From the Ludwig Institute for Cancer Research,
Stockholm Branch, Karolinska Intitutet, SE-17177 Stockholm, Sweden,
the
Ludwig Institute for Cancer Research, Uppsala Branch,
SE-75124 Uppsala, Sweden, and the ** Department of Cell and
Molecular Biology, Karolinska Institutet,
SE-17177 Stockholm, Sweden
Received for publication, June 14, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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The Coxsackievirus and adenovirus receptor (CAR)
functions as a virus receptor, but its primary biological function is
unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR.
LNX harbors several protein-protein interacting domains, including four
PDZ domains, and was previously shown to bind to and regulate the
expression level of the cell-fate determinant Numb. CAR was able to
bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif
in the C terminus of CAR but also upstream sequences. The CAR binding
region in LNX was mapped to the second PDZ domain. CAR and LNX were
also shown to colocalize in vivo in mammalian cells.
We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.
For more than a decade, adenovirus has been used as vector to
transfer genes of interest into several tissues in both animals and
humans. It was long thought that adenovirus-based vectors could
transfer genes promiscuously into almost all tissues and cell types.
This optimistic view has, however, been revised; several obstacles for
efficient gene transfer, including inaccessibility or complete lack of
the primary virus receptor on certain cell types, have been identified.
A more detailed investigation regarding the presence, regulation, and
normal cellular function of the virus receptor in different tissues is
therefore a prerequisite for efficient gene transfers.
The primary virus receptor was cloned in 1997 (1, 2). The same receptor
is shared between two distinct virus groups, Coxsackie B and
adenoviruses, and it has therefore been named CVADR (Coxsackie Virus
and ADenovirus Receptor) or
CAR.1 CAR is a member of the
CTX subfamily of the large Ig superfamily characterized by one V-type
and one C-type immunoglobulin domain in the extracellular domain, one
membrane-spanning region, and an intracellular tail of varying length
(for a review on CAR, see Ref. 3). Because of differential splicing,
CAR is expressed as at least two isoforms, here termed CAR-1 and CAR-2
(4). The two isoforms differ only in the extreme C terminus located in
the intracellular tail of the protein.
The cellular function for CAR is unknown, and no CAR-specific ligand
has yet been identified. CAR might serve as a cell adhesion molecule
and has been shown to mediate homophilic interaction with CAR molecules
on neighboring cells (5, 6). One report suggests that CAR might
function as a tumor suppressor, because the level of CAR expression
inversely correlated with tumorigenicity (7). In polarized epithelial
cells, CAR is located at the basolateral surface and specifically
concentrates at tight junctions where it colocalizes with the PDZ
domain protein ZO-1 (6). A large variation in CAR expression between
tissues has been reported (1, 8, 9). CAR also seems to be
developmentally regulated. Although high levels of CAR are expressed in
the developing brain, expression is very limited in the adult (5, 8).
CAR expression is also down-regulated in skeletal muscle during
maturation and in oropharyngeal epithelium during differentiation (10,
11). Conditions such as differentiation of erythroid and myeloid cells, increased cell density of endothelial cells, and inflammation in heart
muscle involve signals that up-regulate CAR levels in the adult,
indicating that CAR expression is tightly regulated (12-14).
As a step toward understanding the normal function of CAR, we have
searched for proteins interacting with the intracellular tail of CAR.
Both CAR-1 and CAR-2 harbor in their respective C termini possible
binding sites for PDZ domain-containing proteins (4, 15). We used the
complete CAR-1 intracellular tail from mouse in a yeast two-hybrid
screen and identified the Ligand-of-Numb protein-X (LNX) as an
interacting partner with CAR. LNX is a multi-PDZ-containing protein
previously identified as a binding partner for Numb, a protein
implicated in asymmetric cell division and regulation of Notch activity
in the developing nervous system in Drosophila (16, 17). We also
analyzed the regions in CAR and LNX involved in the interaction, and we
demonstrated colocalization of CAR and LNX in mammalian cells.
