Targeting of GLUT1-GLUT5 Chimeric Proteins in the Polarized Cell Line Caco-2

Kouichi Inukai, Kuniaki Takata, Tomoichiro Asano, Hideki Katagiri, Hisamitsu Ishihara, Mitsuhiro Nakazaki, Yasushi Fukushima, Yoshio Yazaki, Masatoshi Kikuchi and Yoshitomo Oka

Institute for Adult Disease (K.I., H.I., M.N., M.K.) Asahi Life Foundation Nishishinjuku, Shinjuku-ku, Tokyo 160 Japan
Third Department of Internal Medicine (T.A., H.K., Y.F., Y.Y.) Faculty of Medicine University of Tokyo Hongo, Bunkyo-ku, Tokyo 113 Japan
Laboratory of Molecular and Cellular Morphology (K.T.) Institute for Molecular and Cellular Regulation Gumma University Showa-machi, Maebashi, Gumma 371 Japan
Third Department of Internal Medicine (Y.O.) Yamaguchi University School of Medicine Kogushi, Ube, Yamaguchi 755 Japan


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Caco-2, a human differentiated intestinal epithelial cell line, is a promising model for investigating the mechanism of polarized targeting of apical and basolateral membrane proteins. We stably transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells with an expression vector. Immunohistochemical study revealed that the GLUT5 protein expressed was localized at apical membranes and that the GLUT1 expressed was present primarily in the basolateral membranes of cells grown on permeable support. Next, to investigate the domain responsible for determining apical vs. basolateral sorting in glucose transporters, we prepared several GLUT1-GLUT5 chimeric cDNAs and transfected them into Caco-2 cells. A GLUT1 [N terminus~sixth transmembrane domain (TM6)]-GLUT5 [intracellular loop (IL)~C terminus] chimera was observed exclusively at the apical membrane, while GLUT1 (N terminus~IL)-GLUT5 (TM7~C terminus) and GLUT1 (N terminus~TM12)-GLUT5 (C-terminal domain) chimeras were observed mainly at the basolateral membrane, a localization similar to that of GLUT1. Moreover, using a recombinant adenovirus expression system, we expressed a GLUT5 (N terminus~TM6)-GLUT1(IL)-GLUT5(TM7~C-ter-minus)chimera, which was observed at the basolateral membrane. Based on these results, the C-terminal domain does not determine isoform-specific targeting of GLUT1 and GLUT5. Rather, it is the intracellular loop in glucose transporters that appears to play a pivotal role in apical-basolateral sorting signals in Caco-2 cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucose is the primary source of metabolic energy for most mammalian cells, and glucose transport across the plasma membrane is the first step in glucose utilization (1). Molecular cloning studies have revealed a family of facilitated glucose transporters, GLUTs, which share a high degree of amino acid and structural homology (2, 3, 4, 5, 6, 7, 8). These transporters have been designated GLUT1/erythrocyte, GLUT2/liver, GLUT3/brain, GLUT4/muscle-fat, and GLUT5/small intestine. Among them, three different GLUTs are of special interest with regard to intestinal absorption. GLUT1 is present in colonocytes and cell lines derived from intestinal epithelial cells and has been found in the basolateral membranes of these cells (2, 10). GLUT2 is also restricted to the basolateral side of enterocytes (11, 12). In contrast, GLUT5, which has recently been demonstrated to mediate mainly fructose transport (13, 14, 15), is localized to the brush-border membranes of human enterocytes (16). Thus, different glucose transporter isoforms are observed to be targeted to one or the other cell surface in a polarized epithelial cell, but which domains determine the apical/basolateral targeting of these glucose transporter proteins remains to be clarified.

Caco-2, a human differentiated intestinal epithelial cell line, is an excellent model for investigating the mechanisms underlying the polarized targeting of apical and basolateral membrane proteins (17, 18). This cell line is derived from a colonic adenocarcinoma, but resembles small intestine enterocytes in many respects including the expression of a Na+-dependent glucose transporter system (19). Caco-2 cells form tight monolayers when grown on permeable support, and a number of domain-specific membrane proteins such as several brush border hydrolases, Na+-K+ ATPase, and basolateral glycoproteins have been identified using peptide-specific antibodies or monoclonal antibodies (18). Recent studies have revealed GLUT1 to be localized to the basolateral membranes and GLUT5 to primarily be present in the apical membranes of fully differentiated Caco-2 cells, a distribution similar to that of GLUTs in normal intestinal epithelial cells (10, 20).

