 6 ~ 3 2 C;lrhohydr;w (57.0. 58.4)

Lahclmg (n=4) (n=2) (n.4)

Cylochal;!sln B ( ob ) h I 0 0 55.5+Y.4 mean 184 55.253.4

Lahellng ( ob (202.166)

56.1 234.Y 38.6 (n=4) (n=2) (n=3)

“ Ratio of the value for GLUT1-transfected cells to that for non- transfected CHO-K1 cells. Results are means +. S.E., except where n = 2 and individual results are shown.

* Percentage of the value for mutant GLUT1-transfected cells to that for wild-type transfected cells. Results are means f S.E., except where n = 2 and individual results are shown.

‘Values are corrected for expression level obtained by Western blotting.

n represents number of individual experiments.

: 0 0 v , 0 2 4 6 8 1 0 1 2

[SI (mM)

FIG. 2. Kinetic parameters of 2-deoxy-~-glucose net uptake in mutant Q282L GLUTl clone. Cells of the wild-type (0) and Q282L (0) clones were grown to confluence in 35-mm culture dishes. The net uptake of 2-deoxy-~-[2,6-~H]glucose was measured for 1 min at the indicated extracellular concentrations. Results shown are from a single experiment representative of two similar experiments. K, and Vmax values were determined by nonlinear regression (weighted for relative error) of the Michaelis-Menten equation.

Q282L cells, respectively). Some nonlinearity was observed in the reciprocal kinetic plots of 2-deoxy-D-glucose transport in the Q282L mutant; and a t high substrate concentrations, Q282L showed ~ 2 - f o l d slower net influx. We have confirmed that the Q282L transporter is expressed at the cell surface by oxidation of the cell-surface carbohydrate with galactose ox- idase followed by labeling with tritiated sodium borohydride and subsequent immunoprecipitation with a GLUTl anti- COOH-terminal peptide antibody (Fig. 3). Both wild-type and Q282L transporters were efficiently labeled by this procedure, whereas nontransfected cells showed very low labeling.

The substitution of asparagines 288 and 317 by isoleucine resulted in only 35 and 51% decreases respectively, in trans- port of 100 PM 2-deoxy-~-glucose when corrected for the expression level as measured by Western blotting (Table I). Table I also shows that both these mutant transporters were

Mutagenesis of GLUTl Erofacial Binding Site 17505

1 . 1 . . I . I " i ' - ~ ~ 4 6 10 12 Gel Sltce Number

Q282L GLUTl clone. Cells of the wild-type (0), Q282L (O), and FIG. 3. Cell-surface carbohydrate labeling of mutant

nontransfected (A) CHO-K1 clones were grown to confluence in 35- mm culture dishes. Cells were simultaneously treated with galactose oxidase and neuraminidase at 18 "C, and then 2 mCi of tritiated sodium borohydride were added. The cells were maintained at 18 "C for 10 min, washed four times in PBS, and then solibilized in CnE9 detergent buffer. Immunoprecipitation of the labeled transporters was then carried out using COOH-terminal peptide antiserum. The labeled transporters were then analyzed by electrophoresis. The po- sitions of the radioactive peaks in the gel lanes were compared with molecular weight markers (arrowheads). Results shown are from a single experiment representative of four similar determinations from two experiments as shown in Table I.

3 4 1 1 1 1 16K 97K 66K

0

4 5 K

1

n low 0 0 2 4 6 8 1 0

Gel Slice Number

FIG. 4. Cytochalasin B labeling of mutant Q282L GLUTl transporter. 200 pg of crude plasma membranes in 400 p1 of TES from nontransfected cells (A) and the wild-type (0) and Q282L (0) clones were labeled with 1.4 pCi of [4-3H]cytochalasin B by irradiation for 45 s in a Rayonet photoreactor. The membranes were washed twice and subjected to electrophoresis. The positions of the radioac- tive peaks in the gel lanes were compared with molecular weight markers (arrowheads). Results shown are from a single experiment representiatve of four similar experiments as shown in Table I.

expressed at the cell surface as detected by labeling with tritiated sodium borohydride.

