• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of mbcLink to Publisher's site
Mol Biol Cell. Apr 1999; 10(4): 819–832.
PMCID: PMC25204

Protein Interactions with the Glucose Transporter Binding Protein GLUT1CBP That Provide a Link between GLUT1 and the Cytoskeleton

Guido Guidotti, Monitoring Editor

Abstract

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin–Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, α-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

INTRODUCTION

Glucose transporters are required for efficient movement of glucose across the plasma membrane of mammalian cells. GLUT1–GLUT5 and GLUT7 are six functional isoforms that comprise the family of facilitative glucose transporters (Mueckler, 1994 blue right-pointing triangle). Despite the high degree of sequence homology, each can be subject to distinct modes of regulation. GLUT1 is a ubiquitously expressed transporter, and like that of other members of this transporter family, its rate of synthesis, subcellular localization, and activity each contribute to the regulation of the rate of glucose uptake by cells expressing this isoform.

As a mode of regulation, insulin-induced translocation of GLUT1 to the plasma membrane occurs but is less pronounced than is that for GLUT4. GLUT1 is more commonly regulated via alterations in the level of transporter expression or via translocation-independent changes in the activity of the transporter residing in the plasma membrane. For example, no changes in the plasma membrane concentration of GLUT1 are noted when glucose transport is stimulated by protein synthesis inhibitors in 3T3 fibroblasts (Clancy et al., 1991 blue right-pointing triangle), by anoxia in Clone 9 cells (Shetty et al., 1993 blue right-pointing triangle), or by glucose starvation in 3T3-L1 adipocytes (Fisher and Frost, 1996 blue right-pointing triangle). Recently, it was shown that GLUT1 in erythrocyte ghosts could be activated by agents that disrupt the actin cytoskeleton (Zhang and Ismail-Beigi, 1998 blue right-pointing triangle). Although no GLUT1-specific regulatory proteins have been identified to date, it is likely that GLUT1 binding proteins exist that could either directly alter GLUT1 activity or mediate interactions with other GLUT1 regulatory factors.

Although a unique insulin-regulated pattern of membrane targeting exists for GLUT4 in muscle and fat tissues, specialized targeting of GLUT1 occurs in other cell types. For example, GLUT1 expression is polarized in cells comprising the blood–brain barrier (Pardridge et al., 1990 blue right-pointing triangle), the blood–placental barrier (Takata et al., 1994 blue right-pointing triangle), peripheral nerve cell sheaths (Muona et al., 1992 blue right-pointing triangle), kidney proximal tubules (Heilig et al., 1995 blue right-pointing triangle), mammary glands (Camps et al., 1994 blue right-pointing triangle), the rat oviduct (Tadokoro et al., 1995 blue right-pointing triangle), and the intestine (Boyer et al., 1996 blue right-pointing triangle). In Caco-2 and Madin–Darby canine kidney (MDCK) cells, which are model systems for intestinal and kidney epithelia, respectively, GLUT1 is localized to the basolateral plasma membrane (Harris et al., 1992 blue right-pointing triangle; Pascoe et al., 1996 blue right-pointing triangle). GLUT1 is localized to glial fibrillary acidic protein (GFAP)1-positive processes in primary astrocytes (Maher, 1995 blue right-pointing triangle), and in the heart, GLUT1 is localized to the specialized membrane surfaces of the intercalated discs (Doria-Medina et al., 1993 blue right-pointing triangle). Thus, in vivo, GLUT1 subcellular localization serves an important and presumably regulated role to facilitate transcellular interchange of glucose between vascular and cellular compartments.

The mechanism(s) by which GLUT1 achieves a polarized distribution within certain cells is unknown. In the parasitic protozoan Leishmania enriettii, association of glucose transporters with the cytoskeleton is a critical determinant for their proper intracellular targeting (Snapp and Landfear, 1997 blue right-pointing triangle), and a critical requirement of microtubules and motor proteins for targeting viral proteins in polarized cells has been demonstrated (Lafont et al., 1994 blue right-pointing triangle). Targeting motifs have been identified that direct some integral membrane proteins to either the apical or basolateral membrane in polarized epithelial cells (Mostov et al., 1992 blue right-pointing triangle; Brown and Stow, 1996 blue right-pointing triangle). Although GLUT1 may contain such motifs, these signals have not been identified.

The cytosolic, C-terminal domain of GLUT1 is 42 amino acids in length and is functionally important because mutant GLUT1 molecules that lack the C terminus exhibit significantly reduced activity (Oka et al., 1990 blue right-pointing triangle; Muraoka et al., 1995 blue right-pointing triangle; Dauterive et al., 1996 blue right-pointing triangle). One source of seizures and delayed development in humans can be directly attributed to the loss of the GLUT1 C terminus and the resulting impaired sugar movement across the blood–brain barrier (De Vivo et al., 1991 blue right-pointing triangle). The C-terminal domain of GLUT1 is conserved between species but is dissimilar in sequence to that of other GLUT isoforms. Therefore, the C terminus is a potential binding site for isoform-specific regulatory and/or targeting proteins. The observations that antibodies against the GLUT1 C terminus stimulate GLUT1 transport activity (Tanti et al., 1992 blue right-pointing triangle) and that peptides derived from the GLUT1 C terminus block azide-induced regulation of GLUT1 activity in Clone 9 cells (Shi et al., 1995 blue right-pointing triangle) both provide indirect evidence supporting the existence of such regulatory proteins, because their normal interactions with the C terminus and subsequent alteration of GLUT1 activity would be blocked by these reagents. The unique sequence and functional importance of the C terminus of GLUT1 and indirect evidence that it serves as a regulatory target prompted a search for proteins capable of interacting with this unique domain.

Using the yeast two-hybrid system, we isolated a cDNA clone whose polypeptide product, GLUT1 C-terminal binding protein (GLUT1CBP), binds to the GLUT1 C terminus. GLUT1CBP is unique among previously reported GLUT1 binding proteins (Lachaal et al., 1990 blue right-pointing triangle; Lachaal and Jung, 1993 blue right-pointing triangle; Liu et al., 1995 blue right-pointing triangle; Shi et al., 1995 blue right-pointing triangle) because of its demonstrated binding specificity and the presence of a novel PDZ domain. This study examines the characteristics of GLUT1CBP, which include the binding specificity of its PDZ domain, patterns of mRNA and protein expression, self-association, and interaction with cytoskeletal motor proteins. The data support a model in which GLUT1CBP links GLUT1 to cytoskeletal motor proteins, thereby facilitating targeting of GLUT1 to specific subcellular sites.