In the present study, we transfected rat GLUT5 cDNA and rabbit GLUT1 cDNA into Caco-2 cells. We found that the GLUT5 protein expressed was localized at the apical membranes and that the GLUT1 expressed was present primarily in the basolateral membrane, the same cellular localizations as those of endogenous GLUT1 and GLUT5. These results strongly suggest that GLUT5 and GLUT1 follow the same sorting pathways as endogenous GLUT5 and GLUT1, two isoforms with distinct apical/basolateral sorting in Caco-2 cells. The Caco-2 cell line is thus a useful system for studying the molecular basis of the differential targeting of GLUT1 and GLUT5 glucose transporters. We therefore constructed and expressed chimeric GLUT1–5 glucose transporters and analyzed the targeting of these chimeras.

This is the first attempt, to our knowledge, to investigate the apical/basolateral sorting system of GLUTs employing molecular manipulation. Our results indicate that the intracellular loop of glucose transporters plays a pivotal role in the apical/basolateral sorting system operating in Caco-2 cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cDNA constructs were prepared for the expression of GLUT1, GLUT5, and three GLUT1–5 chimeras and introduced into Caco-2 cells. The three chimeras had the following compositions: G1–5a was constructed from GLUT1 (N terminus~TM6) and GLUT5 (intracellular loop~C-terminal), G1–5b from GLUT1 (N terminus~intracellular loop) and GLUT5 (TM7~C terminus), and G1–5c from GLUT1 (N terminus~TM12) and GLUT5 (C-terminal region). These swapping sites are shown in Fig. 1Go. We screened transfectants by Northern blotting, and GLUT transcripts were detected in several clones. These clones were selected, and one clone from each cDNA construct was designated clone G5 (wild type GLUT5), clone G1–5a (G1–5a chimera), clone G1–5b (G1–5b chimera), clone G1–5c (G1–5c chimera), and clone G1 (wild type GLUT1). One of the clones transfected with pCXN alone was designated clone D. Northern blots of these clones, probed with rabbit GLUT1 cDNA (bp 955-2477), rat GLUT5 cDNA (bp 21–656), and rat GLUT5 cDNA (bp 1540–2170) are shown in Fig. 2Go, A, B, and C, respectively. As shown in Fig. 2AGo, we detected small amounts of the GLUT1 signal in clone D, clone G5, clone G1–5a, and clone G1–5b, presumably due to cross-hybridization with the intrinsic human GLUT1 signal expressed in Caco-2 cells. We detected abundant GLUT1 signals in clone G1–5c and clone G1 (Fig. 2AGo, lanes 5 and 6), suggesting that these two clones express their transfected glucose transporter proteins. Hybridization was also performed with two different GLUT5 probes. As shown in Fig. 2BGo, the signal amount was significant only in clone G5 when hybridized with the N-terminal half region of rat GLUT5 cDNA. The size of GLUT5 transcripts in clone G5 was approximately 2.8 kb, consistent with the predicted size for the GLUT5 transcripts produced using the expression vector pCXN. Endogenous human GLUT5 was not observed in clone D, or in other clones transfected with chimeric cDNA, which means that we identified only transfected GLUT5 transcripts, although it is possible that a small amount of endogenous GLUT5 exists in Caco-2 cells. When hybridized with a GLUT5 cDNA fragment corresponding to the C-terminal region of GLUT5 (Fig. 2CGo), significant signal amounts were detectable in four clones that had been transfected with cDNA containing the sequence corresponding to the C-terminal region of GLUT5 and 3'-noncoding region of GLUT5 cDNA. These results demonstrate that GLUT5 and chimeric cDNAs were successfully transcribed in Caco-2 cells.



View larger version (28K):
[in this window]
[in a new window]
 
Figure 1. A Model for the Orientation of the Chimeric Glucose Transporter

The putative membrane-spanning domains are numbered 1–12 from the NH2 terminus to the COOH terminus. Three chimeric glucose transporters were prepared from combinations of GLUT1 and GLUT5, and termed G1–5a, G1–5b, and G1–5c. The amino acid sequences of these chimeras are indicated beside each chimeric transporter.

 


View larger version (55K):
[in this window]
[in a new window]
 
Figure 2. Expression of the Transfected Glucose Transporter mRNA in Caco-2 Cells

Twenty micrograms of total RNA were separated by electrophoresis on 1% agarose-formaldehyde gels, blotted onto a nylon membrane, and probed with (A) rabbit GLUT1(bp 955-2477), (B) rat GLUT5(bp 21–656), (C) rat GLUT5(bp 1540–2170). Hybridization was performed in a solution containing 50% deionized formamide, 5x Denhardt’s solution, and 0.1 mg/ml salmon sperm DNA at 42 C. Blots were each washed twice for 10 min at room temperature in 2x SSC, 0.1% SDS, and twice each for 10 min at 55 C in 0.1x SSC, 0.1% SDS. The following RNA sources were used: Lane 1, clone D; lane 2, clone G5; lane 3, clone G1–5a; lane 4, clone G1–5b; lane 5, clone G1–5c; lane 6, clone G1.

