From the Division of Immunologic and Infectious Diseases, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318
Received for publication, November 18, 2000, and in revised form, March 29, 2001
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ABSTRACT |
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The coxsackievirus and adenovirus receptor (CAR)
mediates attachment and infection by coxsackie B viruses and many
adenoviruses. In human airway epithelia, as well as in transfected
Madin-Darby canine kidney cells, CAR is expressed exclusively on the
basolateral surface. Variants of CAR that lack the cytoplasmic domain
or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We
have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that
ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a
putative tyrosine-based sorting signal. In addition, we have examined
the targeting of two murine CAR isoforms, with different C-terminal
sequences. The results suggest that multiple regions within the CAR
cytoplasmic domain contain information that is necessary for
basolateral targeting.
The coxsackievirus and adenovirus receptor
(CAR)1 mediates attachment
and infection by coxsackie B viruses as well as by many human
adenoviruses (1-3). Human CAR (hCAR) is a 46-kDa cell surface glycoprotein composed of an extracellular region with two
immunoglobulin-like domains, a typical hydrophobic transmembrane
region, and a cytoplasmic domain of 107 amino acids. A murine homolog
of the human receptor has also been characterized (2, 4), and homologs
in the rat, pig, dog (5), and zebrafish (6) have been reported. The
murine and human proteins are very similar (91% amino acid identity
within the extracellular domain, 77% within the transmembrane domain,
and up to 95% identity within the cytoplasmic domain). Two forms of
mouse CAR, which differ only at the C terminus, and which most likely
result from alternative splicing, have both been shown to function in
virus infection (4). Similar variant isoforms have also been identified
for rat and human CAR (5).
CAR expression is a major determinant of a cell's susceptibility to
adenovirus-mediated gene transfer. In human airway epithelium, hCAR is
localized to the basolateral surface (7-10), where it is inaccessible
to adenovirus delivered to the apical (or luminal) surface. Basolateral
receptor expression has thus been a significant barrier to the use of
adenovirus vectors in gene therapy for cystic fibrosis (11).
To establish and maintain cell surface polarity, epithelial cells
depend on the selective sorting of proteins to specific plasma
membranes and the subsequent retention of these proteins at the correct
cellular domain. Sequences in the transmembrane region (12),
N- or O-glycans in the extracellular region (13, 14), or linkage to a glycosylphosphatidylinositol (GPI) anchor (15-17)
are important signals for targeting to the apical membrane. Basolateral
targeting is associated with signals within the cytoplasmic domain.
These often include dileucine motifs, or the tyrosine-based motifs
NPXY or YXXO (where X is any amino
acid and O is any aliphatic amino acid) (18-23). A growing
number of basolateral sorting signals have been identified that have no
sequence similarities to any of these motifs (24-26). In addition,
interaction between membrane proteins with hydrophobic C-terminal
peptides and scaffolding proteins containing PDZ domains (reviewed in
Ref. 27) have also been implicated in polarized targeting in epithelial
cells (28, 29).
When expressed in Madin-Darby canine kidney (MDCK) cells, hCAR is
localized exclusively to the basolateral surface, as it is in airway
epithelium (10). GPI-anchored hCAR, and hCAR from which the cytoplasmic
domain has been deleted (tailless hCAR), are expressed on both the
apical and basolateral surfaces of transfected MDCK cells, consistent
with our overall view that CAR basolateral targeting depends on
sequences within the cytoplasmic domain.
We have examined the expression of CAR mutants with successive
truncations of the cytoplasmic domain, as well as of CAR with mutations
that ablate a potential PDZ interaction motif and a putative
tyrosine-based sorting signal. In addition, we have studied the
targeting of the two mouse isoforms of CAR, one of which has a
C-terminal amino acid sequence distinctly different from that of both
human CAR and the other mouse isoform. The results suggest that
multiple regions within the CAR cytoplasmic domain contain information
that is necessary for basolateral targeting.
