©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ATP-sensitive Binding of a 70-kDa Cytosolic Protein to the Glucose Transporter in Rat Adipocytes (*)

(Received for publication, November 14, 1994; and in revised form, January 19, 1995)

Hongzhi Liu Shuhui Xiong Yanwei Shi Sam Jayanth Samuel Mohsen Lachaal Chan Y. Jung (§)

From the Biophysics Laboratory, Veterans Administration Medical Center, and the Department of Biophysical Sciences, State University of New York, Buffalo, New York 14215

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have identified a 70-kDa cytosolic protein (GTBP70) in rat adipocytes that binds to glutathione S-transferase fusion proteins corresponding to the cytoplasmic domains of the facilitative glucose transporter isoforms Glut1, Glut2, and Glut4. GTBP70 did not bind to irrelevant fusion proteins, indicating that the binding is specific to the glucose transporter. GTBP70 binding to the glucose transporter showed little isoform specificity but was significantly subdomain-specific; it bound to the C-terminal domain and the central loop, but not to the N-terminal domain of Glut4. The GTBP70 binding to Glut4 was not affected by the presence of 2 mM EDTA, 2.4 mM Ca, or 150 mM K. The binding was inhibited by ATP in a dose-dependent manner, with 50% inhibition at 10 mM ATP. This inhibition was specific to ATP, as ADP and AMP-PCP (adenosine 5`-(beta,-methylenetriphosphate)) were without effect. GTBP70 did not react with antibodies against phosphotyrosine, phosphothreonine, or phosphoserine, suggesting that it is not a phosphoprotein. The binding of GTBP70 to Glut4 was not affected by the pretreatment of adipocytes with insulin. When these experiments were repeated using rat hepatocyte cytosols, no ATP-sensitive 70-kDa protein binding to the glucose transporter fusion proteins was evident, suggesting that either GTBP70 expression or its function is cell-specific. These findings strongly suggest the possibility that GTBP70 may play a key role in glucose transporter regulation in insulin target cells such as adipocytes.


INTRODUCTION

A family of intrinsic membrane proteins (facilitative glucose transporters) catalyze the uptake and release (transport) of glucose in animal cells(1, 2) . Six isoforms differing in tissue distribution and sensitivity to regulation have been identified in this family(2) . In muscle and adipose tissues, the transporter function is further subjected to hormonal and metabolic regulation determining the rate of cellular glucose utilization(3, 4) . Rat epididymal adipocytes, for example, express largely Glut4 (^1)(90%) with a small amount of Glut1(5) . The two isoforms in adipocytes differ in their relative abundance between the plasma membrane and the intracellular storage pool. In basal adipocytes (in the absence of insulin), only 5-6% of the cellular Glut4, but as much as 40% of Glut1, are at the cell surface and functional, the rest being stored in microsomal pools(6, 7) . In the presence of insulin, the steady-state cell surface pool size of Glut4 increases 6-20-fold, while that of Glut1 increases about 2-fold, the former accounting for the most of the insulin-induced glucose transport stimulation(7, 8) .

By labeling cell surface pool of Glut4 with an impermeable glucose analog, [^3H]1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl 4-benzoylbenzoate, and studying the kinetics of the label mixing at the steady state, we (9) have shown previously that Glut4 in both basal and insulin-stimulated adipocytes constantly and rapidly recycles between the plasma membrane and the intracellular pool, and that this process is describable essentially by two first order rate constants, one for internalization (k), and the other for externalization (k). The study has also revealed that insulin modulates both of these rate constants, reducing k and increasing k by about 3-fold each. In similar experiments, we (10) have also shown that okadaic acid, a well known serine/threonine phosphatase inhibitor and an insulin mimicker, increases k about 3-fold without affecting k, suggesting an involvement of protein phosphorylation in Glut4 externalization pathway. The biochemical mechanisms that determine these rate constants are yet to be elucidated.