DNA Constructs
Yeast Expression Plasmids--
pGBT9 and pACT-2 were purchased
from Clontech. pGal4DBD Mammalian Expression Plasmids--
pHA/tLNX, pHA/tLNX-PDZ (1,
2), pHA/tLNX-PDZ (1) were constructed by standard PCR amplification
using yeast pHA/tLNX as template. A common 5'-primer, No. 88, was used
with 3'-primers, Nos. 89, 92, and 91, respectively. PCR fragments were
digested with XhoI and cloned into pBKCMV vector
(Stratagene) treated with EcoRI/Klenow/XhoI.
pHA/tLNX-PDZ (3,4) was constructed by treating pHA/tLNX with
BspHI/Klenow/XhoI and inserting a 0.7-kb fragment into the vector part of pHA/tLNX-PDZ (1) treated with
BbsI/mungbean nuclease/XhoI. pHA/tLNX-PDZ (2) was
constructed by self-ligation of pHA/tLNX-PDZ (1, 2) treated with
BbsI/mungbean nuclease/PmlI. pHA/Wt p80 was
constructed by nested RT-PCR on RNA prepared from mouse lung. RT
reaction was performed using the SuperScript First Strand System
(Invitrogen) according to the manufacturer's directions. Primer
pairs were Nos. 141 and 142 and Nos. 123 and 91. The resulting 1.1-kb
fragment was digested with EcoRI/PmlI and ligated
into pHA/tLNX treated likewise. pHA/Wt p70 was cloned by
excision of Wt p70 from pWt p70 by
EcoRI/XhoI/mungbean nuclease treatment and
ligation downstream of an HA-tag in a pcDNA3.1- derived vector plasmid. pWt p80 was constructed by RT-PCR using RNA prepared from
mouse lung as template. RT reaction was performed using the SuperScript
First Strand System according to the manufacturer's directions. PCR
primers were Nos. 141 and 89. The resulting 2.3-kb fragment was
digested with XhoI and ligated to pcDNA3.1/Zeo(+) vector
(Invitrogen) digested with EcoRV/XhoI. To
construct pWt p70, RT-nested PCR was performed using total RNA from
mouse lung with the following primer pairs, A, Nos. 17 and 16; B, Nos.
122 and 91. The amplified fragment was digested with
EcoRI/PmlI and cloned into pHA/tLNX digested
likewise. pBKCMVmCAR expresses the entire mouse CAR-1 cDNA and has
been described (pRTMR) (1).
GST Plasmids--
pGST was purchased from Amersham Biosciences
(pGEX-2T). pGST·CAR, pGST·CAR
The sequence of all PCR primers can be found at www.licr.ki.se/. The
relevant portions of all new plasmid constructs were sequenced to
verify correct DNA sequences.
Yeast Two-hybrid and cDNA Library Screening
The screen, transformation, and selection procedures were all
performed according to instructions by the manufacturer
(Clontech, Matchmaker Gal4 two-hybrid system).
Briefly, a pACT2 vector-based mouse 17-day embryo cDNA library was
purchased from Clontech. The bait pGal4DBD Mammalian Cell Lines, Culture Conditions, and Transfection
-293 cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 10% heat-inactivated fetal calf serum,
2 mM L-glutamine, 100 units/ml penicillin, and
100 units/ml streptomycin (all from Invitrogen). Cells were cultured at
37 °C in a CO2-humidified incubator. LipofectAMINE 2000 reagent (Invitrogen) was used for plasmid transfection of cells using
conditions that were recommended by the manufacturer.
Expression of GST Fusion Proteins
GST fusion constructs were expressed in Escherichia
coli BL21-competent cells using conditions recommended by the
manufacturer (Amersham Biosciences). Equal amounts of purified GST
fusion proteins were separated on a 10% SDS-PAGE gel under reducing
conditions and visualized by staining with 0.25% Coomassie
Brilliant Blue (BioRad).
Preparation of Mammalian Protein Extract, GST Pull-down Assay,
and Western Blot
293 cells were transfected with 10 µg of LNX plasmid in 10-cm
petri dishes. 24 h later, proteins were extracted by incubating cells in 500 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 137 mM NaCl, 1% Triton X-100, EDTA-free 1× complete
protease inhibitor mixture tablets (Roche)) on ice for 15 min. Lysates
were centrifuged for 10 min at maximum speed in an Eppendorf centrifuge
at 4 °C. 250 µl of supernatant was then diluted with 250 µl of
lysis buffer and incubated with GST fusion protein in the presence of
glutathione-Sepharose 4B at 4 °C for 90 min. After several washes in
lysis buffer, bound proteins were separated on a 10% SDS-PAGE gel
under reducing conditions and transferred to Protean nitrocellulose
transfer membrane (Schleicher & Schuell). Membrane was incubated in
blocking buffer (standard phosphate-buffered saline with 0.1% Tween 20 (1× phosphate-buffered saline-T) and 5% dry milk) overnight at
4 °C, treated with an HA-specific polyclonal antibody
(Clontech) diluted 1:100 in blocking buffer at room
temperature for 1 h, washed in 1× phosphate-buffered saline-T,
incubated with HRP-labeled anti-rabbit Ig (Amersham Biosciences)
diluted 1:4000 in blocking buffer at room temperature for 1 h, and
washed again. Peroxidase activity was detected using enhanced
chemiluminescence and Hyperfilm ECL (Amersham Biosciences).