Cell Culture--
MDCK type II cells were grown in Dulbecco's
modified Eagle's medium with 10% fetal calf serum in 10%
CO2. For immunofluorescence and adenovirus infection,
3 × 105 cells/well were plated on 12-mm diameter
polyester filters with a pore size of 0.4 µm (Transwell clears,
Corning-Costar Corp., Cambridge, MA); for biotinylation experiments,
1 × 106 cells/well were seeded onto Transwell 24-mm
diameter filters. In both cases, the MDCK cells were cultured for 3-5
days at which point the cell monolayer was polarized, as demonstrated
by "tight" transepithelial resistances (>700 ohms cm2)
measured with an epithelial voltohmmeter (World Precision
Instruments, Inc., Sarasota, FL).
Expression Vectors Encoding Deletion and Chimeric Mutants of
CAR--
hCAR cytoplasmic deletion and substitution mutants were made
using polymerase chain reaction (PCR)-based strategies to modify coding
sequences cloned in the eukaryotic expression plasmid pcDNA3.1 (Invitrogen, Carlsbad, CA). Tailless hCAR and GPI hCAR were previously described (30). For generation of deletion mutants, a forward primer
was designed to anneal to sequences 5' to a unique restriction site,
BsiWI, in the hCAR cDNA. The reverse mutagenic
primers contained termination codons ~200-300 nucleotides downstream
of the forward primer, as well as the unique restriction site
XbaI. Mutants were named for the final three amino acids
encoded by the truncated cDNA, as well as for the position of the
final amino acid (Fig. 1). Stop codons were introduced so that the
terminal amino acid was lysine 315 in YSK315, asparagine 344 in APN344,
glycine 349 in RMG349, and serine 359 in AQS359. PCR products were
digested with BsiWI and XbaI, then inserted into
the hCAR pcDNA3.1 plasmid cut with the same enzymes. The
PCR-derived portion of each construct was sequenced to confirm that the
correct mutation had been introduced.
Constructs encoding chimeric proteins consisting of the hCAR
extracellular and transmembrane domains fused to the cytoplasmic domain
of each mCAR isoform were generated by splice overlap extension PCR
(31). The constructs for Y318A and LSRM(A4) were also generated by
splice overlap extension PCR, with primers designed to encode an
alanine rather than a tyrosine at position 318 for Y318A or four
alanine residues in place of the amino acids LSRM for LSRM(A4).
Cell Transfection and Isolation of CAR Expressing Cell
Lines--
Mutant cDNA constructs were transfected into MDCK cells
by electroporation (Bio-Rad, Hercules, CA) or with LipofectAMINE 2000 (Life Technologies, Inc., Gaithersburg, MD), and stably transfected cells were selected with 500 µg/ml Geneticin (Life Technologies, Inc.). Cell populations with surface hCAR expression were isolated by
two or three rounds of fluorescence-activated cell sorting with the
anti-hCAR monoclonal antibody RmcB and fluorescein
isothiocyanate-conjugated goat antibody to murine immunoglobulin
(Sigma-Aldrich, St. Louis, MO). The murine myeloma protein mineral oil
plasmacytoma 195 (Sigma-Aldrich) was used instead of RmcB as a
negative control.
Immunofluorescence and Confocal Microscopy--
To test for
apical expression using immunofluorescence, polarized cells were fixed
in paraformaldehyde (1% in PBS) for 30 min, washed, and then stained
only on the apical surface with RmcB followed by a fluorescein
isothiocyanate-conjugated goat antibody to murine immunoglobulin
(Sigma-Aldrich). To examine the distribution of hCAR throughout a cell,
polarized cultures were fixed in 1% paraformaldehyde, washed,
permeabilized with 0.2% Triton X-100, and stained from both the apical
and basal surfaces with RmcB, followed by
tetramethylrhodamine-conjugated goat antibody to murine immunoglobulin
(Sigma-Aldrich). Cells were then examined either by conventional
immunofluorescence using a Nikon Eclipse 800 epifluorescence microscope
or by confocal microscopy in both XY and XZ planes using a Leica TCS 4D
confocal microscope. All immunofluorescence experiments were performed at least three times.