We have examined the importance of glutamine 282 and asparagines 288 and 317 for binding and photolabeling by cytochalasin B, a permeant reagent that is thought to bind the region of GLUTl in the proximity of the inner glucose- binding site (27-31). Fig. 4 shows that the Q282L and N317I mutant transporters bound cytochalasin B at levels that were =40-50% of the wild-type level.

We next examined whether the mutant glucose transporters were able to interact with the impermeable reagent ATB- BMPA, which binds to GLUTl in the proximity of the outer glucose-binding site (19). Fig. 5 shows that clones N288I and N317I had very high levels of ATB-BMPA photolabeling that were comparable to those of the wild-type clone. However, the substitution of glutamine 282 by leucine resulted in a large reduction in ATB-BMPA photolabeling. The labeling was (5% of that observed in the wild-type clone and was not

1 I R K 45K 29K

I 2 4 6 8 10 12 14 Gel Sl~ce Number

FIG. 5. ATB-BMPA labeling of mutant GLUTl transport- ers. Cells of the wild-type (O), Q282L (X) , N288I (m), N317I ( 0 , and nontransfected (A) clones in 35-mm culture dishes were labeled with 100 pCi of ATB-[2-3H]BMPA in 250 pl of PBS at 18 "C by irradiation for 1 min in a Rayonet photochemical reactor. Following irradiation, the cells were washed five times in PBS and solubilized in CI2E9 detergent buffer. Protein was then precipitated with chlor- form/methanol and subjected to electrophoresis. The positions of the radioactive peaks were compared with molecular weight markers (arrowheads). Results shown are from a single experiment represent- ative of two similar experiments.

6

5

4

2 3 >

2

1

o ! . , . , . , . , . , . 1

0 10 20 30 40 50 60 [Ethylidene Glucose] (mM)

FIG. 6. Ethylideneglucose inhibition of transport activity of mutant Q282L GLUTl transporter. Cells of the wild-type (0) and Q282L (0) clones were grown to confluence in 35-mm culture dishes. The net uptake of 100 p~ 2-deo~y-D-[2,6-~H]glucose was determined at the indicated concentrations of 4,6-O-ethylidene-~- glucose. Results shown are from a single experiment representative of two similar experiments. The K, was determined by nonlinear regression (weighted for relative error) of the equation u/u, = K,/(K, + I).

significantly different from that observed in the nontrans- fected CHO-K1 cells. This result suggests that residue 282 is very important for outside site-specific ligand binding. Fur- thermore, it suggests that outside site-specific ligand binding can be completely abolished without major perturbation of the 2-deoxy-~-glucose transport activity and cytochalasin B binding activity.

To further characterize the Q282L mutant, we examined the ability of 4,6-O-ethylidene-D-glucose to inhibit P-deoxy- D-glucose transport in this mutant and the wild-type clone. Fig. 6 shows that 4,6-0-ethylidene-~-glucose is a very potent inhibitor of transport activity in the wild-type clone. The apparent K, was 11.7 & 0.77 mM, and this value is similar to that reported for inhibition of glucose transport activity in erythrocytes (12), where GLUTl is the predominant isoform, and in insulin-stimulated rat adipocytes (30), where GLUT4 is the predominant isoform. However, in the Q282L clone, 4,6-O-ethylidene-~-glucose was a very poor inhibitor of 2- deoxy-D-glucose transport, and the apparent K; was increased by 10-fold to 121 & 20 mM.

17506 Mutagenesis of GLUT1 Exofacial Binding Site

DISCUSSION

Mueckler et al. (18), in their analysis of the sequence of GLUT1, predicted that the transporter has 12 transmembrane helical strands and that the transporter is divided into two halves by a cytoplasmic central loop region. Both NH2- and COOH-terminal segments were predicted to be cytoplasmic, and this was subsequently confirmed by binding studies using polyclonal antibodies carried out in either right side-out or inverted vesicles containing human erythrocyte GLUTl (31).