MATERIALS AND METHODS

The Yeast Two-Hybrid System

Coding sequences for the following transporter C-termini were PCR amplified and inserted downstream of the coding sequence for the Gal4 DNA binding domain (Gal4 DBD) in pGBT9 (Clontech, Palo Alto, CA): from mouse GLUT1, amino acids 451–492 (GT1), 451–468 (GT1Δ24), 451–488 (GT1Δ4), and 451–492 with a valine(492)-to-alanine mutation (GT1V492A); from human GLUT3, amino acids 475–522 (HGT3); or from mouse GLUT4, amino acids 464–509 (GT4). The PCR-amplified portions of all constructs were sequenced to ensure that no PCR-induced errors were present. The entire GLUT1CBP cDNA was excised from pACT with BglII and ligated into the BglII site of pGBT9 for two-hybrid screening. The coding sequence for amino acids 107–247 of GLUT1CBP (the PDZ domain) was PCR amplified and inserted into pGBT9 or pGAD10. Other domains of GLUT1CBP were subcloned using available restriction sites. Construction of the rat brain cDNA library has been described (Brondyk et al., 1995 blue right-pointing triangle). Two-hybrid screens were performed as detailed in the Clontech Matchmaker Library protocol PT1020-1 and the Clontech Matchmaker two-hybrid system protocol PT1265-1. To exclude false positives, we cotransformed DNA (in pACT) from positive clones into HF7c with either pGBT9 (Gal4 DBD alone) or pSE1112 (Snf-1 fused to Gal4 DBD). These cotransformants failed to demonstrate LacZ activity or growth on minus-histidine plates above background levels.

Sequencing of GLUT1CBP

The BglII fragment of the GLUT1CBP cDNA (in pACT) was subcloned into pBSKII (Bluescript II SK+; Stratagene, La Jolla, CA), and the sequence was determined at the DNA sequencing and synthesis facility at Iowa State University (Ames, IA).

Northern Blot Analysis

RNA purification (guanidinium thiocyanate extraction and sedimentation through CsCl) and Northern blot analysis were performed as described (Sambrook et al., 1989 blue right-pointing triangle). Poly(A)+ RNA was purified by oligo(dT)-cellulose chromatography. Ethidium bromide staining confirmed that an equal amount of RNA was loaded in each lane.

Preparation of His6–GLUT1CBP, Glutathione S-Transferase (GST)–GLUT1CBP(249–333), and GST–GLUT Fusion Proteins

GLUT1CBP was subcloned into the XhoI site of pET30a(+) (Novagen, Madison, WI). The resulting fusion protein contained His6- and S-tag sequences attached to the N terminus of GLUT1CBP. This construct was transformed into Escherichia coli BL21 (DE3)pLysS. Cells were induced, and the fusion protein was purified by Ni affinity chromatography from inclusion bodies using guanidine HCl as described by the manufacturer, except that 1 M sodium chloride was included to maintain the protein in solution during the removal of guanidine HCl. The concentration of GLUT1CBP was estimated assuming A2800.1% = 1. cDNA’s encoding glucose transporter C-termini were subcloned from pGBT9 vectors into pGEX-4T-1 (Pharmacia, Piscataway, NJ). The coding sequence for amino acids 249–333 of GLUT1CBP was inserted into pGEX-4T-1. Bacterial cells were grown and GST fusion protein synthesis was induced as described by the manufacturer.

Protein Overlay Assay

Cell pellets from bacteria expressing GST fusion proteins were dissolved in gel-loading buffer containing 50 mM Tris, pH 6.8, 2 mM EDTA, 6 M urea, 2% SDS, and bromophenol blue. The proteins were separated by SDS-PAGE using 10% gels and transferred to nitrocellulose membranes. The overlay assay was performed as described (Li et al., 1992 blue right-pointing triangle). However, buffer A in their procedure was modified to contain 10 mM HEPES-NaOH, pH 7.5, 400 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 40 nM purified His6–GLUT1CBP as a probe. Bound His6–GLUT1CBP was detected by enhanced chemiluminescence (ECL) using horseradish peroxidase (HRP)-labeled S-protein that recognizes the S-tag sequence present in His6–GLUT1CBP.

Production of Antibody against GLUT1CBP

For antibody LSU43, peptide I, Cys-Gln-Arg-Ser-Ser-Gly-Gly-His-Pro-Gly-Ser-Gly-Pro-Gln-Leu-(amide), corresponding to amino acids 223–236 of GLUT1CBP (Figure (Figure1,1, dashed line), was coupled to keyhole limpet hemocyanin with m-maleimidobenzoyl-N-hydroxysuccinimide ester as described (Sambrook et al., 1989 blue right-pointing triangle), dialyzed against PBS (minus KCl), and injected into New Zealand White rabbits (Cocalico Biologicals, Reamstown, PA). The antibody (LSU43) was purified and concentrated by adsorption to peptide I coupled to Sulfolink Gel (Pierce, Rockford, IL) according to the manufacturer’s instructions. Antibody was eluted from the washed gel with glycine buffer, pH 3.0. The eluate was immediately neutralized with 1 M Tris, pH 9.5. The antibody was precipitated in 50% ammonium sulfate, resuspended, and dialyzed against PBS.

Figure 1
The nucleotide and deduced amino acid sequences of GLUT1CBP. The amino acid and nucleotide sequence numbering is indicated on the right. A box surrounds the four-proline repeat present in the proline-rich N terminus. The PDZ domain (amino acid residues ...

Antibody GAb(249–333) against the purified GST fusion protein to amino acids 249–333 of GLUT1CBP was raised in goat (Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA).

Western Blot Analysis of the Cellular and Tissue Distribution of GLUT1CBP

Tissues from one male Sprague Dawley rat (Harlan Sprague Dawley, Indianapolis, IN) were isolated, quick frozen in liquid nitrogen, and stored at −80°C overnight. Frozen tissues (0.6–3.0 g) were disrupted with a Polytron 10N homogenizer in a 50-ml conical tube containing 2–4 ml of homogenization buffer (15% glycerol, 100 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.2 mM EDTA, 10 μg/ml leupeptin, 1 mM PMSF) (Kain et al., 1994 blue right-pointing triangle). Samples were transferred to a glass–teflon homogenizer and further homogenized 10 times (on ice). Aliquots of the homogenate were frozen at −80°C. Cell monolayers were scraped directly into gel-loading buffer containing 4% SDS and 10 mM DTT. Protein content was determined by bicinchoninic acid analysis (Pierce) using bovine serum albumin as a standard. One hundred micrograms of protein from tissue or cellular extracts were diluted into gel-loading buffer, heated at 80°C for 5 min, separated by SDS-PAGE in 10% gels, and then transferred to nitrocellulose membranes in Tris-glycine buffer containing 20% (vol/vol) methanol. Membranes were blocked overnight in buffer containing 5% dry milk and 0.1% Tween 20 and then incubated with a 1:750 dilution of GAb(249–333). Membranes were washed, and bound GAb(249–333) was detected with HRP-conjugated monoclonal anti-goat IgG (Sigma, St. Louis, MO) and ECL.