In Vitro Transcription/Translation
The TNT T7 quick-coupled transcription/translation system
(Promega) was used according to instructions.
[35S]Met-labeled Wt p80 protein was prepared by adding
mammalian pWt p80 directly to the reaction mix. To prepare labeled Wt
p70, PCR amplification using mammalian pWt p70 as template was first done to introduce a T7 promoter. Primers were Nos. 131 and 89. For a
description of the primers, see www.licr.ki.se. Labeled protein extract
was diluted to 1 ml in lysis buffer (see above), and 500 µl was used
in GST pull-down assays performed as described above.
Production of a Polyclonal Antiserum toward CAR-1
A peptide encompassing the last 13 amino acids of human CAR-1
(FKYPYKTDGITVV) was coupled to Imject Maleimide activated mcKLH carrier
(Pierce) via a Cys residue added to the N terminus and used to immunize
rabbits according to standard protocols. The resulting antiserum
(RP194) cross-reacts with the mouse CAR-1 homologue.
Indirect Immunofluorescence
293 cells grown on 6-cm petri dishes were transfected with 0.25 µg of pBKCMVmCAR and/or 1.7 µg of LNX construct. Six hours post-transfection, cells were split and transferred to coverslips in
6-well plates. At 24 h post-transfection, immunofluorescence on
permeabilized cells was performed as described in Ref. 18 except that
the antibody incubation time was increased to 1 h. Primary
antibodies were rabbit polyclonal CAR-1-specific RP194 (see above) and
monoclonal clone HA-7 (Sigma), both diluted 1:500. Secondary antibodies
were fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma)
and Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes), both
diluted 1:200. Stained proteins were analyzed in a Zeiss Axiophot
fluorescence microscope.
Identification of a Protein Interacting with the Cytoplasmic Tail
of CAR--
To identify proteins interacting with the intracellular
tail of mouse CAR-1 (Fig. 1C),
a yeast two-hybrid screen was performed. The complete intracellular
tail of CAR-1 was expressed as a fusion protein with the DNA binding
domain (DBD) of the yeast transcription factor Gal4 by cloning into the
yeast expression vector pGBT9. In the resulting bait plasmid (DBD/CAR),
Gal4 DBD is located in the 5'-end to maintain a free C terminus of
CAR-1 in the expressed fusion protein. DBD/CAR was cotransformed with a
17-day mouse embryo cDNA library in the yeast strain AH109.
Proteins expressed from the pACT-2-derived library all contained an
HA-tag in the N terminus. Of the 3 × 106 clones
screened, 31 cDNAs were identified as positive based on growth on
selective media and activation of the lacZ reporter. Library-derived
plasmids were rescued and retransformed together with bait plasmid in
AH109 to verify the phenotype. Yeast transformed with cDNA only
that was able to grow on high-stringency selection plates, as well as
yeast transformed with cDNAs that expressed proteins that bound to
the Gal4 DNA binding domain, were eliminated from the screen.
Sequencing of the four remaining cDNAs identified two of the clones
as LNX and the remaining two clones as the closely related protein
LNX2. In this report we have focused on LNX. None of the four clones
was full-length cDNA. The N-terminal truncated LNX was therefore
called HA/tLNX (Fig. 1A). Fig.
2A, right
panel shows that the interaction between HA/tLNX and CAR was
specific because growth of blue colonies on high-stringency-selective
plates containing X-
Wild-type LNX is expressed as two isoforms, Wt p70 and Wt p80, that
differ only in the N terminus (Fig. 1A). Both Wt p70 and p80
harbor four PDZ domains and an LDNPAY sequence that previously was
shown to be important for binding to the Numb protein (16). In
addition, Wt p80 harbors a RING finger domain. All of these conserved
protein domains have in other proteins been shown to mediate
protein-protein interactions. To see whether full-length LNX could
interact with CAR, Wt p80 and p70 were cloned in pACT-2 and transformed
in yeast as was done for HA/tLNX. The resulting transformants were
tested for growth on selective and nonselective plates (Fig. 2,
B and C, respectively). Both LNX isoforms showed the same phenotype as HA/tLNX. As an additional control, we performed the same experiment using a cDNA encoding the activation domain of
the transcription factor RXR (Fig. 2D). No growth on
selective media was seen when cotransformed with DBD/CAR, indicating
that CAR does not exhibit unspecific binding to any protein containing a transcription activation domain. Together these experiments showed
that mouse HA/Wt p70, HA/Wt p80, and HA/tLNX all specifically bind to
the intracellular tail of mouse CAR-1 in vivo in yeast.