Many cell surface proteins and receptors recycle via endocytosis and exocytosis(11) , and these processes are known to be controlled by a set of specific cytosolic proteins or adaptors that bind to the cytoplasmic domain of these proteins(11, 12) . Glut4 recycling may also be regulated by a specific cytosolic protein or proteins that interact with glucose transporters at their cytoplasmic domain. Regarding the insulin action on glucose transport, the biochemical/molecular pathway linking the insulin signal generated at the receptor to the glucose transporter is currently unknown. A direct interaction of a cytosolic protein with the glucose transporter may be the key step in this transduction pathway being directly responsible for the modulation of glucose transporter recycling kinetics. Evidence indicates that insulin also causes a significant increase in the intrinsic activity of the glucose transporters(13) . An importance of the C-terminal cytoplasmic domain for Glut1 intrinsic activity has been noted(14) , and this intrinsic activity regulation may also be mediated by a direct interaction of a cytosolic protein with the cytoplasmic domain of glucose transporters. This putative protein could be an enzyme such as a protein kinase or a protein phosphatase, as is the case in the regulation of glycogen synthase activity by insulin(15) .

The present study is an attempt to identify a protein that binds to the glucose transporter and modulates glucose transporter recruitment or intrinsic activity. We made GST fusion proteins corresponding to the N terminus, the central loop, and the C terminus of the cytoplasmic domains for Glut4, Glut1, and Glut2, and we studied whether any cytosolic proteins bind in vitro to these fusion proteins. We found a 70-kDa cytosolic protein in rat adipocytes that binds to these fusion proteins but not to irrelevant fusion proteins. The binding was displaced by ATP, but not by ADP and AMP-PCP. This binding was not evident in hepatocyte cytosols, suggesting that the 70-kDa protein expression or its function is specific to adipocytes. The role, if any, of this protein in glucose transporter regulation is yet to be determined.


EXPERIMENTAL PROCEDURES

Construction, Expression, and Purification of GST Fusion Proteins

The specific cDNA fragments encoding the cytoplasmic domains of rat glucose transporters were generated by PCR amplification. Rat GLUT1, GLUT4, and GLUT2 cDNA plasmids were supplied by Drs. M. Birnbaum and H. F. Lodish of Boston, MA. Seven pairs of sense and antisense primers were synthesized: rG1C, 5`-CAGGATCCTCAAAGTTCCTGAGACCAAAGGC-3` and 5`-CGAATTCACACTTGGGAGTCAGCCCCCAG-3`; rG2C, 5`-CAGGATCCTTAAAGTTCCAGAAACC-3` and 5`-CGAATTCACACAGTCTCTGATGA-3`; rG4C, 5`-CAGGATCCTAAGAGTGCCTGAAACCAGA-3` and 5`-GGAATTCAGTCATTCTCATCTGGCCCTAA; rG1L, 5`-GTGGGATCCTCTGCCCTGAGAGCCCCCGCTTC-3` and 5`-AGAATTCTGGGCTGGCGGTAGGCGGGTGA-3`; rG2L, 5`-ACGGATCCTCTGTCCAGAAAGCCCCAGA-3` and 5`-AGAATTCTGGGCTGCCGATAATTCGG-3`; rG4L, 5`-TGAATTCCTGTCCCGAGAGCCCCCGCTAC-3` and 5`-AGAATTCGGGGCTGCCGGTGGGTACGGCT; rG4N, 5`-TTGGATCCAGATGCCGTCGGGTTTCCAG-3` and 5`-GGAATTCCCAGTGTCCCAGTCACTCG-3`. Each sense primer contains a 5` BamHI site, and each antisense primer incorporates a 3` EcoRI site. The PCR products were purified and digested with BamHI and EcoRI, and subsequently subcloned in frame and downstream of Glutathione S-transferase into the pGEX.3X expression vector (Pharmacia Biotech Inc.). The fidelity of the construction was verified by restriction analysis. The plasmids encoding the fusion proteins were transformed into DH5alpha Escherichia coli strain (Life Technologies, Inc.). Fusion proteins were induced by isopropyl-1-thio-beta-D-galactopyranoside and affinity-purified with glutathione-agarose beads (Sigma) as described (16) . Proteins were separated in SDS-PAGE gels and visualized with Coomassie Blue staining. The amount of GST fusion proteins on glutathione-agarose beads was determined by protein assay after elution with 2% formic acid.