 
The immunoblotting of cellular homogenates of these transfectants with an antibody raised against the COOH-terminal peptide of rat GLUT5 showed the presence of large amounts of the expressed proteins in clone G5, clone G1–5a, clone G1–5b, and clone G1–5c (Fig. 3Go), while the signal was barely observable in clones D and G1. The apparent molecular mass of the proteins detected was 45~80 kDa. These results suggest that GLUT5 and chimeric proteins were expressed in Caco-2 cells and that our antibody detected only transfected proteins in these clones.



View larger version (91K):
[in this window]
[in a new window]
 
Figure 3. Immunoblot Analysis of Glucose Transporters in Caco-2 Cells

For membrane preparation, differentiated cells (15 days postconfluence) grown on plastic dishes were used. One hundred micrograms of brush border membranes (lanes 2 and 3) and crude homogenates (lanes 1, 4, 5, and 6) were subjected to SDS-PAGE (10%) and transferred onto nitrocellulose filters. Immunoblotting was performed using antisera raised in rabbits against the synthesized peptide corresponding to the COOH-terminal domain of GLUT5 (residues 490–502), as has been described in detail previously (15). The filters were incubated with [125I]protein A (Amersham) and subjected to autoradiography. The following membrane sources were employed: Lane 1, clone D; lane 2, clone G5; lane 3, clone G1–5a; lane 4, clone G1–5b; lane 5, clone G1–5c; lane 6, clone G1.

 
Immunofluorescence labeling of Caco-2 monolayers revealed marked differences in the cellular distributions of GLUT1 and GLUT5 (Fig. 4Go). GLUT5 was observed on the apical side in clone G5 (Fig. 4aGo). In double-labeling experiments, the GLUT5 labeling was colocalized with the intense labeling of F-actin, a marker of the brush border (Fig. 4bGo), indicating that GLUT5 is expressed in the brush border of the apical membrane. We also investigated two other clones expressing rat wild type GLUT5 and obtained results similar to those of clone G5 (data not shown). Almost no GLUT5 signal was observed in clone D (Fig. 4gGo). In contrast to the distribution of GLUT5, GLUT1 was observed predominantly on the lateral and basolateral sides in clone G1, which overexpressed rabbit GLUT1, while weak signals were observed on the apical side (Fig. 4dGo). We also observed a similar distribution of GLUT1 in clone D, which expressed endogenous GLUT1 (data not shown). The cellular distributions of the GLUT1 and GLUT5 expressed were compatible with those seen in previous studies on the distribution of endogenous GLUT1 and GLUT5 (10, 20).



View larger version (59K):
[in this window]
[in a new window]
 
Figure 4. Immunofluorescence Localization of Glucose Transporters in Clone G5 (upper), Clone G1 (middle), and Clone D (lower)

Wild type rat GLUT5 cDNA, wild-type rabbit GLUT1 cDNA, and pCXN vector alone were respectively transfected into these clones. For immunofluorescence microscopy, Caco-2 cells (15 days postconfluence) grown on Transwell filters were fixed in 3% formaldehyde/PBS. Semithin frozen sections of 1 µm thickness were made and incubated with either antipeptide antibody against the COOH-terminal domain of GLUT1 (d) and antipeptide antibody against the COOH-terminal domain of GLUT5 (a and g). The sections were then incubated with lissamine rhodamine-labeled affinity-purified donkey anti-rabbit IgG. For staining of F-actin, a brush border marker, fluorescein-phalloidin in PBS was added to the secondary antibody (b, e, and h). The arrowheads point to the apical sides of Caco-2 cells, which are rich in F-actin and thus intensely stained. Nomarski differential interference-contrast images are also shown (c, f, and i). Bar = 10 µm.

 
For comparison with the distribution of wild type GLUT1 or GLUT5, stable transfectants expressing GLUT1–5 chimeric protein were likewise studied. Figure 5Go shows the results of immunofluorescence studies of clone G1–5a, clone G1–5b, and clone G1–5c, obtained using an anti-GLUT5 C-terminal antibody. Interestingly, in clone G1–5a, the chimeric glucose transporter composed of GLUT1 (N terminus~TM6) and GLUT5 (intracellular loop~C-terminal) was localized to the apical membrane, a distribution similar to that of the wild type GLUT5. In sharp contrast, the G1–5b chimeric transporter, composed of GLUT1 (N terminus~intracellular loop) and GLUT5 (TM7~C terminus), and the G1–5c chimeric glucose transporter, composed of GLUT1 (N terminus~TM12) and GLUT5 (C-terminal region), were observed primarily on the basolateral sides of Caco-2 cells. The distribution of these two constructs was essentially the same as that of expressed, i.e. intrinsic, GLUT1.