Selective Biotinylation of Polarized MDCK Cells--
For
biotinylation experiments, polarized cultures of MDCK were labeled for
30 min at 4 °C, either apically or basolaterally, with 1 mg/ml
sulfo-N-hydroxysuccinimidobiotin (Pierce, Rockford, IL).
After labeling, the cells were washed with 1 mg/ml glycine in
Dulbecco's modified Eagle's medium to quench unreacted biotin. The
filters were excised to remove unpolarized cells from the edge, and
cells were lysed at 4 °C in PBS containing 1% Triton X-100, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
and 0.15 trypsin inhibitor units/ml aprotinin. The lysate was
centrifuged at 4 °C for 30 min at top speed in a microcentrifuge and
the supernatant was precleared twice, for 2 h at 4 °C, with
mineral oil plasmacytoma 195 bound to protein G beads. To
immunoprecipitate CAR protein, the precleared supernatant was agitated
overnight at 4 °C with 15 µl of protein G beads covalently linked
to RmcB antibody. The beads were washed on ice with PBS containing 1%
Triton X-100 and 2 mM EDTA, then boiled for 5 min in 30 µl of Laemmli buffer. Beads were separated by brief centrifugation,
then supernatant was run on a 10% sodium dodecyl
sulfate-polyacrylamide gel and transferred to a polyvinylidene
difluoride membrane. For detection of biotinylated CAR, membranes were
blocked overnight at 4 °C in PBS containing 2% bovine serum
albumin, exposed for 20 min at room temperature in a 1:2000 dilution of
horseradish peroxidase-conjugated streptavidin (Pierce) in PBS 0.1%
Tween-20, then developed with ECL reagents (Amersham Pharmacia Biotech,
Arlington Heights, IL) and an exposure was made to film. These
experiments were performed at least two times for each cell line.
Infection of Polarized Monolayers with Adenovirus--
Polarized
monolayers of cells expressing mutant hCAR constructs were treated with
neuraminidase type II (Sigma-Aldrich) to remove the glycocalyx as
previously described (10). 1010 particles of adenovirus
type 5 encoding green fluorescence protein (AdVGFP, a kind gift from
Erik Falck-Pedersen, Cornell University) were added to the apical
chamber of each well and incubated for 2 h at 37 °C. Monolayers
were washed, incubated 48 h at 37 °C, then examined for GFP
expression by epifluorescence microscopy. These experiments were
performed at least three times for each cell line.
Basolateral Sorting Information between Residues 315 and
349--
Full-length hCAR is targeted to the basolateral surface of
MDCK cells, whereas hCAR lacking a cytoplasmic domain (tailless) or
bound to the cell by a GPI anchor is expressed on both the apical and
basolateral surfaces (10). To define the sequences responsible for
basolateral localization, we generated a series of CAR mutants with
truncations within the cytoplasmic domain (Fig.
1).
Mutant constructs were stably expressed in MDCK cells, which were
selected for hCAR surface expression. Transfected cells were grown to
confluence as polarized cultures, and examined by both fluorescence and
confocal microscopy (Fig. 2). As
previously observed (10), expression of full-length hCAR was restricted to the basolateral membranes of the polarized MDCK cells, with expression being highest on the lateral surfaces. This was evident by
the characteristic cobblestone pattern of RmcB immunoreactivity with
conventional immunofluorescence, the lack of apical hCAR staining in
unpermeabilized cells exposed to anti-hCAR antibody at the apical
surface, and the primarily lateral localization of hCAR in the XZ plane
observed by confocal microscopy. In contrast, tailless and GPI-linked
hCAR were expressed at both the apical and basolateral surfaces of the
polarized cells (Fig. 2).