LNX PDZ Domain No. 2 Interacts with the Intracellular Tail of CAR
in Mammalian Cell Extracts--
The yeast experiments suggested that
the domain of LNX responsible for binding to CAR was located in one or
more of the PDZ domains because HA/Wt p70, HA/Wt p80, as well as
HA/tLNX bound CAR. To verify the interaction in mammalian cells and to
map the interacting domain in LNX, HA/Wt p70 as well as HA/tLNX and PDZ deletions thereof were recloned together with the pACT-2-derived N-terminal HA-tag in a mammalian expression vector (Fig.
1A). The different HA-tagged LNX constructs were transfected
into 293 cells. Protein extracts were prepared and used for in
vitro binding to GST or to a GST·CAR fusion protein (Fig.
1B). Bound proteins were isolated, separated on SDS protein
gels, transferred to filters, and visualized by Western analysis using
an HA-specific antibody (Fig. 3). Unbound
protein extract (input) was included as a control for
transfection efficiency and to show that proteins of expected size were
expressed. The HA-antibody recognized a 70-kDa protein in untransfected
293 cell extract (lane 1). This nonspecific band, however,
disappeared following binding to the GST proteins (lanes 2 and 3). Only HA/LNX-specific signals therefore remained in
the binding experiments when transfected cell extracts were used.
HA/tLNX bound to GST·CAR, thus verifying the interaction seen in the
yeast system (Fig. 3, lane 9). Constructs harboring either PDZ 1 and 2 (HA/tLNX-PDZ (1, 2), lane
15) or PDZ 2 alone (HA/tLNX-PDZ (2), lane
18) retained binding to GST·CAR. In contrast, constructs harboring only PDZ 3 and 4 (HA/tLNX-PDZ (3, 4),
lane 12) or PDZ 1 alone (HA/tLNX-PDZ (1), lane
21) did not bind to GST·CAR. To verify binding to full-length
LNX, HA/Wt p70 were tested and also found to bind GST·CAR (lane
6). No binding to GST alone was seen with any of the HA/LNX
constructs (lanes 5, 8, 11,
14, 17, 20). Similar amounts of GST
fusion proteins were used in all binding reactions (Fig.
4B, upper bands).
Together these results confirmed the interaction between LNX and CAR in
mammalian cell extracts and also identified PDZ domain 2 as being
important for binding to the intracellular tail of CAR.
Sequences in CAR-1 Involved in Binding to LNX--
We next wanted
to identify the region in CAR involved in the interaction with LNX, and
GST·CAR was therefore truncated at two positions (Fig.
1B). PDZ domains of the class I type have previously been
shown to interact mainly with the extreme C terminus of target proteins
that ends with the consensus sequence T/S-X-bulky amino
acid, where X is any amino acid (15). Consistent with this
observation, the binding seen between GST·CAR and HA/tLNX was almost
completely abolished when the binding was instead carried out with
GST·CAR Wild-type, Untagged LNX p70 and LNX p80 Bind to the Intracellular
Tail of CAR--
The in vivo binding experiments carried
out in yeast as well as the in vitro binding experiments
using mammalian cell extracts were all done with LNX proteins harboring
an HA-tag in the N terminus. To investigate whether untagged LNX
proteins could interact with CAR, both full-length LNX proteins were
expressed from a T7 promoter in rabbit reticulocyte lysate in the
presence of [35S]Met. Labeled proteins were used in a GST
pull-down assay, separated on an SDS protein gel, and visualized by
autoradiography (Fig. 5, A and
B). As can be seen in the input control lanes, only one band
of the expected size was synthesized (lane 1). No labeled band was seen in the absence of DNA template in the coupled in vitro transcription/translation reaction (data not shown). Both Wt
p70 and Wt p80 bound to GST·CAR with comparable efficiencies (lane 3). The binding was specific because no binding was
seen to GST alone (lane 2). This experiment showed
that both isoforms of untagged full-length LNX proteins were able to
specifically bind to CAR. Both in vitro-synthesized
full-length LNX proteins showed considerable less binding to
GST·CAR Full-length LNXp70, LNXp80, and tLNX Colocalize with CAR in 293 Cells--
CAR has previously been reported to localize to the plasma
membrane and to accumulate at cell-cell contacts (6). To investigate whether LNX and CAR colocalized in vivo, 293 cells were
cotransfected with plasmids expressing CAR-1 and HA-tagged LNX. The
proteins were localized by indirect immunofluorescence using CAR-1- and HA-specific antibodies, respectively (Fig.