View larger version (94K):
[in this window]
[in a new window]
 
Figure 5. Immunofluorescence Localization of Glucose Transporters in Clone G1–5a (upper), Clone G1–5b (middle), and Clone G1–5c (lower)

Ultrathin frozen sections were incubated with antipeptide antibody against the COOH-terminal domain of GLUT5 (a, c, and e). The sections were then incubated with rhodamine-labeled affinity-purified donkey anti-rabbit IgG. The arrowheads point to the apical sides of Caco-2 cells. Nomarski differential interference-contrast images are also shown (b, d, and f).

 
To clarify the role of the GLUT intracellular loop domain, we investigated the targeting of another chimeric glucose transporter, which has two reciprocal swapping sites; GLUT5 (N terminus~TM6, amino acid residues 1–211)-GLUT1 (intracellular loop domain, amino acid residues 207–271)-GLUT5 (TM7~C-terminus, amino acid residues 277–502), designated G5–1-5. We prepared the cDNA constructs encoding G5 and G5–1-5 cDNA and transfected them transiently into Caco-2 cells with a recombinant adenovirus expression system. Expression of these proteins was confirmed by Western blotting with the antibody against GLUT5 (data not shown). On immunohistochemical study, G5–1-5 protein was mainly targeted to the basolateral membrane, while the wild type GLUT5 was mainly targeted to the apical membrane (Fig. 6Go).



View larger version (80K):
[in this window]
[in a new window]
 
Figure 6. Immunofluorescence Localization of G5 (middle), G5–1-5 (lower), and lacZ (upper) Expressed Using a Recombinant Adenovirus Expression System

Recombinant adenoviruses for expressing wild type GLUT5, G5–1-5, and lacZ were transfected into Caco-2 cells. Semithin frozen sections, 1 µm in thickness, were made and incubated with antipeptide antibody against the COOH-terminal domain of GLUT5 (a, d, and g). The sections were then incubated with lissamine rhodamine-labeled affinity-purified donkey anti-rabbit IgG. The apical sides of Caco-2 cells are rich in F-actin and thus intensely stained (b, e, and h). Nomarski differential interference-contrast images are also shown (c, f, and i). Bar = 10 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Most studies on GLUT sorting have focused on the unique sequestration of GLUT4. A variety of molecular manipulations, such as site-directed mutagenesis and chimeric formation, have been employed to examine the GLUT4 molecule, and the mechanism of its intracellular sequestration has been clarified to some extent (21, 22, 23). In polarized epithelial cells, recent studies have shown that glucose transporter isoforms are sorted to either the apical or the basolateral surface (10, 11, 12, 16, 20). However, the domains responsible for this apical/basolateral sorting have not been determined. In the present study, we first confirmed that GLUT1 and GLUT5, when expressed with an expression vector, show the same cellular localization as endogenous GLUT1 and GLUT5. We then prepared chimeric proteins combining these two GLUT isoforms and investigated their targeting in polarized cells.

The human colon carcinoma cell line Caco-2 exhibits, at late confluence, the same morphological characteristics as differentiated small-intestinal enterocytes (24). The facilitative glucose transporters GLUT1 and GLUT3 and the fructose transporter GLUT5 are expressed in these cells (10). However, the expression of GLUT5 in Caco-2 cells is considered to be controversial. A clonal difference was previously suggested as to the level of expression of intrinsic GLUT5, based on the observation that GLUT5 was expressed only in low-glucose-consuming clones (25). Moreover, considerable developmental variations were observed in GLUT5 expression. In particular, an increase in GLUT5 expression was observed after confluence had been reached, and the expression level also depends on the passage number of the Caco-2 cells (20). For instance, GLUT5 was detected in only 40% of Caco-2 cells (passage 60) at 15 days after confluence. These earlier observations suggest that Caco-2 cells show heterogeneity and that the regulation of GLUT5 expression depends on the degree of differentiation in Caco-2 cells.

In the present study, we ligated rat GLUT5 cDNA into the expression vector pCXN and transfected rat GLUT5 into Caco-2 cells. This vector has the actin promoter and CMV enhancer sequences (26). Thus, the regulation system for expression of transfected GLUT5 is assumed to be considerably different from that of endogenous GLUT5. We found that the transfected GLUT5 was expressed even in the the early postconfluence stage (data not shown) and was observed most abundantly on the apical side in essentially all cells at 15 days after reaching confluence. These results were obtained with expressed GLUT5 and are somewhat inconsistent with those of a previous report on endogenous GLUT5, indicating that clone G5 has the property of homogeneity and that its GLUT5 expression is regulated by a vector program.