Metabolic Labeling of Rat Adipocytes with [S]Methionine orP(i)and Isolation of Cytosols

Adipocytes were isolated from epididymal fat pads of Sprague-Dawley male rats weighing 180-220 g by collagenase digestion methods as described(9) . For S labeling, cells were washed with methionine/cysteine-deficient Dulbecco's modified Eagle's medium (ICN) supplemented with 2% bovine serum albumin, 4 mML-glutamine, and 20 mM Hepes, and incubated in the same medium for 60 min. Then cells (final cytocrit of 35-40%) were added 0.2-0.4 mCi of TranS-label (ICN)/ml and incubated for an additional 3 h. Bovine serum albumin and free S label were removed by washing with homogenizing buffer (50 mM Hepes, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl(2), 10% glycerol, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 4 mg/ml aprotinin, 5 mg/ml leupeptin, 2 mg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride, at pH 7.4). Cells were then resuspended in an equal volume of homogenizing buffer and homogenized in 10 strokes using a glass-Teflon homogenizer (Thomas Scientific, Philadelphia, PA). Cytosols and membranes were separated by centrifugation (200,000 times g, for 90 min) at 4 °C. For P labeling, the procedures were similar to those above for S labeling, except that cells were washed and incubated with phosphate-deficient Dulbecco's modified Eagle's medium (ICN) and labeled with 0.2-0.4 mCi of phosphorus-32 (ICN)/ml of cells for 2 h. Insulin (Sigma), when specified, was added to cell suspension prior to termination of metabolic labeling and also to subsequent washing with homogenizing buffer for total of 20 min of treatment. Similar procedures were also used for S labeling of rat hepatocytes. Incorporation of radioactivity in protein was estimated by counting radioactivity in trichloroacetic acid precipitates.

Assay of Cytosolic Protein Binding to GST Fusion Proteins

GST fusion proteins were purified and immobilized using glutathione-agarose beads (5-10 mg of protein/ml of resin). S- and P-labeled rat adipocyte cytosols (200-500 µg for each set) were precleared with glutathione-agarose beads, and incubated with GST fusion proteins (40-100 µg for each set) at 4 °C for 6 or 18 h with end-over-end rotation. In some experiments, 1% CE(8) (octalthylene glycol dodecyl ether) was added to the cytosols to reduce nonspecific binding to the agarose beads. Following the incubation, the beads were washed three time with homogenizing buffer or phosphate-buffered saline (PBS), and subsequently eluted with 5 mM glutathione in 50 mM Tris-HCl, pH 8.0. The eluted proteins were mixed with Laemmli's sample solubilizer and then separated by SDS-PAGE. After Coomassie Blue staining and destaining, the gels were treated with EN^3HANCE(TM) (DuPont NEN), dried, and subjected to fluorography for 3-7 days at -70 °C.

Immunoblotting

The procedure for immunoblotting is described in detail elsewhere(9) . The enhanced chemiluminescence reagent (Amersham Corp.) was used to visualize the immunoreactive proteins. Rabbit anti-Glut4 polyclonal sera were raised against two synthetic peptides corresponding to the C-terminal sequence 443-483 or the N-terminal sequence 1-12 of rat Glut4, and used after affinity purification(9) . Affinity-purified rabbit anti-Glut1 C-terminal peptide antibody was a generous gift from Dr. E. Gibbs (Pfizer Central Research). Rabbit anti-Glut2 C-terminal antibody was purchased from Eastacres (Southbridge, MA). Horseradish peroxidase-conjugated recombinant anti-phosphotyrosine antibody (RC20) was from Transduction Laboratories (Lexington, KY). Monoclonal anti-phosphothreonine, monoclonal anti-phosphoserine, monoclonal anti-heat shock protein 70 (HSP70), and anti-mouse IgG peroxidase-conjugated antibody were products of Sigma. Protein A-peroxidase was obtained from Zymed Laboratories (San Francisco, CA).