Cell Culture

Chinese hamster ovary (CHO)-K1-HIR cells were seeded into 6-cm Falcon dishes (Lincoln Park, NJ) in Ham’s F-12 media and harvested at confluence. Clone 5 MDCK cells (a generous gift of Dr. Mike Roth, Southwestern Medical School, Dallas, TX) were either maintained in Dulbecco’s modified Eagle’s medium in 10-cm Falcon dishes and harvested at confluence (nonpolarized cells) or maintained in polycarbonate transwell inserts (Corning, Cambridge, MA) and harvested 10 d after confluence (polarized cells). Caco-2 cells (a generous gift of Dr. Xavier Alvarez, Louisiana State University Medical Center [LSUMC], Shreveport, LA) were cultured on polycarbonate transwells as described for MDCK cells. Polarization and formation of tight junctions in MDCK and Caco-2 cells were assessed by measuring transepithelial resistance. 3T3-L1 preadipocytes were seeded into 6-cm Falcon dishes containing Dulbecco’s modified Eagle’s medium and grown to confluence. Differentiation was induced 2 d after confluence by exposure to isobutylmethylxanthine, dexamethasone, and insulin for 2 d followed by 4 d of incubation with insulin-containing media as described previously (Zuber et al., 1985 blue right-pointing triangle). Insulin was removed, and the cells were used after 2 additional days of incubation in insulin-free media. 3T3-L1 preadipocytes were cultured in parallel without exposure to insulin, isobutylmethylxanthine, or dexamethasone. Neurons and astrocytes were provided by Dr. Judson Chandler (LSUMC, Shreveport, LA). Pheochromocytoma (PC)-12 cells were provided by Dr. Donard Dwyer (LSUMC, Shreveport, LA).

Native Protein Binding Assays

His6–GLUT1CBP and rabbit IgG were coupled to CNBr-activated Sepharose CL-4B (Pharmacia) according to the manufacturer’s instructions at a density of 1 μg of protein per microliter of beads. Before use, the beads were blocked for 1 h at room temperature in PBS + 1% Triton X-100 (lysis buffer) + 1% BSA. For GLUT1 pull-down assays, one portion of beads was preincubated with 0.2 mg/ml peptide A (GLUT1 C-terminal 12 amino acids) for 1 h before the extract was added. Cell extracts were prepared by scraping confluent 10-cm dishes of CHO or MDCK cells into 0.5 ml/plate of ice-cold PBS containing 1% Triton X-100 and protease inhibitors (0.2 mM PMSF, 1 μg/ml leupeptin, 2 μg/ml soybean trypsin inhibitor, 0.096 trypsin inhibitor unit/ml aprotinin). Lysates were treated eight times with 10 strokes each in a glass–teflon homogenizer with a total incubation time of 1 h on ice and then were centrifuged at 100,000 × g for 1 h at 4°C. One milliliter of clear supernatant was added to 100 μl of GLUT1CBP– or rabbit IgG–Sepharose beads and incubated for 2 h at 4°C. The beads were washed for 5 min at 37°C once with 1 ml of lysis buffer, twice with 1 ml of PBS containing 0.1% Triton X-100, and once with 1 ml of PBS. Bound proteins were eluted in 100 μl of gel-loading buffer containing 4% SDS by heating for 30 min at 37°C and 5 min at 80°C. Thirty microliters of the supernatant were resolved on an SDS-PAGE gel, and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were probed with RE11 (anti-GLUT1 C terminus, 1:2000 dilution), LSU43 (anti-GLUT1CBP, 1.5 μg/ml), anti-myosin VI (1 μg/ml, kindly provided by Dr. Tama Hasson), or anti-α-actinin (Sigma A 5044, 1:500 dilution). Detection was with 125I-protein A for RE11, LSU43, and anti-myosin VI or with horse anti-mouse IgG (heavy + light) for anti-α-actinin followed by ECL.

RESULTS

GLUT1CBP Cloning

Approximately 500,000 yeast colonies were obtained by cotransformation with plasmids expressing both a Gal4 DNA binding domain–GLUT1 C terminus fusion protein (DBD–GT1) and the Gal4 activation domain fused to a library of proteins encoded by rat brain cDNAs (ACT–cDNA). Three colonies survived after selection for growth on synthetic media lacking histidine. ACT–cDNA plasmids from the three colonies were isolated, sequenced, and found to contain identical 1607-bp inserts. The sequence for the cDNA clones, designated GLUT1CBP, is presented in Figure Figure1.1. β-Galactosidase assays demonstrated that the activation of the LACZ reporter gene required the presence of both DBD–GT1 and ACT–GLUT1CBP plasmids (our unpublished results).

The cDNA contains two extended open reading frames that have coding potential for a protein containing either 333 or 289 amino acids. Two altered GLUT1CBP cDNAs were created to determine the authentic translation start codon (Figure (Figure2A).2A). In vitro translation of mRNA derived from the full-length cDNA produced a protein product migrating at 39 kDa as determined by SDS-PAGE (Figure (Figure2B,2B, WT). This is close to the predicted size of 36.1 kDa for a protein initiated from the first AUG and is identical in size to GLUT1CBP protein detected in Western blots of various tissue and cell extracts (see Figure Figure7).7). Translation of a truncated GLUT1CBP mRNA containing only the second AUG (missing sequence upstream of nucleotide 150, the SmaI site in the cDNA) produced shorter protein products (Figure (Figure2B,2B, TRUNCATED), indicating that the first AUG was used in the full-length construct. Mutation of the first AUG to an AAG codon, which does not initiate translation, significantly reduced the amount of full-length protein produced and induced the appearance of a shorter protein (Figure (Figure2B,2B, AUG-AAG). The doublets that appear with both mutants most likely arise from less efficient initiation from in-frame CUG codons that are used when the upstream AUG is absent.

Figure 2
Identification of the authentic translation start site in GLUT1CBP mRNA. (A) Diagram of the cDNA constructs used for in vitro transcription and translation. Each indicated cDNA insert in pBSKII was transcribed in vitro with T3 RNA polymerase. (B) In vitro ...
Figure 7
Analysis of GLUT1CBP protein expression in various tissues and cells. Tissue and cell extracts were prepared as described in MATERIALS AND METHODS. One hundred micrograms of each sample (40 μg for 3T3-L1 preadipocytes and neurons) were separated ...