The distributions of GLUT1, GLUT5, and the three chimeric proteins used herein showed two distinct patterns. The wild type GLUT5 and the G1–5a chimeric protein were localized on the apical side, while the wild type GLUT1 and the G1–5b and G1–5c chimeric proteins were observed mainly on the lateral and basolateral sides. These results suggest that the intracellular loop region of these glucose transporter isoforms determines apical/basolateral sorting in Caco-2 cells. These chimeric transporters were properly sorted to the plasma membrane in a fashion similar to that of the wild type in CHO cells (27), suggesting that the secondary structure of chimeric proteins was not markedly impaired.

To further elucidate the role of the GLUT’s intracellular loop region, we studied the targeting of G5–1-5 protein, GLUT5, in which the intracellular loop domain had been replaced with that of GLUT1. First, we prepared G5–1-5 cDNA and transfected it into Caco-2 cells with an expression vector using the calcium phosphate method. However, we could not obtain a clone expressing an adequate amount of G5–1-5 protein for immunofluorescence study. The reason for this is not clear. We then employed a recombinant adenovirus expression system and obtained adequate amounts of the expressed proteins. Interestingly, G5–1-5 protein was targeted to the basolateral membrane, while the wild type GLUT5 was mainly targeted to the apical membrane (Fig. 6Go).

It is possible that GLUT1 contains the basolateral sorting signal in its intracellular loop region. Several basolateral sorting signals, such as the tyrosine motif and the di-leucine motif, have been identified in another polarized model system, MDCK cells (28, 29, 30). A recent report indicated, however, that a specific sorting signal is not likely to be required for intracellular vesicle traffic to the basolateral membrane in Caco-2 cells (31). Thus, an alternative, and quite plausible, explanation is that GLUT5 contains the apical sorting signal in its intracellular loop region and that the delivery of GLUT1 to the basolateral membrane represents the default pathway.

A recent study has shown that newly synthesized proteins in Caco-2 cells are sorted to the apical membrane via two different routes, an exocytic route and an endocytic-transcytotic route (18). Regarding the GLUT5 traffic, we have not analyzed which route is taken by GLUT5. Based on the observation that GLUT5 is also present in the basolateral membranes of some undifferentiated Caco-2 cells (10), it is tempting to speculate that fully differentiated Caco-2 cells, in which the transcytotic route is well developed, can effectively carry GLUT5-containing vesicles from the basolateral endosome to the apical plasma membrane. In this context, it is reasonable to speculate that the intracellular loop domain of GLUT5 actually contains the transcytotic and apical signal. The basolateral sorting of G5–1-5 protein is consistent with the idea that the apical sorting signal exists in the intracellular loop domain of GLUT5, and not in that of GLUT1. Although further study is expected to clarify in greater detail the transporting mechanism of GLUT5, our results indicate that the intracellular loop domains of GLUT1 and GLUT5 play pivotal roles in the apical/basolateral sorting in the polarized cell line Caco-2.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Chimeric cDNA Constructs
Chimeric cDNAs were produced according to previously described methods (32), which allowed us to prepare chimeric cDNAs at any swapping site. A rabbit GLUT1 clone (4) and a rat GLUT5 clone (15) were used as templates for PCR. Three chimeras in which the N-terminal region was GLUT1 and the C-terminal region was GLUT5 (G1–5a, G1–5b, G1–5c) were constructed. Fragments prepared by PCR from the wild type rabbit GLUT1 cDNA and the rat GLUT5 cDNA were fully sequenced and observed to have no unexpected mutations. As shown in Fig. 1Go, the three chimeras had the following compositions: G1–5a was constructed from GLUT1 (N terminus~TM6) and GLUT5 (intracellular loop~C-terminal), G1–5b from GLUT1 (N terminus~intracellular loop) and GLUT5 (TM7~C terminus), and G1–5c from GLUT1 (N terminus~TM12) and GLUT5 (C-terminal region).