The hCAR C-terminal peptide, SIV, closely resembles C-terminal motifs
((T/S)X-hydrophobic) responsible for interaction with PDZ
domains (27, 32). Because such PDZ interactions determine basolateral
localization for some proteins (33), we tested a construct, AQS359,
from which the putative PDZ recognition motif had been removed (Fig.
1). Like full-length hCAR, AQS359 was expressed exclusively at the
basolateral surface, suggesting that PDZ interactions were not required
for CAR targeting (Fig. 3). Mutant
RMG349, which lacked 16 C-terminal residues, was also expressed
exclusively on the basolateral surface, indicating that residues
350-365 do not contain essential targeting information (Fig. 3).
We next generated a mutant, YSK315, from which approximately half of
the cytoplasmic domain had been deleted (Fig. 1). YSK315 was expressed
on both the apical and basolateral surfaces of polarized cells (Fig.
3), suggesting that basolateral targeting information must be contained
within the distal portion of the CAR cytoplasmic domain, between amino
acids 315 and 349.
To define more precisely where this sorting information is located, we
generated an additional deletion mutant, APN344, which lacked 21 C-terminal residues. Apical expression of APN344 was evident by both
confocal and epifluorescence microscopy of transfected MDCK cells (Fig.
3). This suggested that the amino acids LSRMG, which are present in
RMG349 but not APN344, might contain basolateral sorting information.
We then replaced the LSRM residues with alanine residues to generate
mutant LSRM(A4). This was also expressed on the apical surface of
polarized MDCK cells (Fig. 4), indicating that these amino acids function in basolateral targeting.
Expression of CAR on the basolateral surface of polarized cells is not
sufficient to permit adenovirus entry from the apical surface (8, 10).
As another measure of apical CAR expression, we examined the
susceptibility of the transfected cell lines to adenovirus-mediated
gene delivery. Polarized monolayers were grown on Transwell plates, and
adenovirus-encoding green fluorescence protein (AdVGFP) was added to
the apical chamber. After 48 h, monolayers were examined for GFP
expression. Only an occasional GFP-positive cell was found in those
cell lines expressing full-length hCAR, RMG349, or AQS359 (Fig.
5). In contrast, cells expressing tailless CAR, YSK315, APN344, or LSRM(A4) all showed bright
fluorescence 2 days after apical exposure to AdVGFP. Consistent with
the results obtained by microscopy, these observations indicate that
APN344, YSK315, and LSRM(A4) are expressed on the apical surface of
polarized MDCK cells but AQS359, RMG349, and wild-type hCAR are
not.
Additional Sorting Information between Residues 261 and
315--
We also used selective biotinylation to examine the
distribution of CAR between the apical and basolateral surfaces.
Polarized monolayers were exposed to a biotinylating agent at the
apical or basolateral surfaces, CAR was immunoprecipitated from cell lysates, and biotinylated CAR was detected with streptavidin. Consistent with the immunofluorescence results shown above, full-length hCAR was detected only on the basolateral membranes of MDCK monolayers (Fig. 6). Two protein bands were visible
when biotinylated full-length CAR, and several of the mutant
constructs, were immunoprecipitated from MDCK cells (Fig. 6) as well as
from transfected Chinese hamster ovary cells (data not shown). Western
blot analysis of the immunoprecipitated protein revealed that both
bands are forms of hCAR (data not shown), and the smaller form may
represent a degradation product.
Mutants AQS359 and RMG349 were found only on the basolateral surface of
polarized MDCK cells, which is in good agreement with the data obtained
by immunofluorescence and adenovirus infection. Apical expression of
APN344 was detectable, but the expression level, when compared with
basolateral expression, was clearly lower than that seen in cells
transfected with YSK315 (Fig. 6). This is similar to what was seen by
immunofluorescence, where apical staining of APN344 appeared dimmer
than that of YSK315 (Fig. 3). Although residues between 345 and 349 (LSRMG) are required for basolateral targeting, additional information
is contained between residues 315 and 345.