An 82-amino-acid domain of the predicted 333-amino-acid GLUT1CBP protein product (Figure (Figure1,1, shaded region, residues 132–213) is homologous to the PDZ domains in several proteins retrieved via a homology search of the GenBank database (Figure (Figure3).3). Another notable feature is a proline-rich N terminus in which proline represents 11 of the first 56 amino acids (20%), with a four-proline repeat within this region (Figure (Figure1,1, open box).

Figure 3
Alignment of the GLUT1CBP PDZ domain with PDZ domains present in other proteins. An amino acid alignment of the PDZ domain of GLUT1CBP with PDZ domains from three representative proteins is shown. Shaded residues mark regions in which the chemical character ...

The PDZ Domain of GLUT1CBP Is Sufficient and the Four-Amino-Acid PDZ Recognition Motif of GLUT1 Is Required for the GLUT1CBP–GLUT1 Interaction

To delineate the region of GLUT1CBP that directs binding to the GLUT1 C terminus, we inserted various domains of the protein into pACT vectors. Using growth on minimal medium minus histidine as a qualitative assay for interactions, we tested the domains for their ability to bind to the Gal4 DBD fusions to the GLUT1 C terminus (residues 451–492) in the two-hybrid system. The results presented in Figure Figure44 demonstrate that the solitary C-terminal domain (250–333) of GLUT1CBP failed to bind to the C terminus of GLUT1, while truncated forms of GLUT1CBP (33–347 and 107–247) retained binding activity equivalent to that of native GLUT1CBP (1–333). This illustrates that neither the proline-rich N-terminal domain nor the C terminus of GLUT1CBP is required for binding to GLUT1 and that amino acids 107–247 (the PDZ domain) are sufficient for GLUT1CBP to recognize and interact with the GLUT1 C terminus.

Figure 4
Interactions of GLUT1 with various domains of GLUT1CBP. Yeast HF7c was cotransformed with plasmids encoding various Gal4 DBD and Gal4 ACT fusion proteins. Transformations were plated onto synthetic dextrose plates lacking tryptophan and leucine. Three ...

To confirm the two-hybrid results and to analyze the binding specificity of GLUT1CBP in more detail, protein overlay assays were performed using GST fusions of various transporter C-terminal constructs (Figure (Figure5A).5A). GLUT1CBP recognizes the GLUT1 C terminus (Figure (Figure5B,5B, left, GT1). The interactions are specific for the membrane distal one-half of the GLUT1 C terminus, because no binding to the homologous membrane proximal region of either the GLUT4 or GLUT1Δ24 C-termini was observed (Figure (Figure5B,5B, left, GT4 and GT1Δ24). Deletion of this motif to form the GLUT1Δ4 mutant abolishes GLUT1CBP binding (Figure (Figure5B,5B, left, GT1Δ4), further localizing the binding site to the GLUT1 PDZ recognition motif. The gel mobility and level of expression of the GST fusion proteins were similar (Figure (Figure5B,5B, right).

Figure 5
Analysis of His6–GLUT1CBP binding specificity and ability to bind endogenous GLUT1. (A) Alignment of the C-terminal regions of GST fusion proteins containing the C-termini of GLUT1 (GT1), GLUT4 (GT4), GLUT1Δ24 (GT1Δ24), human GLUT3 ...

The PDZ motifs of some PDZ-containing proteins have a specific requirement for a C-terminal valine in the PDZ recognition motif. For instance, a valine-to-alanine mutation at the C terminus of the Kv 1.4 potassium channel abolishes its interaction with the PDZ domains of postsynaptic density (PSD)-95 (Kim et al., 1995 blue right-pointing triangle). However, a valine-to-alanine mutation in the GLUT1 C terminus increased the amount of GLUT1CBP bound to GLUT1 (Figure (Figure5B,5B, left, GT1V492A). The GLUT1CBP PDZ domain was unable to bind to human GLUT3, which possesses a potential PDZ recognition motif at the C terminus (Figure (Figure5B,5B, left, HGT3), thereby indicating further sequence discrimination. These characteristics of GLUT1CBP binding were confirmed in the two-hybrid system (our unpublished results). Thus, the GLUT1CBP PDZ domain exhibits distinct differences in sequence binding specificity compared with the specificity of other PDZ domains.

GLUT1CBP Binds to Endogenous GLUT1

Although indicative of direct interactions between GLUT1CBP and the C terminus of GLUT1, the two-hybrid system and protein overlay assay do not address the ability of GLUT1CBP to interact with the native, full-length GLUT1 molecule. GLUT1CBP must be capable of binding the C terminus of GLUT1 resident in the native transporter for the interaction to have potential physiological importance.

This capability was tested using a technique in which purified His6–GLUT1CBP was covalently attached to Sepharose beads, and the beads were subsequently incubated with MDCK cell lysates prepared with the nondenaturing detergent Triton X-100. After extensive washing of the beads, bound GLUT1 was detected by Western analysis using a GLUT1-specific antibody. The endogenous glucose transporter was bound to GLUT1CBP beads but not to rabbit IgG beads (Figure (Figure5C).5C). Preincubating the GLUT1CBP beads with a peptide consisting of the C-terminal 12 amino acids of GLUT1 eliminated >70% of GLUT1 binding, providing further confirmation of binding specificity. This indicates that GLUT1CBP can interact with the C terminus of GLUT1 in the context of the native, full-length transporter.

Comparison of the Tissue-specific Expression of GLUT1CBP and GLUT1 mRNA

Functional interactions between GLUT1CBP and GLUT1 require that both proteins be expressed in the same tissue and cell types in vivo. As a first step toward addressing this issue, Northern blots of poly(A)+ RNA were performed to measure the level of expression of mRNA for each gene (Figure (Figure6).6). A GLUT1CBP antisense RNA probe hybridized to a 1.6-kb RNA from all tissues examined (Figure (Figure6,6, top). A GLUT1 antisense RNA probe hybridized to a 2.6-kb RNA that was also present in most tissues examined (Figure (Figure6,6, bottom). Among the tissues examined, the highest amount of both RNA species was observed in brain, and the lowest of both species was in liver, in accordance with previously published data regarding GLUT1 expression (Mueckler, 1994 blue right-pointing triangle). GLUT1CBP mRNA was also present in MDCK, Caco-2 (our unpublished results), CHO, and 3T3-L1 (preadipocyte and adipocyte) cells. Only the MDCK and Caco-2 cell lines lack the 1.6-kb form of GLUT1CBP message, which is replaced by a slightly larger 2.0-kb form. GLUT1 mRNA was present at very high levels in MDCK cells and 3T3-L1 adipocytes and at lower levels in CHO and 3T3-L1 preadipocytes.