Cell Culture and Transfection
The cell line Caco-2 (at 50 passages) was kindly provided by Dr. M. Shimizu (Faculty of Agriculture, University of Tokyo). The cells were cultured in a CO2/air (1:19) atmosphere at 37 C in DMEM supplemented with 10% FCS, 1% nonessential amino acids (GIBCO, Glasgow, Scotland), 4 mM L-glutamine, penicillin (50 units/ml), and streptomycin (50 µg/ml). Cells were grown in 10-cm plastic dishes (Corning, Corning, NY) and passaged at 60% confluence. For immunofluorescence experiments, cells were grown on Transwell filters (Costar, Cambridge, MA). GLUT1, GLUT5, and the three chimeric cDNAs were ligated into the expression vector pCXN (26) at the EcoRI site and subsequently transfected into Caco-2 cells with calcium phosphate precipitation as previously described (33). Caco-2 cells were observed to be most susceptible to transfection at near confluence. Transfections were performed in 6-cm dishes using 5 µg DNA. Cells were selected on the basis of their resistance to 600 µg/ml of the neomycin derivative G418, (GIBCO, Grand Island, NY). Cell lines expressing GLUT1, GLUT5, or any of the chimeras were isolated and used in this study.

Northern Blotting
For RNA preparation, differentiated cells (15 days postconfluence) grown on plastic dishes were used. Twenty micrograms of total RNA isolated with Isogen (Nippon Gene, Toyama, Japan) were separated by electrophoresis on 1% agarose-formaldehyde gels, blotted onto Biodyne nylon membranes (Pall, East Hills, NY), and hybridized with probes labeled by the Megaprime labeling system (Amersham, Buckinghamshire, UK). Hybridization was performed in a solution containing 50% deionized formamide, 5x Denhardt’s solution (1x Denhardt’s solution = 0.2 g/liter polyvinylpyrrolidone, 0.2 g/liter BSA, 0.2 g/liter Ficoll 400), 6x SSPE (1 M NaCl, 60 mM NaH2PO4, 6 mM EDTA), and 0.1 mg/ml salmon sperm DNA at 42 C. Blots were washed twice each for 10 min at room temperature in 2x NaCl-sodium citrate (SSC), 0.1% SDS, and twice each for 10 min at 55 C in 0.1x SSC, 0.1% SDS.

Membrane Preparation and Western Blotting
For membrane preparation, differentiated cells (15 days postconfluence) grown on plastic dishes were used. Cells were harvested in cold homogenization buffer consisting of 2 mM Tris-HCl, 50 mM mannitol, and 1 mM phenylmethyl sulfonyl fluoride, pH 7.0, and homogenized in a Potter-Elvehjem glass-Teflon type homogenizer at 4 C. Brush border-enriched fractions were prepared by the standard Ca2+ precipitation method (34). In brief, the homogenate was incubated for 15 min on ice with the addition of CaCl2 to a final concentration of 10 mM. Subsequently, the homogenate was centrifuged at 1,000 x g for 15 min at 4 C, and the resulting supernatant was centrifuged at 30,000 x g for 30 min at 4 C. The pellet was then resuspended, using a syringe with a 26 gauge needle, in the suspension buffer (0.25 M sucrose, 50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, pH 7.4). This suspension was homogenized with 10 strokes of a motor-driven Teflon pestle in a volume of the suspension buffer that was 10 times the weight of the original tissue. The homogenate was centrifuged at 1,000 x g for 15 min at 4 C, and the resulting supernatant was centrifuged at 30,000 x g for another 30 min at 4 C. The final pellet containing purified brush border membranes was resuspended in the same buffer. Protein determination was performed with a BCA protein assay (Pierce, Rockford, IL). Brush border membranes (100 µg protein) were subjected to SDS-PAGE (10%) and transferred onto nitrocellulose filters. Immunoblotting was performed using antisera raised in rabbits against the synthesized peptide corresponding to the COOH-terminal domain of GLUT5 (residues 491–503) as described previously in detail (15). Finally, the filters were incubated with [125I]protein A (Amersham, Buckinghamshire, UK) and subjected to autoradiography.

Transient Expression of Glucose Transporters with a Recombinant Adenovirus Expression System
The cassette cosmid for constructing recombinant adenovirus, pAdex1wt, was the generous gift of Dr. Izumi Saito (Institute of Medical Science, University of Tokyo). The cDNAs encoding GLUT5 and GLUT5 (N terminus~TM6)-GLUT1 (intracellular loop domain)-GLUT5 (TM7~C terminus) were ligated into the SwaI sites of pAdex1wt. Recombinant adenoviruses were obtained as previously described (35). Caco-2 cells were infected with these viruses for 1 h, then grown for 72 h. As a negative control, we prepared the adenovirus expressing lacZ.