GPI-anchored CAR was detected predominantly on the apical surface, and
tailless CAR was distributed equally between the apical and basolateral
membranes. In contrast, YSK315 showed significantly higher expression
on the basolateral than the apical surface (Fig. 6). This suggests that
there is also sorting information contained between residues 261 and
315, in the region deleted from tailless CAR but not from YSK315.
Tyrosine 318 Is Important for Basolateral Targeting--
As noted
earlier, the amino acids between positions 315 and 345 appear to
contain one or more basolateral sorting determinants. An examination of
this region revealed the presence of a potential tyrosine-based sorting
signal (YNQV) beginning at amino acid 318. To see if this sequence
plays a role in hCAR sorting, the tyrosine residue at position 318 was
changed to an alanine (Fig. 1). As seen in Fig.
7, Y318A could be detected on both the
apical and basolateral membranes of polarized MDCK cells as determined
by fluorescence and confocal microscopy and by selective biotinylation. Polarized cells expressing Y318A were easily infected by AdVGFP that
had been added to the apical chamber of a Transwell filter, confirming
that Y318A is expressed on the apical membrane (Fig. 7). Thus, Y318 is
involved in basolateral targeting.
CAR Isoforms mCAR1 and mCAR2 Are Both Sorted to the Basolateral
Membranes of MDCK Cells--
The two isoforms of murine CAR (mCAR)
have cytoplasmic domains very similar to that of human CAR, although
the C-terminal peptide of mCAR2 is distinctly different from that of
hCAR (Fig. 8) (2, 4). To determine
whether the cytoplasmic domains of both isoforms contained basolateral
targeting signals, chimeras were constructed that combined the
extracellular and transmembrane regions of hCAR with the cytoplasmic
domains of mCAR1 (hm1) or mCAR2 (hm2). Both chimeras were expressed
solely on the basolateral membranes of MDCK cells as determined by
immunofluorescence and biotinylation (Fig. 8).
Previous work demonstrated that wild-type hCAR is sorted solely to
the basolateral membrane of polarized MDCK cells, and that essential
sorting information is contained within the CAR cytoplasmic domain
(10). In these experiments we have delineated CAR sequences involved in
basolateral sorting. Our data suggest that multiple regions within the
CAR cytoplasmic domain are required for basolateral targeting. The
amino acids YNQV, beginning at amino acid 318, comprise a previously
described basolateral sorting signal, and changing the tyrosine within
this motif led to apical expression of hCAR. Deletion or mutation of
the sequence LSRMG between residues 345 and 349 led to apical
expression of hCAR, indicating that these amino acids may also be a
distinct sorting motif. There also appears to be additional sorting
information contained between residues 261 and 315 that functions
independently of these other potential signals.
We found that the deletion of a putative PDZ interaction motif (SIV)
from the hCAR C terminus did not alter the exclusively basolateral
expression pattern, indicating that interaction with PDZ proteins is
not essential for basolateral sorting of hCAR. This contrasts with
evidence that C-terminal PDZ interaction motifs (with the consensus
sequence (T/S)X-hydrophobic) are involved in polarized
expression of such proteins as the cystic fibrosis transmembrane
conductance regulator (29) and the receptor tyrosine kinase let 23 (33). However, our results are similar to those obtained with the
An examination of the cytoplasmic domain of hCAR does not reveal other
known sorting signals. Nevertheless, there is likely to be additional
targeting information contained within the cytoplasmic domain. Tailless
hCAR was expressed in equal amounts on the apical and basolateral
membranes of polarized MDCK cells. In contrast, mutant YSK315, which
lacks approximately half the cytoplasmic domain and contains neither
tyrosine residue 318 nor the potential basolateral sorting sequence
LSRMG between amino acids 345 and 349, was preferentially, but not
exclusively, distributed to the basolateral surface. This suggests that
other basolateral sorting information is contained within the proximal
half of the hCAR cytoplasmic domain. Although these regions are distant
from each other in the linear amino acid sequence of hCAR, they may be
in close proximity with one another in the tertiary structure of this
protein. Therefore, deletion or mutation of any one of these regions
may impair the interaction of the hCAR cytoplasmic domain with cellular
sorting machinery.