Purification and Identification of GTBP70

Adipocyte cytosol (16 mg of protein obtained from about six rats) and S-labeled adipocyte cytosol (1 mg of protein) were pooled in a final concentration of 20 mg of protein/ml in 1% CE(8), and precleared by passing through 200 µl of glutathione-agarose beads. One milligram of GST or GST-G4C fusion protein, immobilized on 120 µl of glutathione-agarose beads, was packed into a 1-ml capacity pipette tip plugged with glass wool to prepare GST and GST-G4C column, respectively. The GST column was connected to the GST-G4C columns in tandem. Precleared cytosol was passed through the columns at 0.03 ml/min. Once all cytosol was passed through, the GST-G4C column was disconnected from the GST column, and the GST-G4C column was washed sequentially, at a speed of 0.1 ml/min, with 5 ml of 1% CE(8) in PBS, 5 ml of 0.5% CE(8) in PBS, 1 ml of PBS, and 1 ml of 50 mM Tris-HCl, pH 8.0. Proteins on the column were then eluted at a speed of 0.03 ml/min with 1 ml of 10 mM ATP in 10 mM MgCl(2), 50 mM Tris-HCl, pH 8.0. Column fractions were collected, concentrated using the Centricon concentrators (Amicon), and subjected to SDS-PAGE separation in 10% acrylamide slab gels. The protein bands were visualized and quantitated by rapid Coomassie staining (Diversified Biotech, Boston, MA) and fluorography.


RESULTS

GST fusion proteins corresponding to the C terminus and central loop of Glut1, Glut2, and Glut4, and to the Glut4 N terminus, were made using rat glucose transporter sequences (Fig. 1A). SDS-PAGE revealed that each of the fusion proteins migrated as a protein-staining band at the predicted molecular weight (Fig. 1B). The C- and N-terminal fusion proteins were stable. The central loop fusion proteins, however, were unstable; about 50% of each fusion protein was degraded during purification, giving extra protein bands of smaller molecular mass on SDS-PAGE. The C- and N-terminal fusion proteins were further verified based on reactivities in immunoblot using isoform and domain-specific antibodies (not illustrated).


Figure 1: Construction, expression, and purification of GST fusion proteins. The specific cDNA fragments encoding the N-terminal, central loop, and C-terminal cytoplasmic domains of rat Glut1, Glut2, and Glut4 were amplified by PCR and subcloned into the pGEX.3X expression vector. The corresponding GST fusion proteins were expressed in E. coli and purified by glutathione-agarose affinity chromatography as detailed (see ``Experimental Procedures''). A, schematic structure of GST fusion proteins: GST alone (GST), Glut1 C terminus (G1C), Glut1 central loop (G1L), Glut2 C terminus (G2C), Glut2 central loop (G2L), Glut4 N terminus (G4N), Glut4 C terminus (G4C), and Glut4 central loop (G4L). B, SDS-polyacrylamide gel electrophoretic separation of purified GST fusion proteins revealed by Coomassie Blue staining.



Using these glucose transporter fusion proteins, interactions of the rat adipocyte cytosolic proteins with the glucose transporter cytoplasmic domains were studied. Glut4 C-terminal GST fusion proteins immobilized on glutathione-agarose beads were incubated with S-labeled adipocyte cytosol for 6 or 18 h at 4 °C. After intensive washing with buffer to eliminate nonspecific binding, bound proteins were eluted with 5 mM glutathione as described under ``Experimental Procedures.'' SDS-PAGE analysis of the glutathione eluate revealed a S-labeled band with an estimated molecular mass of 70 kDa (Fig. 2). This 70-kDa labeled band was not present in glutathione eluate from the beads bearing GST alone (Fig. 2), indicating that this cytosolic protein binds to Glut4 C terminus but not to GST. The binding specificity of this 70-kDa protein to the glucose transporter was further confirmed in control experiments using three irrelevant GST fusion proteins (Fig. 2). TK7 (provided by Dr. J. G. Koland, University of Iowa), a GST fusion protein containing the cytoplasmic domain of epidermal growth factor receptor including tyrosine autophosphorylation sites, did not bind any 70-kDa protein. FP1 and FP2, two irrelevant GST fusion proteins derived from a mouse hepatocyte protein, also failed to bind any 70-kDa protein (Fig. 2). The GST fusion protein obtained by expressing reversed nucleotide sequence of the Glut4 C-terminal downstream of GST also gave no 70-kDa binding (data not shown). It is clear in these experiments that the binding of this 70-kDa protein is specific to the glucose transporter. We refer to this protein as GTBP70 (glucose transporter-binding protein, 70 kDa).