Studies in which the GLUTl transporter was photolabeled by bismannose derivatives (19, 29), by cytochalasin B (19, 32), and by forskolin derivatives (33) all suggests that the COOH-terminal half of the protein (the COOH-terminal do- main) contains the ligand-binding sites. In each case, trypsin cleavage of the photolabeled transporters in the central loop region clearly showed that labeling occurs in the 18-kDa proteolytic fragment derived from the COOH-terminal half of the protein, but not in the NHz-terminal 22-kDa fragment. In addition, these photolabeling experiments have identified the regions of the protein that are labeled by the exofacial bismannose ligands as TM8 (34) and TM9 (29). In discussing the labeling obtained by the bismannose compound (29), we suggested that the hydrogen-bonding hydroxyls of the sugar moiety of the probe could interact with the neighboring am- phipathic regions of helix 7. In contrast with this exofacial labeling, cytochalasin B (29, 32) and forskolin (33) were shown to label in a region at the bottom of TMlO and TM11.

We have now studied the structural separation of the out- side and inside ligand-binding sites revealed in labeling stud- ies by site-directed mutagenesis of glutamines and asparagines in the COOH-terminal half of the protein. In this study, we have shown that glutamine 282 in helix 7 is important for binding the bismannose compound ATB-BMPA. A mechan- istically very interesting finding is that exofacial ligand bind- ing is selectively perturbed. In addition, it is clear that gluta- mine 282, asparagine 288, and asparagine 317 are not absolute requirements for transport or for binding the inside site- specific ligand cytochalasin B. The transport activity of the N288I mutant was slightly reduced (by 50% of the wild-type level), but this is a surprisingly small reduction in view of the absolute conservation of this residue in all the glucose trans- porter sequences so far analyzed (1-11).

In the two-dimensional model for GLUTl (18), glutamine 282 is located in the center of the helix in TM7. Thus, we consider that TM7 may move up in the membrane to accept exofacial ligands. This may involve some unfolding of the TM7 helix near the top. Studies in which the transporter was proteolyzed provide additional evidence to support this sug- gestion. It has been found that the exofacial bismannose photolabels 2-N-(4-azidosalicoyl)-1,3-bis(~-mannos-4-yloxy)- 2-propylamine (29) and ATB-BMPA (19) render the trans- porter resistant to proteolysis by trypsin and thermolysin at the bottom of helix 7, suggesting that a large conformational change occurs that moves this region upward so that it is not accessible to the protease. A similar effect was shown when erythrocyte GLUTl was maintained in the presence of 4,6-0- ethylidene-D-glucose and treated with trypsin (35). In con- trast with the protection from proteolysis afforded by the exofacial ligands, the cytochalasin B-labeled transporter was rapidly proteolyzed, suggesting that this inside site-specific ligand stabilizes a conformation of the transporter where the protease site is accessible (29,36). Helix 12 may also move up in the membrane to accept exofacial ligands. A COOH-ter- minal truncated GLUTl transporter has been shown to bind cytochalasin B normally, but shows reduced binding of the exofacial label ATB-BMPA (37). Similarly, Clark and Hol-

man (19) showed that the 18-kDa fragment of erythrocyte GLUT1, which is also truncated at the COOH terminus, binds cytochalasin B, but not ATB-BMPA. These results were interpreted as being consistent with the truncated mutant and the erythrocyte GLUTl trypsin fragment adopting an inward-facing conformation with reduced transport activity (19, 37). We suggest here that the results obtained with the truncated mutant and the erythrocyte GLUTl trypsin frag- ment are consistent with a close association of helixes 7 and 12. We propose that this occurs because the six helixes of the COOH-terminal half of the protein are arranged concentri- cally, with helix 7 next to helix 12, to form a channel and are lined by hydrogen-bonding residues. We are currently exam- ining this structure by molecular graphics techniques.' We suggest that the alternating conformational change that ex- poses the external site involves both TM7 and TM12 moving up in the membrane and that truncation of the COOH- terminal segment of TM12 prevents this upward helix move- ment. Studies in which cysteine residues at the top of helix 12 were labeled by impermeable thiol reagents provide addi- tional support for this postulate. May (38) has shown that the binding of cytochalasin B results in a decrease in the cell- surface accessibility of the top of helix 12 (consistent with this helix moving down in the membrane), whereas the bind- ing of exofacial ligands such as maltose increases the acces- sibility (consistent with the top of this helix moving up).