Figure 6
Comparison of GLUT1 and GLUT1CBP mRNA expression in various tissues and cells. Poly(A)+ RNA (2 μg) isolated from each of the indicated rat tissues or cell monolayers was resolved on a denaturing agarose gel and then transferred to a nylon ...

Analysis of GLUT1CBP Protein Expression

Tissue proteins reactive with GAb(249–333) antibody were detected by Western blot (Figure (Figure7A,7A, left). All tissues examined, with the exception of the small intestine, possessed a 39-kDa protein that was reactive with GAb(249–333). This protein was present at highest levels in brain, testis, and lung. LSU43 antibody also recognizes an identical pattern of expression for the 39-kDa GLUT1CBP (our unpublished results). However, LSU43 recognizes additional high molecular weight immunoreactive proteins. These apparently are unrelated to GLUT1CBP because they are not recognized by GAb(249–333).

To confirm that immunoreactive bands were specific for the GLUT1CBP epitope, we incubated the antibody with Sepharose beads or covalently coupled His6–GLUT1CBP–Sepharose beads before use in Western blot analysis (Figure (Figure7A,7A, left and right, respectively). Preincubation of GAb(249–333) with beads did not affect the ability to detect any of the immunoreactive species on Western blots. However, preincubation of GAb(249–333) with GLUT1CBP beads eliminated most immunoreactive bands, indicating the presence of a conserved epitope between these proteins and GLUT1CBP. Some nonspecific interactions are evident in the diaphragm sample. The immunoreactive triplet evident below GLUT1CBP in the kidney sample, the high molecular weight singlet in 3T3-L1 preadipocytes, and the higher molecular weight doublet in diaphragm (Figure (Figure7A)7A) are most likely proteins unrelated to GLUT1CBP that contain epitopes recognized by GAb(249–333) because they are not recognized by antibody LSU43 (our unpublished results). Furthermore, the smaller triplet of proteins detected in kidney does not appear to represent proteolytic products of GLUT1CBP. Fragments of GLUT1CBP missing the epitope recognized by LSU43 and retaining that recognized by GAb(249–333) would be smaller than 12 kDa.

The 39-kDa GAb(249–333)-reactive protein was also present in primary cultures of rat cortical neurons, astrocytes, and human umbilical vein-endothelial (HUVEC) and artery-endothelial cells (HUAEC) (Figure (Figure7,7, B and C, left). Several cell lines including 3T3-L1, CHO, PC-12, MDCK, and Caco-2 possess the 39-kDa GAb(249–333)-reactive protein. The relative level of expression of GLUT1CBP by neurons and 3T3-L1 preadipocytes is underrepresented in Figure Figure7B7B because only 40 μg of protein extract was applied to the gel, rather than the 100 μg used for all other cells and tissues presented. Interestingly, upon differentiation of preadipocytes to adipocytes, 3T3-L1 cells cease the expression of GLUT1CBP. Furthermore, as demonstrated with the tissue extracts, immunoreactive protein bands observed in the primary cultures and cell lines are not observed when GAb(249–333) is preincubated with GLUT1CBP beads (Figure (Figure7,7, B and C, right).

Identification of Additional Proteins That Bind GLUT1CBP

PDZ-containing proteins often harbor additional sites for protein interactions outside the PDZ domain. To gain a better understanding of the protein interactions mediated by GLUT1CBP, we used the full-length protein in the two-hybrid system to screen a brain library for additional interacting proteins. Twenty-three independent ACT–cDNA clones were isolated, of which only a subset was identifiable by a search for homology to proteins in the sequence databases (Figure (Figure8).8). Interestingly, GLUT1CBP was isolated in the screen, indicating the potential of this protein to form homo-multimers. Also, GLUT1CBP interacted with the proteins myosin VI, an unconventional myosin; the kinesin superfamily protein 1B (KIF-1B), a kinesin-like monomeric microtubule motor; and α-actinin-1, an actin cross-linking protein.

Figure 8
Interactions of GLUT1CBP with cellular proteins. Yeast HF7c was cotransformed with plasmids encoding the indicated Gal4 DBD and Gal4 ACT fusion proteins and assayed as described in Figure Figure4.4. Yeast SNF-1 and SNF-4 interact in this system ...

We next determined which of the interacting proteins bound to GLUT1CBP via its PDZ domain. Whereas Gal4 ACT fusions with myosin VI, KIF-1B, α-actinin-1, and GLUT1CBP support growth of yeast colonies expressing a Gal4 fusion to full-length GLUT1CBP, only KIF-1B and α-actinin-1 Gal4 ACT fusions interact with a Gal4 DBD fusion to amino acids 107–247 of GLUT1CBP (Figure (Figure8).8). This indicates that, in addition to GLUT1, only KIF-1B and α-actinin-1 are capable of binding via the GLUT1CBP PDZ domain (Figure (Figure8).8). The failure of myosin VI and GLUT1CBP to bind the PDZ domain indicates the presence of at least one other protein interaction domain in GLUT1CBP.

These interactions were tested outside the yeast two-hybrid environment. Purified His6–GLUT1CBP was first assayed for its ability to bind endogenous GLUT1CBP from detergent-solubilized CHO cells (Figure (Figure9A).9A). In the absence of cell extract, only a portion of the larger molecular weight His6–GLUT1CBP that escaped cross-linking to the beads is detected by antibody (LSU43) against GLUT1CBP (Figure (Figure9A,9A, left lane). Association of the lower molecular weight native endogenous GLUT1CBP with the His6–GLUT1CBP cross-linked beads is detected when the beads are incubated with extract (Figure (Figure9A,9A, middle lane). This association is specific because no native GLUT1CBP binds to IgG cross-linked beads (Figure (Figure9A,9A, right lane). In addition, His6–GLUT1CBP–Sepharose beads precipitated myosin VI and α-actinin-1 from detergent-solubilized MDCK cells (Figure (Figure9,9, B and C, respectively). Native protein binding assays have not been performed with KIF-1B because of our inability to obtain the antibody to this protein. The ability of GLUT1CBP to bind the intact, endogenous proteins suggests that these interactions are important physiologically. The fact that GLUT1CBP, KIF-1B (Nangaku et al., 1994 blue right-pointing triangle), myosin VI (Hasson and Mooseker, 1994 blue right-pointing triangle), and α-actinin-1 (Puius et al., 1998 blue right-pointing triangle) are ubiquitously expressed further supports the possibility of functional interactions among these proteins.

Figure 9
Interaction of purified GLUT1CBP with cellular GLUT1CBP, myosin VI, and α-actinin-1. Native protein binding assays were performed essentially as described in Figure Figure5C.5C. Extracts were prepared from CHO cells for endogenous GLUT1CBP ...