Figure 2: Binding of a 70-kDa, cytosolic protein to the Glut4 C-terminal GST fusion protein. S-Labeled rat adipocyte cytosol (300 µg of protein in 350 µl/set) were precleared with glutathione-agarose beads and incubated with beads bearing the Glut4 C-terminal GST fusion protein (40 µg/set) or beads bearing the irrelevant fusion proteins (40-60 µg/set), at 4 °C overnight with end-over-end rotation. Following the incubation, the beads were washed three times with phosphate-buffered saline and eluted with 150 µl of 5 mM glutathione in 50 mM Tris-HCl, pH 8.0. The eluate was treated with Laemmli's sample solubilizer, and then electrophoresed by SDS-PAGE. After Coomassie Blue staining and destaining, the gels were treated with EN^3HANCE, dried, and exposed for 3 days at -70 °C. Molecular size markers are as indicated. GST, GST protein alone; G4C, Glut4 C-terminal fusion protein; TK7, the cytoplasmic domain containing tyrosine autophosphorylation site of epidermal growth factor receptor; FP1 and FP2, the C-terminal segment and the full length of an immunosuppressive protein, respectively. The lane labeled 1% CE(8) at the bottom illustrates the binding experiment done in the presence of CE(8). Similar results were obtained in four independent experiments.



GTBP70 binding to the glucose transporter fusion proteins was significantly domain-specific, but only slightly isoform-specific (Fig. 3). For each isoform tested, GTBP70 binding was evident with the central loop and the C terminus, but the intensity of binding was much greater with the central loop than the C terminus. The binding was virtually absent with Glut4 N terminus. No significant difference in GTBP70 binding to the central loops were observed among three isoforms tested. For the C-terminal segments, GTBP70 bound slightly more to Glut4 than Glut1 and Glut2. Since the central loop fusion proteins are rather unstable and degraded during the purification and the storage, the degraded, partial central loop fusion protein was tested for GTBP70 binding; only intact central loop was found to bind GTBP70, while degraded central loop fusion protein lost its binding with GTBP70 (data not shown).


Figure 3: Domain and isoform specificity of GTBP70 binding. The binding of adipocyte cytosolic proteins to GST fusion proteins corresponding to the C terminus and the central loop of Glut1, Glut2, and Glut4, and the N-terminal domain of Glut4, using S-labeled cytosol (500 µg protein/set in 300 µl) and in the presence of 1% CE(8). The experiments were otherwise identical to those described in Fig. 3. Top, fluorogram, representative of two experiments. Bottom, relative staining intensities of GTBP70 binding to different domains of different isoforms calculated from two independent sets of experiments, normalized with GTBP70 binding to the Glut4 central loop.



We looked for factors that would regulate GTBP70 binding to glucose transporters. The inclusion of KCl (150 mM), CaCl(2) (2.4 mM), or EGTA (2 mM) in cytosols did not affect GTBP70 binding to the Glut4 C-terminal GST fusion protein (Fig. 4). The presence of ATP (20 mM) in cytosols, however, reduced GTBP70 binding in these experiments (Fig. 4). This inhibition of GTBP70 binding by ATP was dose-dependent; it was significant at 5 mM ATP and was about 50% of maximum at 10 mM ATP (Fig. 5). Furthermore, ADP or AMP-PCP (Boehringer Mannheim), a non-hydrolyzable ATP analog, had no effect in these experiments (Fig. 6), indicating that the inhibition is specific to ATP. When S-labeled cytosol of higher specific radioactivity was applied in the binding assay, a S-labeled band with apparent molecular mass of 73 kDa was also evident in these experiments, although its intensity was much less compared to GTBP70. The binding of the 73-kDa protein was also inhibited by ATP ( Fig. 4and Fig. 6).