Whereas it is clear that many of the hexose-binding residues are deep in the helix bundle structure of the transporter, the structural separation of the outer and inner ligand binding function and transport function revealed by our study of Q282L, N2881, N3171, and other mutants led us to propose that outside (ATB-BMPA-binding) and inside (cytochalasin B-binding) sites are arranged contiguously. The outside site would be occupied by upward movement of helixes 7 and 12. In this conformation of the protein, the binding of a nontrans- ported exofacial probe would be very dependent on glutamine 282. However, a transported substrate such as P-deoxy-~- glucose would be rapidly relayed from the exofacial site to the inside site by a cluster of hydrogen-bonding residues in the center of the protein. In this position in the center of the protein, a single amino acid substitution would be less critical; and therefore, 2-deoxy-D-glucose binding and transport would be less dependent on glutamine 282. However, as the Q282L mutation alters specificity at the exofacial site, the transport of hexoses other than 2-deoxy-D-glucose may be perturbed, and we are currently investigating the kinetics of equilibrium exchange transport in this mutant with additional transported substrates.

A relay mechanism of the type proposed here has been described for the movement of lactose and H' through the E. coli lac permease (39). A mechanism such as this would also account for the perturbation of transport activity without a corresponding loss of external or internal ligand binding as reported for the mutant GLUTl clone in which tryptophan 412 was substituted by leucine (40). The tryptophan may be located at the relay point between outer and inner sites.

The studies on the Q282L mutant show that GLUTl helix 7 constitutes part of the outside ligand-binding site and that the outside site is structurally separate from the inside site. Our results suggest an alternating conformational model for transport in which the outside and inside binding sites are occupied by an alternating conformational change involving upward and downward movements of helixes 7 and 12 in

P. A. Hodson, D. J. Osguthorpe, A. E. Clark, G . D. Holman, M. Hashiramoto, T. Kadowaki, and M. Kasuga, unpublished data.

Mutagenesis of GLUTl Exofacial Binding Site 17507

which a transported substrate is relayed between outside and inside sites.

Acknowledgment-We are grateful to Dr. G. I. Bell for very kindly providing us with human GLUTl cDNA.

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REFERENCES

1. Gould, G. W., and Bell, G. I. (1990) Trends Biochem. Sci. 1 5 , 18-22 e Zhang, C. C., Durand, M. C., Jeanjean, R., and Joset, F. (1989) Mol.

Maiden, M. C. J., Davis, E. O., Baldwin, S. A,, Moore, D. C. M., and

Maiden, M. C. J., Jones-Mortimer, M. C., and Henderson, P. J. F. (1988)

Barnell, W. O., Yi, K. C., and Conway, T. (1990) J. Bacteriol. 172 , 7227-

Microbiol. 3 , 1221-1229

Henderson, P. J. F. (1987) Nature 325,641-643

J. Biol. Chem. 263,8003-8010

Szkutnicka, K., Tschopp, J. F., Andrews, L., and Cirillo, V. P. (1989) J .

Chang, Y. D., and Dickson, R. L. (1988) J. Biol. Chem. 263,16696-16703 Celenza, J. L., Marshall-Carlson, L., and Carlson, M. (1988) Proc. Natl.

Sauer, N., and Tanner, W. (1989) FEBS Lett. 259,43-46 Cairns, B. R., Collard, M. W., and Landfear, S. M. (1989) Proc. Natl. Acad.

Sauer, N., Friedlander, K., and Graml-Wicke, U. (1990) EMBOJ. 9,3045-

Barnett. J. E. G.. Holman. G. D.. and Mundav, K. A. (1973) Biochem. J.