Immunofluorescence Study
For immunofluorescence microscopy, Caco-2 cells (15 days postconfluence) grown on Transwell filters were fixed in 3% formaldehyde/PBS. Semithin 1 µm thick frozen sections were made and incubated with either an antipeptide antibody against the COOH-terminal domain of GLUT1, for the detection of GLUT1, or an antipeptide antibody against the COOH-terminal domain of GLUT5, for the detection of GLUT5, and three chimeric transporters (15, 35). The sections were then incubated with lissamine rhodamine-labeled affinity-purified donkey anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA) (37, 38). For F-actin staining, fluorescein-phalloidin (1:50 dilution, Molecular Probes, Eugene, OR) in PBS was added to the secondary antibody. After being washed with PBS, the sections were mounted in Perma Fluor Aqueous mountant (Lipshaw, Pittsbugh, PA). Specimens were observed with an Olympus BX-50 microscope equipped with epifluorescence and Nomarski differential-interference-contrast optics.


    FOOTNOTES
 
Address requests for reprints to: Yoshitomo Oka, M.D., Third Department of Internal Medicine, Yamaguchi University School of Medicine, 1144 Kogushi, Ube, Yamaguchi 755 Japan.

This work was supported by Grant-in-Aid for Scientific Research on Priority Areas No. 8268235 (to Y.O.) from the Ministry of Education, Science and Culture of Japan.