Figure 4: Effects of KCl, CaCl(2), EGTA, and ATP on GTBP70 binding. 150 mM potassium chloride, 2.4 mM calcium chloride, 2 mM EGTA supplemented with or without 20 mM ATP (all in final concentration) were added to S-labeled rat adipocyte cytosol, then the cytosols were incubated with Glut4 C-terminal fusion protein. The binding experiments were otherwise identical to those in Fig. 3. Top, fluorogram for the detection of S-labeled binding protein. Bottom, Coomassie Blue protein staining of SDS-PAGE corresponding to the fluorograph above. Molecular size markers shown are, from the top down, 116, 97, 66, 45, and 29 kDa. Results similar to these were obtained in two independent experiments.




Figure 5: Dose-dependent inhibition by ATP of GTBP70 binding. ATP was added to S-labeled rat adipocyte cytosol at the final concentration of 0, 1, 5, 10, and 20 mM, respectively; then, these cytosols were used for GTBP70 binding to the Glut4 C-terminal fusion protein. The binding assay, SDS-PAGE separation, and fluorography were otherwise similar to those described in Fig. 3and Fig. 5. Top, fluorogram; bottom, protein stain corresponding to the fluorography above. The results were reproduced in two other similar experiments.




Figure 6: Effects of ATP, ADP, and AMP-PCP on GTBP70 binding. S-Labeled rat adipocyte cytosol (260 µg of protein/set in 500 µl) was incubated with Glut4 C-terminal fusion protein (100 µg/set) immobilized on glutathione-agarose beads overnight at 4 °C to allow GTBP70 binding. The beads were then washed and incubated without (CON) or with 10 mM ATP (ATP), ADP (ADP), or AMP-PCP (AMP-PCP) in a buffer containing 10 mM MgCl(2), 50 mM Tris-HCl, and either 150 mM NaCl (NaCl) or 150 mM KCl (KCl), at room temperature with end-over-end rotation for 30 min. Then the beads were washed again and subsequently eluted with 5 mM glutathione. The eluted proteins were separated on SDS-PAGE, and the gel was subjected to fluorography for GTBP70 (top) and protein staining of the GST fusion proteins with Coomassie Blue (bottom). The results presented are representative of three experiments. Molecular size markers shown are (from the top down) 66, 45, and 29 kDa.



Possible effects of insulin treatment on GTBP70 binding activity were measured using glucose transporter C-terminal GST fusion proteins (Fig. 7). For all three isoforms tested, there were no significant differences in GTBP70 binding activity between cytosols obtained from basal adipocytes and insulin-treated (100 nM, for 20 min, at 20-37 °C) adipocytes. The insulin treatment clearly increased phosphorylation of both the insulin receptor beta subunit and the insulin receptor substrate 1 in these experiments (not shown), suggesting that insulin does not affect GTBP70 binding to the glucose transporters.


Figure 7: Insulin effect on GTBP70 binding to the glucose transporter. S-Labeled adipocytes were incubated without (- insulin) or with (+ insulin) 100 nM insulin for total 20 min. For insulin-treated cells, insulin was present throughout washing and homogenization. Basal and insulin-treated cytosols were used for GTBP70 binding assays. In each set, 230 µg of cytosol protein in 500 µl were applied to 100 µg each of Glut1, Glut2, and Glut4 C-terminal GST fusion protein beads and GST beads, respectively, in the presence of 1% CE(8). SDS-PAGE separation of eluate with 5 mM glutathione, followed by fluorography for GTBP70 detection (top) and protein staining for fusion protein quantitation (bottom) were performed as described in Fig. 3. Results similar to these shown were obtained in two independent sets of experiments.



To test if GTBP70 is a phosphoprotein, GTBP70 isolated from basal and insulin-treated cytosols with Glut4 C-terminal fusion protein was subjected to immunoblots using monoclonal antibodies specific for phosphoamino acids. No decoration of GTBP70 was evident in immunoblots with anti-phosphotyrosine antibody, while a clear increase in tyrosine phosphorylation of insulin receptor substrate 1 and insulin receptor beta subunit in insulin-treated adipocytes was evident in these experiments (not illustrated). Similar negative results were also found in immunoblots with anti-phosphothreonine and anti-phosphoserine antibodies. The reactivities of these two antibodies, however, have not been thoroughly verified. GTBP70 binding to glucose transporter fusion proteins was not detectable by P labeling of adipocytes (data not shown). These findings suggest that GTBP70 is not a phosphoprotein.