7240

Bacteriol. 171 , 4486-4493

Acad. Sci. U. S. A. 85,2130-2134

Sci. U. S. A. 8 6 , 7682-7686

3050

131,’211-221 Gorga, F. R., and Lienhard, G. E. (1981) Biochemist? 20,5108-5113 Walmsley, A. R., and Lowe, A. G. (1987) Biochim. B~ophys. Acta 901,229-

Holman, G. D., and Rees, W. D. (1982) Biochim. Biophys. Acta 6 5 5 , 78-

Baldwin, S. A,, and Henderson, P. J. F. (1989) Annu. Rev. Physiol. 5 1 ,

Quiocho, F. A. (1986) Annu. Reu. Biochem. 5 5 , 287-315 Mueckler, C., Caruso, C., Baldwin, S. A,, Panico, M., Blench, I . , Morris, R.

941-945 H., Allard, W. J. , Lienhard, G. E., and Lodish, H. F. (1985) Science 2 2 9 ,

238

86

459-471

19. Clark, A. E., and Holman, G. D. (1990) Biochern. J. 269,615-622 20. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,488-492 21. Hasegawa, K., Anraku, Y., Kasahara, M., Akamatsu, Y., and Nishijima, M.

(1990) Biochim. Biophys. Acta 1051,221-229 22. Oka, Y., Asano, T., Shibasaki, Y., Kasuga, M., Kanazawa, Y., and Takaku,

F. (1988) J. Biol. Chem. 263,13432-13439 23. Laemmli, U. K. (1970) Nature 227,680-685 24. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U.

25. Calderhead, D. M., and Lienhard, G. E. (1988) J. Biol. Chem. 263 , 12171- S. A. 76,4350-4354

26. Harrison, S. A,, Buxton, J. M., He1 erson, A. L., MacDonald, R. G., 12174

ChlaDowski. F. J., Carruthers, A,, and Czech, M. P. (1990) J. Biol. Chem.

27. Deves, R., and Krupka, R. M. (1978) Biochim. Biophys. Acta 510,339-348 28. Basketter, D. A., and Widdas, W. F. (1978) J. Physlol. (Lond.) 278 , 389-

265; 5793-5801

29. Holman, G. D., and Rees, W. D. (1987) Biochim. Biophys. Acta 897 , 395-

30. Holman, G. D., Pierce, E. J., and Rees, W. D. (1981) Biochim. Biophys.

31. Davies. A.. Meeran. K.. Cairns. M. T.. and Baldwin, S. A. (1987) J. Biol.

401

405

Acta 646,382-388

33.

34.

35.

36.

37.

38. 39.

40.

32. Cairns, M. T., Alvarez, J., Panico, M., Gibhs, A. F., Morris, H. R., Chapman, Chem. 262,9347-9352

D., and Baldwin, S. A. (1987) Biochim. Biophys. Acta 905,295-310 Wadzinski, B. E., Shanahan, M. F., Scamon, K. B., and Ruoho, A. E. (1990)

Biochem. J. 272,151-158 Davies, A. F., Davies, A,, Preston, R. A. J., Clark, A. E., Holman, G. D.,

and Baldwin, S. A. (1991) Abstrfcts of the Meeting “Molecular Bask of Biological Membrane Function, Science and Engineering Research Council, Birmingham, U. K., October 2, 1991

Gihbs. A. F., Chapman. D., and Baldwin, S. A. (1988) Biochem. J. 256 , 421-427

Acta 902,402-405

Akanuma, Y., and Takaku, F. (1990) Nature 345,550-553

Karim, A. R., Rees, W. D., and Holman, G. D. (1987) Biochim. Biophys.

Oka, Y., Asano, T., Shibasaki, Y., Lin, J.-L., Tsukuda, K., Katagiri, H.,

May J. M. (1989) J. Mernbr. Biol. 108,227-233 Kabick, R. H., Bibi, E., and Roepe, P. D. (1990) Trends Biochem. Sci. 15 ,

Katagiri, H., Asano, T., Shibasaki, Y., Lin, J.-L., Tsukuda, K., Ishihara, 309-314

H., Akanuma, Y., Takaku, F., and Oka, Y. (1991) J. Biol. Chem. 266 , 7769-7773