For some proteins, such as the low density lipoprotein and epidermal
growth factor receptors (18, 24), deletion of basolateral targeting
determinants within the cytoplasmic domain results in expression that
is almost exclusively apical, perhaps because of strong apical sorting
determinants in the transmembrane or extracellular domains. In
contrast, deletion of the entire CAR cytoplasmic domain resulted in an
equal distribution of tailless CAR on the apical and basolateral
surfaces, and even GPI-anchored CAR showed significant basolateral
expression; these results suggest that the CAR extracellular domain
does not contain strong apical sorting signals.
There are two isoforms of murine CAR: the cytoplasmic domain of mCAR1
is nearly identical to that of hCAR; mCAR2 differs from hCAR and mCAR1
at the C terminus (2, 4). To see if these differences in the
cytoplasmic domain affected the targeting of these receptors, we
constructed chimeric receptors containing the extracellular portion of
hCAR fused to the cytoplasmic domains of the two mCAR isoforms. This
was necessary, because the anti-CAR monoclonal antibody RmcB recognizes
the extracellular region of hCAR, but not of mCAR. Like hCAR, both
mouse isoforms were targeted exclusively to the basolateral membrane of
polarized MDCK monolayers. We found that deletion of 21 residues from
the C terminus of hCAR interfered with basolateral sorting; these
residues are not present in mCAR2, which lacks 26 C-terminal residues
present in hCAR and mCAR1 but which nonetheless is sorted to the
basolateral surface. The mCAR2 C terminus must contain sorting
information that compensates for loss of information from mCAR1, even
though its sequence is quite dissimilar.
CAR's role in virus infection is well established, but its cellular
function remains to be determined. Recent evidence suggests that CAR
may be involved in homotypic cell adhesion (35). It is concentrated at
sites of cell-cell contact in both polarized (10, present study) and
non-polarized cells.2
Consistent with this, CAR's N-terminal immunoglobulin-like domain forms a homodimer, and residues at the homodimer interface are more
highly conserved in evolution than is the rest of the extracellular domain (6).
The CAR cytoplasmic domain shows greater sequence conservation than
does the extracellular domain. At the C terminus, 23 of 23 residues are
identical in human and zebrafish
CAR.3 This sequence
conservation most likely reflects conservation of biological function.
We find that information required for basolateral sorting is present
within this highly conserved region; although the implicated sequences
are absent from CAR's variant isoform, the basolateral targeting is
preserved. These observations suggest that expression at the
basolateral surface of polarized epithelium may be important to CAR's
primary function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequence of the hCAR cytoplasmic
domain. The first amino acid of the domain is located at position
259, whereas the final amino acid is number 365. The C-terminal residue
of each deletion mutant is indicated by an arrow. Tyrosine
318 is outlined by a box.
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Fig. 2.
Immunolocalization of hCAR expression in
polarized MDCK cells. Cells transfected with wild-type
hCAR, tailless hCAR, or GPI hCAR were cultured on permeable supports,
then fixed with paraformaldehyde, and stained for hCAR with the
monoclonal antibody RmcB. The first column (labeled
permeabilized) shows representative immunofluorescence
images of cell monolayers that were permeabilized with Triton X-100
before staining with RmcB. Monolayers in the next column (labeled
apical staining) were fixed but not permeabilized, then
exposed to RmcB only at the apical surface. The third column (labeled
XZ plane) shows representative confocal images in the XZ
plane of cells that had been permeabilized prior to staining with
anti-hCAR.
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Fig. 3.