DISCUSSION

The PDZ domain, which recognizes C-terminal amino acid motifs, places GLUT1CBP among a unique and interesting category of proteins involved in membrane protein organization. The PDZ designation, previously termed DHR or GLGF repeat, is derived from the names of three proteins initially noted to contain this domain: mammalian PSD protein PSD-95, Drosophila discs large tumor suppressor DLG, and mammalian tight junction protein ZO-1. Proteins that possess PDZ domains participate in a variety of cellular processes. Among these are receptor clustering (Kim et al., 1995 blue right-pointing triangle), organizing signal transduction cascades (Montell, 1998 blue right-pointing triangle), anchoring proteins to the cytoskeleton (Short et al., 1998 blue right-pointing triangle), localizing proteins to specific regions of the plasma membrane (Simske et al., 1996 blue right-pointing triangle), modulating the activity of ion channels (Hall et al., 1998 blue right-pointing triangle), and determining the substrate specificity of some enzymes (Snow et al., 1998 blue right-pointing triangle).

A recent study of PDZ domain binding specificity identified at least two independent groups of PDZ domains that possess divergent recognition motif specificity (Songyang et al., 1997 blue right-pointing triangle). Each of the two groups can be subdivided on the basis of primary sequence determinants within the PDZ domain. Members of the first group, including PSD-95 and DLG, preferentially bind to peptides with a C-terminal sequence Xxx-Ser/Thr/Tyr-Xxx-Val/Ile, where Xxx represents any amino acid. The second group, which includes p55 and Lin2, bind preferentially to Xxx-Phe/Tyr-Xxx-Val/Phe/Ala. C-terminal sequences of proteins known to require the PDZ domain of GLUT1CBP for interaction are included in Figure Figure10.10. An analysis of the recognized sequences indicates that the specificity of the PDZ domain of GLUT1CBP is most closely related to the first group of PDZ domains because it recognizes a C-terminal hydrophobic valine, alanine, or leucine, with a serine or threonine two amino acids upstream (position −2) in GLUT1, GLUT1V492A, KIF-1B, TAX, or α-actinin-1. It should be noted that the binding of GLUT1CBP to internal sequences in α-actinin-1 cannot be excluded, because PDZ domains can bind to the spectrin repeats of α-actinin-2 (Xia et al., 1997 blue right-pointing triangle).

Figure 10
C-terminal sequences of proteins tested for their ability to bind to the PDZ domain of GLUT1CBP. The results reflect a summary of protein interactions tested in this article with the exception of TAX that was reported (Rousset et al., 1998 blue right-pointing triangle) to interact ...

These observations suggest that the GLUT1CBP PDZ domain can exhibit some plasticity in binding specificity as described previously for other PDZ domains (Songyang et al., 1997 blue right-pointing triangle). Such plasticity allows PDZ-containing proteins to participate in a variety of cellular functions by interacting with a limited, but not unique, class of partners. Nevertheless, the PDZ domain of GLUT1CBP can discriminate between other prospective targets within the same recognition group, as evidenced by its failure to bind to the C terminus of human GLUT3, which ends with the sequence Thr-Thr-Asn-Val. Furthermore, the GLUT1 C terminus is not spuriously recognized by PDZ domains because the two-hybrid screen with this domain failed to isolate any of the other known PDZ-containing proteins, such as PSD-95. This characteristic is consistent with the observation that a PDZ domain from one subgroup is not able to bind to a peptide specifically designed for another member of the same subgroup (Songyang et al., 1997 blue right-pointing triangle). These observations suggest that additional determinants within the PDZ domain and sequences adjacent to the terminal recognition motif may further refine the specificity of PDZ domain interactions (Shieh and Zhu, 1996 blue right-pointing triangle).

This study demonstrates that GLUT1CBP can interact with multiple cellular proteins. GLUT1 is a strong candidate because of the overlapping expression of both mRNAs and protein. Brain, heart, and kidney tissue showed high relative levels of expression of both GLUT1 and GLUT1CBP mRNA species. However, all tissues examined possessed a detectable signal for both mRNAs with the exception of liver, which lacked detectable levels of GLUT1. The wide tissue distribution observed for GLUT1CBP in this study is supported by the numerous homologous GenBank expressed sequence tag sequences identified in diverse human and mouse tissues. The function of GLUT1CBP may therefore be important in a number of different cellular environments.

The significance of the larger form of GLUT1CBP mRNA observed in canine MDCK and human Caco-2 cells is not yet understood. This may reflect species- or tissue-specific variations in the length of the 5′- or 3′-untranslated regions or alternative splicing. In either case, the modifications do not significantly alter the size of the resultant GLUT1CBP detected by the goat anti-GLUT1CBP antibody.

High levels of GLUT1CBP mRNA expression correlate with high GLUT1CBP protein expression in heart, lung, and brain tissue, whereas low mRNA levels correlate with lower GLUT1CBP protein expression in liver tissue. However, the contrasting high message level and low protein levels exhibited in gastrocnemius and kidney tissue suggest that an altered translational and/or posttranslational regulatory state exists for these two tissues. Similar contrasts in translational and/or posttranslational control of GLUT1CBP expression apparently occur during the differentiation of 3T3-L1 preadipocytes. As illustrated in Figure Figure7B,7B, upon differentiation of 3T3-L1 preadipocytes into adipocytes, the expression of GLUT1CBP protein is lost with only a slight decrease in the level of message (Figure (Figure66).

What may be the function of GLUT1CBP? The membrane topology of GLUT1 and the wide tissue distribution of the cytosolic, soluble proteins GLUT1CBP, KIF-1B, α-actinin-1, and myosin VI suggest the formation of dimeric (or multimeric) complexes characteristic of PDZ-containing proteins. Such linkages have been reported for ion channels and other membrane proteins and may be important for localizing GLUT1 to specialized membrane sites or for regulating transport activity. GLUT1CBP binds to GLUT1, KIF-1B, and α-actinin-1 via its PDZ domain and to myosin VI or another molecule of GLUT1CBP via one or more adjacent domains. Although GLUT1CBP has only one PDZ domain, a dimeric form of the protein may exist because the protein interacts with itself. Furthermore, because the interaction is not via the PDZ domain, the dimer would expose two PDZ domains for binding to other cellular proteins. These characteristics make GLUT1CBP an ideal candidate to serve as a bridging protein between GLUT1 monomers or between GLUT1 and cytoskeletal elements (Figure (Figure11,11, A and B, respectively). By cross-linking GLUT1 monomers, GLUT1CBP could stabilize GLUT1 within specific domains of the plasma membrane, which is one proposed mechanism for localizing proteins to specialized membrane domains. Alternatively, linkage of GLUT1 monomers may affect the transport activity of GLUT1. There is a possibility for regulation of GLUT1 activity to occur via GLUT1CBP-mediated colocalization with an as yet unidentified regulatory protein present in its native cellular environment. Another mechanism for localizing GLUT1 to a specific membrane site may involve tethering the transporter to cytoskeletal motor proteins such as myosin VI and/or KIF-1B, which would then direct GLUT1-containing vesicles toward their proper location in the cell. Tethering GLUT1 to α-actinin-1 may be yet another way to restrict GLUT1 to specific membrane domains (Figure (Figure11C),11C), and disruption of this interaction could serve to regulate GLUT1 activity. This is consistent with the recent observation that disruption of the actin cytoskeleton in erythrocytes leads to activation of GLUT1 (Zhang and Ismail-Beigi, 1998 blue right-pointing triangle). Importantly, the topology of the GLUT1 transporter in the plasma membrane and intracellular vesicles is appropriate in both cases to permit interaction of its C terminus with cytosolic GLUT1CBP in vivo.

