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
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ABSTRACT
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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.
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INTRODUCTION
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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 GLUT15 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.
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RESULTS
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The cDNA constructs were prepared for the expression of GLUT1,
GLUT5, and three GLUT15 chimeras and introduced into Caco-2 cells.
The three chimeras had the following compositions: G15a was
constructed from GLUT1 (N terminus
TM6) and GLUT5 (intracellular
loop
C-terminal), G15b from GLUT1 (N terminus
intracellular loop)
and GLUT5 (TM7
C terminus), and G15c from GLUT1 (N terminus
TM12)
and GLUT5 (C-terminal region). These swapping sites are shown in Fig. 1
. 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 G15a (G15a chimera), clone G15b
(G15b chimera), clone G15c (G15c 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 21656), and rat GLUT5
cDNA (bp 15402170) are shown in Fig. 2
, A, B, and C,
respectively. As shown in Fig. 2A
, we detected small amounts of the
GLUT1 signal in clone D, clone G5, clone G15a, and clone G15b,
presumably due to cross-hybridization with the intrinsic human GLUT1
signal expressed in Caco-2 cells. We detected abundant GLUT1 signals in
clone G15c and clone G1 (Fig. 2A
, 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. 2B
, 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. 2C
),
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.

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Figure 1. A Model for the Orientation of the Chimeric Glucose
Transporter
The putative membrane-spanning domains are numbered 112 from the
NH2 terminus to the COOH terminus. Three chimeric glucose
transporters were prepared from combinations of GLUT1 and GLUT5, and
termed G15a, G15b, and G15c. The amino acid sequences of these
chimeras are indicated beside each chimeric transporter.
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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 21656), (C) rat
GLUT5(bp 15402170). Hybridization was performed in a solution
containing 50% deionized formamide, 5x Denhardts 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 G15a; lane 4, clone
G15b; lane 5, clone G15c; lane 6, clone G1.
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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 G15a, clone G15b, and clone G15c (Fig. 3
), 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.

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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 490502), 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 G15a; lane 4, clone G15b;
lane 5, clone G15c; lane 6, clone G1.
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Immunofluorescence labeling of Caco-2 monolayers revealed marked
differences in the cellular distributions of GLUT1 and GLUT5 (Fig. 4
). GLUT5 was observed on the apical side in clone G5
(Fig. 4a
). In double-labeling experiments, the GLUT5 labeling was
colocalized with the intense labeling of F-actin, a marker of the brush
border (Fig. 4b
), 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. 4g
). 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. 4d
). 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).

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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.
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For comparison with the distribution of wild type GLUT1 or GLUT5,
stable transfectants expressing GLUT15 chimeric protein were likewise
studied. Figure 5
shows the results of
immunofluorescence studies of clone G15a, clone G15b, and clone
G15c, obtained using an anti-GLUT5 C-terminal antibody.
Interestingly, in clone G15a, 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 G15b
chimeric transporter, composed of GLUT1 (N terminus
intracellular
loop) and GLUT5 (TM7
C terminus), and the G15c 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.

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Figure 5. Immunofluorescence Localization of Glucose
Transporters in Clone G15a (upper), Clone G15b
(middle), and Clone G15c (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).
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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 1211)-GLUT1 (intracellular loop domain, amino
acid residues 207271)-GLUT5 (TM7
C-terminus, amino acid residues
277502), designated G51-5. We prepared the cDNA constructs encoding
G5 and G51-5 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, G51-5 protein
was mainly targeted to the basolateral membrane, while the wild type
GLUT5 was mainly targeted to the apical membrane (Fig. 6
).

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Figure 6. Immunofluorescence Localization of G5
(middle), G51-5 (lower), and lacZ
(upper) Expressed Using a Recombinant Adenovirus
Expression System
Recombinant adenoviruses for expressing wild type GLUT5, G51-5, 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.
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DISCUSSION
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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 G15a
chimeric protein were localized on the apical side, while the wild type
GLUT1 and the G15b and G15c 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 GLUTs intracellular loop region,
we studied the targeting of G51-5 protein, GLUT5, in which the
intracellular loop domain had been replaced with that of GLUT1. First,
we prepared G51-5 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 G51-5 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, G51-5
protein was targeted to the basolateral membrane, while the wild type
GLUT5 was mainly targeted to the apical membrane (Fig. 6
).
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 G51-5
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.
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MATERIALS AND METHODS
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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 (G15a, G15b,
G15c) 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. 1
, the three
chimeras had the following compositions: G15a was constructed from
GLUT1 (N terminus
TM6) and GLUT5 (intracellular loop
C-terminal),
G15b from GLUT1 (N terminus
intracellular loop) and GLUT5 (TM7
C
terminus), and G15c 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 Denhardts solution (1x
Denhardts 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 491503) 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.
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FOOTNOTES
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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.
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REFERENCES
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