S-Labeled rat hepatocyte cytosol was assayed for GTBP70 binding activity using C-terminal GST fusion proteins. When cytosols containing equal amounts of radioactivity and similar amounts of proteins were used, GTBP70 binding was evident with adipocyte cytosol but not with hepatocyte cytosol (Fig. 8). No protein binding that is specific to the transporter fusion proteins was evident in hepatocyte cytosol.


Figure 8: Tissue specificity of GTBP70 binding. Cytosols were prepared from S-labeled rat adipocytes and hepatocytes and assayed for GTBP70 binding to Glut1, Glut2, and Glut4 C-terminal fusion proteins. To compare GTBP70 binding activities directly, cytosols containing equal amounts of radioactivity (1.8 times 10^6 dpm) and similar amounts of proteins (230 µg of adipocyte cytosol protein and 160 µg of hepatocyte cytosol protein, each in 350 µl) were used in binding assay. Fluorogram (top) and corresponding protein staining of gel (bottom) were obtained as in Fig. 4. The results shown were reproduced in two additional experiments.



GTBP70 was purified to homogeneity by GST-G4C affinity column and ATP elution as described under ``Experimental Procedures.'' SDS-PAGE of this GTBP70 preparation showed a single, Coomassie Blue staining band with an estimated molecular mass of 70 kDa, which precisely overlapped the S-labeled GTBP70 in the same gel (not shown). We also identified a 73-kDa protein, the counterpart of the 73-kDa S-labeled band. The recovery of GTBP70 was estimated based on its Coomassie Blue staining intensity calibrated against that of bovine serum albumin standard in SDS-PAGE. The yield of GTBP70 is approximately 100 ng/2 mg of cytosolic protein.


DISCUSSION

One notable feature common to all facilitative glucose transporters is a large (accounting for more than 30% of the entire protein mass), cytoplasmic domain, suggesting its functional importance. This domain is made of three subdomains including both N and C termini and a large central loop(17) . A number of recent observations indeed indicate the importance of these domains as possible molecular determinants for the constitutive regulation of transporter targeting to the plasma membrane. Experiments using Glut1 and Glut4 chimerical proteins revealed the importance of the C terminus and the central loop for the constitutive regulation of glucose transporter distribution between the plasma membrane and the intracellular storage pool(18, 19, 20, 21) . A leucine-leucine motif in the C terminus of Glut4, for example, has been shown to be critical for its retention in the storage pool(22, 23) . The importance of the phenylalanine-based internalization motif at the N terminus of Glut4 in constitutive subcellular distribution is also indicated(24) . Studies with deletion mutants indicated that the intrinsic activity of Glut1 also requires the intactness of at least a part of the C-terminal cytoplasmic domain(14, 25) . How information stored in these domains are translated into protein function and regulation is unknown and can only be speculated at this time. It is quite likely that a specific cytosolic protein or proteins physically interact with transporters at these specific cytoplasmic domains, regulating transporter redistribution or intrinsic activity.

Using GST-glucose transporter cytoplasmic domain fusion proteins, we have identified here a 70-kDa, glucose transporter-binding protein (GTBP70) in rat adipocyte cytosol. This protein bound only to glucose transporter fusion proteins, but not to any of the four irrelevant fusion proteins tested, indicating that the binding is specific to glucose transporters. The GTBP70 binding was not particularly isoform-specific. GTBP70 binds to the C termini and the central loops of the three facilitative glucose transporter isoforms tested, namely Glut1, Glut2, and Glut4. GTBP70 binds to the C terminus of Glut4 with a slight preference over the C terminus of Glut1 or Glut2, but its binding to the central loop shows little preference among isoforms. The binding shows a significant subdomain specificity within the cytoplasmic domains. GTBP70 binds to Glut4 at the C terminus and the central loop, but not at the N terminus. Adipocyte cytosols used here were diluted with buffer 30-fold or more during preparation (see ``Experimental Procedures''), and GTBP70 binding monitored above represents those in the ionic environment of the homogenizing buffer containing 150 mM NaCl and 1.5 mM MgCl(2). The inclusion of 2 mM EGTA, 2.4 mM Ca, or 150 mM K in cytosols, however, did not affect GTBP70 binding to the Glut4 C-terminal fusion protein appreciably, indicating that the binding is neither calcium-dependent nor potassium-dependent.