Figure 11
Models depicting the potential functions of GLUT1CBP. (A) GLUT1CBP tethers GLUT1-containing vesicles to cytoskeletal motor proteins. This particular model invokes dimerization of GLUT1CBP and linkage to KIF-1B and the microtubule. An alternative model ...

ACKNOWLEDGMENTS

The authors thank Drs. Ian Macara and Graeme Bell for the rat brain cDNA library used in the two-hybrid screen and for the human GLUT3 cDNA, respectively, Dr. Tama Hasson for providing anti-myosin VI antibody, Dr. Judson Chandler for providing primary neuronal and astrocyte cultures, Dr. Donard Dwyer for providing PC-12 cells, Dr. Xavier Alvarez for Caco-2 cells, Dr. Mike Roth for MDCK cells, Dr. Steven Alexander for HUVEC and HUAEC cells, Sue Hagius and Drs. Martin Roop and Phil Elzer for help and suggestions in preparing GAb(249–333), and colleagues in the Department of Biochemistry and Molecular Biology at LSUMC (Shreveport, LA) for numerous suggestions. This work was supported by National Institutes of Health grant DK-42647, by a grant-in-aid (GS-14) from the American Heart Association, Louisiana Affiliate, by the Edward P. Stiles Trust Fund and Biomedical Research Foundation of Northwest Louisiana, and in part by a research award from the American Diabetes Association.

Abbreviations used:

CHO
Chinese hamster ovary
DBD
DNA binding domain
ECL
enhanced chemiluminescence
GFAP
glial fibrillary acidic protein
GLUT1CBP
GLUT1 C-terminal binding protein
GST
glutathione S-transferase
HRP
horseradish peroxidase
HUAEC
human umbilical artery-endothelial cells
HUVEC
human umbilical vein-endothelial cells
KIF-1B
kinesin superfamily protein 1B
MDCK
Madin–Darby canine kidney
PC-12 cells
pheochromocytoma-12 cells
PSD
postsynaptic density
PVDF
polyvinylidene fluoride