Received for publication October 10, 1995. Revision received October 28, 1996. Accepted for publication December 31, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Wheeler TJ, Hinkle PC 1985 The glucose transporter of mammalian cells. Annu Rev Physiol 47:503–517[CrossRef][Medline]
  2. Bell GI, Kayano T, Buse JB, Burant CF, Takeda J, Lin D, Fukumoto H, Seino S 1990 Molecular biology of mammalian glucose transporters. Diabetes Care 13:198–207[Abstract]
  3. Birnbaum MJ, Haspel HC, Rosen OM 1986 Cloning and characterization of a cDNA encoding the rat brain glucose transporter protein. Proc Natl Acad Sci USA 83:5784–5788[Abstract]
  4. Asano T, Shibasaki Y, Kasuga M, Kanazawa Y, Takaku F, Akanuma Y, Oka Y 1988 Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development. Biochem Biophys Res Commun 154:1204–1211[Medline]
  5. Thoren B, Sarkar HK, Kaback HR, Lodish HF 1988 Cloning and functional expression in bacteria of a novel glucose transporter present in liver, intestine, kidney, and ß-pancreatic islet cells. Cell 55:281–290[Medline]
  6. Asano T, Katagiri H, Takata K, Tsukuda K, Lin J-L, Ishihara H, Inukai K, Hirano H, Yazaki Y, Oka Y 1992 Characterization of GLUT3 protein expressed in Chinese hamster ovary cells. Biochem J 288:189–193[Medline]
  7. James DE, Strube M, Mueckler M 1989 Molecular cloning and characterization of an insulin-regulatable glucose transporter. Nature 338:83–87[CrossRef][Medline]
  8. Kayano T, Burant CF, Fukumoto H, Gould GW, Fan Y-S, Eddy RL, Byers MG, Shows TB, Seino S, Bell GI 1990 Human facilitative glucose transporters. J Biol Chem 265:13276–13282[Abstract/Free Full Text]
  9. Miyamoto K, Hase K, Taketani Y, Minami H, Oka T, Nakabou Y, Hagihira H 1992 Developmental changes in intestinal glucose transporter mRNA levels. Biochem Biophys Res Commun 183:626–631[Medline]
  10. Harris DS, Slot JW, Geuze HJ, James DE 1992 Polarized distribution of glucose transporter isoforms in Caco-2 cells. Proc Natl Acad Sci USA 89:7556–7560[Abstract]
  11. Cheeseman CI 1993 GLUT2 is the transpoter for fructose across the rat intestinal basolateral membrane. Gastroenterology 105:1050–1056[Medline]
  12. Thorens B, Cheng ZQ, Brown D, Lodish HF 1990 Liver glucose transpoter:a basolateral protein in hepatocytes and intestine and kidney cells. Am J Physiol 259:C279–C285
  13. Burant CF, Takeda J, Laroche EB, Bell GI, Davidson NO 1992 Fructose transporter in human spermatozoa and small intestine is GLUT5. J Biol Chem 267:14523–14526[Abstract/Free Full Text]
  14. Rand EB, Depaoli AM, Davidson NO, Bell GI, Burant CF 1993 Sequence, tissue distribution, and functional characterization of the rat fructose transporter GLUT5. Am J Physiol 264:G1169–1176
  15. Inukai K, Asano T, Katagiri H, Ishihara H, Anai M, Fukushima Y, Tsukuda K, Kikuchi M, Yazaki Y, Oka Y 1993 Cloning and increased expression with fructose feeding of rat jejunal GLUT5. Endocrinology 133:2009–2014[Abstract]
  16. Davidson NO, Hausman AML, Ifkovits CA, Buse JB, Gould GW, Burant CF, Bell GI 1992 Human intestinal glucose transporter expression and localization of GLUT5. Am J Physiol 262:C795–C800
  17. Hof WV, Meer GV 1990 Generation of lipid polarity in intestinal epithelial (Caco-2) cells:sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane. J Cell Biol 111:977–986[Abstract]
  18. Matter K, Brauchbar M, Bucher K, Hauri HP 1990 Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2). Cell 60:429–437[Medline]
  19. Blais A, Bissonnette P, Berteloot A 1987 Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon. J Membr Biol 99:113–125[Medline]
  20. Mahraoui L, Rousset M, Dussaulx E, Darmoul D, Zweibaum,A, Edith Brot-Laroche 1992 Expression and localization of GLUT-5 in Caco-2 cells, human small intestine, and colon. Am J Physiol 263:G312–G318
  21. Asano T, Takata K, Katagiri H, Tsukuda K, Lin JL, Ishihara H, Inukai K, Hirano H, Yazaki Y, Oka Y 1992 Domains responsible for the differential targeting of glucose transporter isoforms. J Biol Chem 267:19636–19641[Abstract/Free Full Text]
  22. Corvera S, Chawla A, Chakrabarti R, Joly M, Buxton J, Czech MP 1994 A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal. J Cell Biol 126:979–989[Abstract]
  23. Piper RC, Tai C, Slot JW, Hahn CS, Rice CM, Huang H, James DE 1992 The efficient intracellular sequestration of the insulin-regulatable glucose transporter (GLUT4) is conferred by the NH2-terminus. J Cell Biol 117:729–743[Abstract]
  24. Hidalgo IJ, Raub TJ, Borchardt RT 1989 Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736–749[Medline]
  25. Mahraoui L, Rodolosse A, Barbat A, Dussaulx E, Zweibaum A, Rousset M, Brot-Laroche E 1994 Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption. Biochem J 298:629–633[Medline]
  26. Niwa H, Yamamura K, Miyazaki J 1991 Gene Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–200[CrossRef][Medline]
  27. Inukai K, Katagiri H, Takata K, Asano T, Anai M, Ishihara H, Nakazaki M, Kikuchi M, Yazaki Y, Oka Y 1995 Characterization of rat GLUT5 and functional analysis of chimeric proteins of GLUT1 glucose transporter and GLUT5 fructose transporter. Endocrinology 136:4850–4857[Abstract]
  28. Matter K, Hunziker W, Mellman I 1992 Basolateral sorting of LDL receptor in MDCK Cells: The cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71:741–753[Medline]
  29. Geffen, Fuhrer C, Leitinger B, Weiss M, Huggel K, Griffiths G, Spiess M 1993 Related signals for endocytosis and basolaterral sorting of the asialoglycoprotein receptor. J Biol Chem 268:20772–20777[Abstract/Free Full Text]
  30. Hunziker W, Fumey C 1994 A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells. EMBO J 13:2963–2969[Abstract]
  31. Rindler MJ, Traber MG 1988 A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells. J Cell Biol 107:471–479[Abstract]
  32. Katagiri H, Asano T, Ishihara H, Tsukuda K, Lin J-L, Inukai K, Kikuchi M, Yazaki Y, Oka Y 1992 Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity. J Biol Chem 267:22550–22555[Abstract/Free Full Text]
  33. Tietze CC, Becich MJ, Engle M, Stenson WF, Mahmood A, Eliakim R, Alpers DH 1992 Caco-2 cell transfection by rat intestinal alkaline phosphatase cDNA increases surfactant-like particles. Am J Physiol 263:G756–G766
  34. Harig JM, Barry JA, Rajendran VM, Soergel KH, Ramaswamy K 1989 D-Glucose and L-Leucine transport by human intestinal brush-border membrane vesicles. Am J Physiol 256:G618–623
  35. Miyake S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C, Saito I 1996 Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA 93:1320–1324[Abstract/Free Full Text]
  36. Asano T, Shibasaki Y, Ohno S, Taira H, Lin J-L, Kasuga M, Kanazawa Y, Akanuma Y, Takaku F, Oka Y 1989 Rabbit brain glucose transporter responds to insulin when expressed in insulin-sensitive Chinese hamster ovary cells. J Biol Chem 264:3416–3420[Abstract/Free Full Text]
  37. Takata K, Kasahara M, Oka Y, Hirano H 1993 Review: Mammalian sugar transporters: Their localization and link to cellular functions. Acta Histochem Cytochem 26:165–178
  38. Takata K, Hirano H 1990 Use of fluorescein-phalloidin and DAPI as a counterstain for immunofluorescence microscopic studies with semithin frozen sections. Acta Histochem Cytochem 23:679–683