One salient feature of GTBP70 binding to the glucose transporter fusion proteins is its inhibition by ATP. This ATP effect was dose-dependent, with an half-maximal inhibition observed at 10 mM ATP, a concentration much too high to be found in adipocyte cytosol. The effect is nevertheless specific to ATP. ADP and AMP-PCP were without effect, suggesting the importance of ATP hydrolysis for inhibition. It is known that ATP or metabolic energy is required for insulin-induced Glut4 translocation and its reversal(26) . In streptolysin-O permeabilized rat adipocytes or 3T3-L1 adipocytes, insulin and GTPS increase plasma membrane Glut4 level(27, 28) , and the magnitude of this effect is considerably reduced in the absence of an exogenous ATP source(28) . Furthermore, removal of ATP perse appears to cause a significant increase in cell surface level of Glut4 (28) . The ATP-sensitive binding of GTBP70 to the glucose transporter may play a role in regulation of glucose transporter targeting in basal or insulin-stimulated adipocytes. On the other hand, ATP and metabolic energy also mediated the regulation of intrinsic activity of the glucose transporter in Clone 9 cells(29) . GTBP70 may serve as a mediator or adaptor in modulating transport activity in these metabolite-induced regulation.

The binding of GTBP70 to Glut4 was not affected by the pretreatment of adipocytes with insulin. This indicates that insulin treatment does not affect the physical binding of this protein to glucose transporters. It is still possible that insulin modulates GTBP70 activity otherwise. If this protein indeed plays a role in insulin-induced glucose transport stimulation, the step(s) subsequent to the binding rather than the binding itself may be modulated by the hormone. GTBP70 may be an enzyme of which Glut4 is a substrate or may be an adaptor specific to Glut4. In either case, GTBP70 would be able to transduce insulin signal to the transporter without changing in its physical binding to transporter. Rat hepatocyte cytosols showed no detectable GTBP70 binding activity, suggesting that either the expression or the function of GTBP70 is cell-specific.

Also described in the present study is another cytosolic protein with apparent molecular mass of 73 kDa that coprecipitates with GTBP70 to glucose transporter fusion proteins. This binding is also inhibited by ATP. This 73-kDa protein binding was quantitatively (5-10 times) less abundant compared to GTBP70 based on the intensity of protein staining in gels and S fluorography. Whether this 73-kDa protein directly binds to the glucose transporter fusion proteins or binds to GTBP70 is not clear at this time.

The chemical identities of GTBP70 and the 73-kDa protein are yet to be determined. GTBP70 did not react with antibodies against phosphotyrosine, phosphothreonine, or phosphoserine in immunoblots, suggesting that it is not a phosphoprotein. GTBP70 is reminiscent of heat shock protein 70 in molecular mass and ATP sensitivity. The adipocyte cytosolic protein bound to Glut4 C-terminal fusion proteins did show a minimal reactivity around 70-73-kDa region in immunoblots with an anti-HSP70 antibody (data not shown). This antibody is known to recognize both constitutive and inducible forms of the heat shock protein. The blot intensity at the 70-kDa position is very weak, and its relationship to GTBP70, if any, is yet to be established. It is more likely that the 73-kDa protein is a heat shock protein similar to HSP70. Nonetheless, the association of HSP70 with glucose transporters remains as an interesting possibility. Partial amino acid sequence determination of GTBP70 and the 73-kDa protein will aid eventual identification of these proteins.


FOOTNOTES

*
This work was supported in part by Grant DK-13376 from the National Institutes of Health and by the Veterans Administration Medical Center, Buffalo, NY. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Veterans Administration Medical Center, 3495 Bailey Ave., Buffalo, NY 14215.

(^1)
The abbreviations used are: Glut, glucose transporter; GTBP, glucose transporter-binding protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; AMP-PCP, adenosine 5`-(beta,-methylenediphosphate); PBS, phosphate-buffered saline.


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