REFERENCES

  • Boyer S, Sharp PA, Debnam ES, Baldwin SA, Srai SKS. Streptozotocin diabetes and the expression of GLUT1 at the brush border and basolateral membranes of intestinal enterocytes. FEBS Lett. 1996;396:218–222. [PubMed]
  • Brondyk WH, McKiernan CJ, Fortner KA, Stabila P, Holz RW, Macara IG. Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A. Mol Cell Biol. 1995;15:1137–1143. [PMC free article] [PubMed]
  • Brown D, Stow JL. Protein trafficking and polarity in kidney epithelium: from cell biology to physiology. Physiol Rev. 1996;76:245–297. [PubMed]
  • Camps M, Vilaro S, Testar X, Palacin M, Zorzano A. High and polarized expression of GLUT1 glucose transporters in epithelial cells from mammary gland: acute down-regulation of GLUT1 carriers by weaning. Endocrinology. 1994;134:924–934. [PubMed]
  • Clancy BM, Harrison SA, Buxton JM, Czech MP. Protein synthesis inhibitors activate glucose transport without increasing plasma membrane glucose transporters in 3T3–L1 adipocytes. J Biol Chem. 1991;266:10122–10130. [PubMed]
  • Dauterive R, Laroux S, Bunn RC, Chaisson A, Sanson T, Reed BC. C-Terminal mutations that alter the turnover number for 3-O-methylglucose transport by GLUT1 and GLUT4. J Biol Chem. 1996;271:11414–11421. [PubMed]
  • De Vivo DC, Trifiletti RR, Jacobson RI, Ronen GM, Behmand RA, Harik SI. Defective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay. N Engl J Med. 1991;325:704–709. [PubMed]
  • Doria-Medina CL, Lund DD, Pasley A, Sandra A, Sivitz WI. Immunolocalization of GLUT-1 glucose transporter in rat skeletal muscle and in normal and hypoxic cardiac tissue. Am J Physiol. 1993;265:E454–E464. [PubMed]
  • Fisher MD, Frost SC. Translocation of GLUT1 does not account for elevated glucose transport in glucose-deprived 3T3–L1 adipocytes. J Biol Chem. 1996;271:11806–11809. [PubMed]
  • Hall RA, et al. The β2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature. 1998;392:626–630. [PubMed]
  • Harris DS, Slot JW, Geuze HJ, James DE. Polarized distribution of glucose transporter isoforms in Caco-2 cells. Proc Natl Acad Sci USA. 1992;89:7556–7560. [PMC free article] [PubMed]
  • Hasson T, Mooseker MS. Porcine myosin-VI: characterization of a new mammalian unconventional myosin. J Cell Biol. 1994;127:425–440. [PMC free article] [PubMed]
  • Heilig C, Zaloga C, Lee M, Zhao XY, Riser B, Brosius F, Cortes P. Immunogold localization of high-affinity glucose transporter isoforms in normal rat kidney. Lab Invest. 1995;73:674–684. [PubMed]
  • Hoskins R, Hajnal AF, Harp SA, Kim SK. The C. elegans vulval induction gene lin-2 encodes a member of the MAGUK family of cell junction proteins. Development. 1996;122:97–111. [PubMed]
  • Jesaitis LA, Goodenough DA. Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein. J Cell Biol. 1994;124:949–961. [PMC free article] [PubMed]
  • Kain SR, Mai K, Sinai P. Human multiple tissue Western blots: a new immunological tool for the analysis of tissue specific protein expression. Biotechniques. 1994;17:982–987. [PubMed]
  • Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M. Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature. 1995;378:85–88. [PubMed]
  • Lachaal M, Berenski CJ, Kim J, Jung CY. An ATP-modulated specific association of glyceraldehyde-3-phosphate dehydrogenase with human erythrocyte glucose transporter. J Biol Chem. 1990;265:15449–15454. [PubMed]
  • Lachaal M, Jung CY. Interaction of facilitative glucose transporter with glucokinase and its modulation by ADP and glucose-6- phosphate. J Cell Physiol. 1993;156:326–332. [PubMed]
  • Lafont F, Burkhardt JK, Simons K. Involvement of microtubule motors in basolateral and apical transport in kidney cells. Nature. 1994;372:801–803. [PubMed]
  • Li M, Jan YN, Jan LY. Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel. Science. 1992;257:1225–1230. [PubMed]
  • Liu HZ, Xiong SH, Shi YW, Samuel SJ, Lachaal M, Jung CY. ATP-sensitive binding of a 70-kDa cytosolic protein to the glucose transporter in rat adipocytes. J Biol Chem. 1995;270:7869–7875. [PubMed]
  • Maher F. Immunolocalization of GLUT1 and GLUT3 glucose transporters in primary cultured neurons and glia. J Neurosci Res. 1995;42:459–469. [PubMed]
  • Makino K, et al. Cloning and characterization of NE-dlg: a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein. Oncogene. 1997;14:2425–2433. [PubMed]
  • Montell C. TRP trapped in fly signaling web. Curr Opin Neurobiol. 1998;8:389–397. [PubMed]
  • Mostov K, Apodaca G, Aroeti B, Okamoto C. Plasma membrane protein sorting in polarized epithelial cells. J Cell Biol. 1992;116:577–583. [PMC free article] [PubMed]
  • Mueckler M. Facilitative glucose transporters. Eur J Biochem. 1994;219:713–725. [PubMed]
  • Muona P, Sollberg S, Peltonen J, Uitto J. Glucose transporters of rat peripheral nerve. Differential expression of GLUT1 gene by Schwann cells and perineural cells in vivo and in vitro. Diabetes. 1992;41:1587–1596. [PubMed]
  • Muraoka A, Hashiramoto M, Clark AE, Edwards LC, Sakura H, Kadowaki T, Holman GD, Kasuga M. Analysis of the structural features of the C-terminus of GLUT1 that are required for transport catalytic activity. Biochem J. 1995;311:699–704. [PMC free article] [PubMed]
  • Nangaku M, Sato-Yoshitake R, Okada Y, Noda Y, Takemura R, Yamazaki H, Hirokawa N. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell. 1994;79:1209–1220. [PubMed]
  • Oka Y, Asano T, Shibasaki Y, Lin J-L, Tsukuda K, Katagiri H, Akanuma Y, Takaku F. C-Terminal truncated glucose transporter is locked into an inward-facing form without transport activity. Nature. 1990;345:550–553. [PubMed]
  • Pardridge WM, Boado RJ, Farrell CR. Brain-type glucose transporter (GLUT-1) is selectively localized to the blood-brain barrier. Studies with quantitative western blotting and in situ hybridization. J Biol Chem. 1990;265:18035–18040. [PubMed]
  • Pascoe WS, Inukai K, Oka Y, Slot JW, James DE. Differential targeting of facilitative glucose transporters in polarized epithelial cells. Am J Physiol. 1996;40:C547–C554. [PubMed]
  • Puius YA, Mahoney NM, Almo SC. The modular structure of actin-regulatory proteins. Curr Opin Cell Biol. 1998;10:23–34. [PubMed]
  • Rousset R, Fabre S, Desbois C, Bantignies F, Jalinot P. The C-terminus of the HTLV-1 Tax oncoprotein mediates interaction with the PDZ domain of cellular proteins. Oncogene. 1998;16:643–654. [PubMed]
  • Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.
  • Shetty M, Loeb JN, Vikstrom K, Ismail-Beigi F. Rapid activation of GLUT-1 glucose transporter following inhibition of oxidative phosphorylation in Clone-9 cells. J Biol Chem. 1993;268:17225–17232. [PubMed]
  • Shi YW, Liu HZ, Vanderburg G, Samuel SJ, Ismail-Beigi F, Jung CY. Modulation of GLUT1 intrinsic activity in clone 9 cells by inhibition of oxidative phosphorylation. J Biol Chem. 1995;270:21772–21778. [PubMed]
  • Shieh BH, Zhu MY. Regulation of the TRP Ca2+ channel by INAD in Drosophila photoreceptors. Neuron. 1996;16:991–998. [PubMed]
  • Short DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem. 1998;273:19797–19801. [PubMed]
  • Simske JS, Kaech SM, Harp SA, Kim SK. LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction. Cell. 1996;85:195–204. [PubMed]
  • Snapp EL, Landfear SM. Cytoskeletal association is important for differential targeting of glucose transporter isoforms in Leishmania. J Cell Biol. 1997;139:1775–1783. [PMC free article] [PubMed]
  • Snow BE, et al. GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain. J Biol Chem. 1998;273:17749–17755. [PubMed]
  • Songyang Z, Fanning AS, Fu C, Xu J, Marfatia SM, Chishti AH, Crompton A, Chan AC, Anderson JM, Cantley LC. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science. 1997;275:73–77. [PubMed]
  • Tadokoro C, Yoshimoto Y, Sakata M, Imai T, Yamaguchi M, Kurachi H, Oka Y, Maeda T, Miyake A. Expression and localization of glucose transporter 1 (GLUT1) in the rat oviduct: a possible supplier of glucose to embryo during early embryonic development. Biochem Biophys Res Commun. 1995;214:1211–1218. [PubMed]
  • Takata K, Kasahara T, Kasahara M, Ezaki O, Hirano H. Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier. Cell Tissue Res. 1994;276:411–418. [PubMed]
  • Tanti JF, Gautier N, Cormont M, Baron V, Vanobberghen E, Lemarchandbrustel Y. Potential involvement of the carboxy-terminus of the Glut-1 transporter in glucose transport. Endocrinology. 1992;131:2319–2324. [PubMed]
  • Xia H, Winokur ST, Kuo WL, Altherr MR, Bredt DS. Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs. J Cell Biol. 1997;139:507–515. [PMC free article] [PubMed]
  • Zhang JZ, Ismail-Beigi F. Activation of Glut1 glucose transporter in human erythrocytes. Arch Biochem Biophys. 1998;356:86–92. [PubMed]
  • Zuber MX, Wang SW, Thammavaram K, Reed DK, Reed BC. Elevation of the number of cell-surface insulin receptors and the rate of 2-deoxyglucose uptake by exposure of 3T3–L1 adipocytes to tolbutamide. J Biol Chem. 1985;260:14045–14052. [PubMed]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...