Immunolocalization of hCAR deletion mutants
in polarized MDCK cells. Cells transfected with AQS359, RMG349,
APN344, or YSK315 were cultured on Transwell filters, then fixed with
paraformaldehyde, and stained for hCAR. The first column shows
representative immunofluorescence images of cell monolayers that were
permeabilized with Triton X-100 before staining with RmcB. Monolayers
in the next column were fixed but not permeabilized, and exposed to
RmcB only at the apical surface. The final column shows representative
confocal images in the XZ plane of cells that had been permeabilized
prior to staining with anti-hCAR.
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Fig. 4.
Immunofluorescence of polarized MDCK cell
monolayers expressing hCAR mutant LSRM(A4). Cells transfected with
LSRM(A4) were cultured on Transwell filters, then fixed with
paraformaldehyde, and stained for hCAR as described for Figs. 2 and
3.
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Fig. 5.
Infection of polarized monolayers with
adenovirus encoding GFP. The apical surfaces of transfected MDCK
cells were exposed to AdVGFP, with transgene expression detected by
fluorescent microscopy 48 h later.
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Fig. 6.
Biotinylation of polarized MDCK
monolayers. Monolayers were biotinylated from either the apical
(A) or basal (B) chamber of Transwell filters.
Monolayers were lysed, and hCAR was immunoprecipitated, subjected to
gel electrophoresis, then transferred to a polyvinylidene difluoride
membrane. Biotin-labeled hCAR was detected with horseradish
peroxidase-conjugated streptavidin and chemiluminescent reagents.
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Fig. 7.
Immunofluorescence, biotinylation, and
adenovirus infection of polarized MDCK cells expressing CAR mutant
Y318A. A, immunofluorescence and confocal microscopy.
B, epifluorescence image of cells that have been infected
from the apical surface by AdVGFP. C, selective
biotinylations. Monolayers were biotinylated from either the apical or
basal chamber of Transwell filters as described under "Experimental
Procedures."
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Fig. 8.
Both isoforms of the CAR
cytoplasmic domain target expression to the basolateral surface.
A, amino acid sequences of the cytoplasmic domains of hCAR
and the murine CAR isoforms mCAR1 and mCAR2 domain. B and
C, polarized MDCK cells expressing CAR chimeras hm1 and hm2.
B, CAR expression determined by immunofluorescence and
confocal microscopy. C, CAR expression determined by
selective biotinylation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid transporter BGT-1, in which deletion of a
PDZ-interacting motif did not prevent localization to the basolateral
membranes of MDCK cells despite the motif's function as a retention
signal (34). It is likely that CAR's PDZ recognition motif is
important for interaction with other cellular proteins, but these
interactions do not drive the polarization of CAR expression.
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ACKNOWLEDGEMENTS |
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We thank Dr. Erik Falck-Pederson for the adenovirus type 5-encoding green fluorescence protein. We are grateful to JenniElizabeth Petrella for excellent technical help, Dr. Peter Bannerman and Susan Puhalla for assistance with confocal microscopy, and Jeffrey Faust and Lester Acosta for their work with the flow cytometry.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants HL 54734 (to J. M. B.) and T32 AI07278 (to C. J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an American Heart Association Established Investigator Award.
Supported by a Pediatric Infectious Diseases Society Fellowship
Award funded by SmithKline Beecham. To whom correspondence should be
addressed: Division of Immunologic and Infectious Diseases, The
Children's Hospital of Philadelphia, Abramson 1202, 3516 Civic Center
Blvd., Philadelphia, PA 19104-4318. Tel.: 215-590-5995; Fax:
215-590-2025; E-mail: Cohenc@email.chop.edu.
Published, JBC Papers in Press, April 20, 2001, DOI 10.1074/jbc.M009531200
2 C. Cohen and J Bergelson, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CAR, coxsackievirus and adenovirus receptor; hCAR, mCAR, human and mouse CAR, respectively; MDCK, Madin-Darby canine kidney; PDZ, PSD95/dlg/ZO-1; GFP, green fluorescence protein; AdVGFP, adenovirus type 5-encoding GFP; GPI, glycosylphosphatidylinositol; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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REFERENCES |
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