6). HA/tLNX as well as HA/Wt p70 and
HA/Wt p80 were all found to colocalize with CAR at cell-cell contacts,
although considerable staining of all three LNX proteins was also seen
in the cytoplasm and in the nucleus (Fig. 6, A,
B, and C, respectively). Cells transfected with
the LNX constructs alone did not localize to cell-cell contacts,
indicating that the amount of endogenous CAR present in these cells was
not enough to visualize colocalization between CAR and LNX (data not shown). No bleed-through between channels was observed. Together these
results showed that CAR is able to recruit LNX to cell-cell contact
sites of the plasma membrane.
In this study we have identified LNX as a protein that binds to
the intracellular tail of CAR-1. The interaction between CAR and LNX
was shown both in vivo in yeast and in vitro, and
the proteins were also shown to colocalize in mammalian cells.
Binding did not appear to be confined to the consensus PDZ
motif-binding region in the extreme C terminus of CAR-1 but was also
found in upstream sequences common to both CAR splice variants.
Although binding to C-terminal sequences appears to be the typical mode of interaction, PDZ domains can also interact with internal amino acid
sequences within ligands (19). Why a deletion of the last 13 amino
acids of CAR-1 retained binding to LNX whereas a truncation of the last
three did not is at present unclear but might be due to masking of the
upstream binding site because of conformational changes in the
CAR LNX was originally isolated as a binding partner to Numb, a protein
implicated in the control of cell fate decisions during development in
Drosophila (16, 17). Numb antagonizes the Notch signaling pathway, a
process that is regulated through asymmetric cell division in which
Numb and Notch segregate to different daughter cells (20-22). Numb and
Notch are both evolutionary conserved proteins. Homologues of the
proteins responsible for asymmetric localization of Numb in Drosophila
have not been found in the mammalian system, and the mechanism for this
process in mammals therefore remains unknown. Efficient binding between
LNX and Numb requires the NPAY sequence motif as well as the first PDZ
domain in LNX. We demonstrate that CAR interacts with the second PDZ
domain of LNX. Because CAR and Numb interact with different domains of
LNX, the possibility exists that these three proteins are present in
the same multi-protein complex. This raises the possibility that one
function of CAR is to regulate the localization of LNX and Numb to
specific sites at the plasma membrane. It is interesting to note that
LNX2 interacts with Numb and that expression of Numb and LNX/LNX2 were
found to overlap in the developing and adult mouse brain (23). The fact
that CAR also interacts with both LNX family members and that CAR is
expressed at high levels in the developing mouse brain further
strengthens the idea that CAR, LNX, and Numb might form a functional
complex. We have so far been unsuccessful in co-precipitating endogenous CAR and LNX. More work is thus needed to unequivocally demonstrate such an interaction in cultured cells or tissues.
LNXp80 was shown to regulate the levels of Numb by acting as an E3
ubiquitin ligase that specifically targets Numb for
ubiquitin-dependent degradation via the proteasome (24).
The E3 ligase activity is confined to the RING finger domain of p80.
The other isoform of LNX, p70, does not harbor a RING finger domain,
suggesting that this protein has a different function. Whether CAR is
also a substrate for LNX-mediated ubiquitylation, or alternatively if
the ubiquitylation activity of LNX is regulated by CAR is currently being investigated.
CAR is localized all over the plasma membrane but concentrates at
intercellular contacts when cells starts to reach confluence (Ref. 6
and this study). In polarized epithelial cells, CAR was found to be
expressed at tight junctions where it contributed to barrier function.
CAR was also found to bind directly or indirectly to ZO-1, a PDZ
motif-containing protein previously shown to be a component of tight
junctions (6). Thus CAR interacts with two PDZ motif-containing
proteins, LNX and ZO-I, a feature which is not uncommon to plasma
membrane receptors (25). Whether the two PDZ proteins interact with
distinct isoforms of CAR has not been investigated. Binding to LNX and
ZO-1 might mediate different functions in different cell types, or the
interactions might be differentially regulated if present in the same
cell. The interaction between a PDZ domain and its ligand has in some
cases been reported to be regulated by phosphorylation at position The multi-domain structure of PDZ-containing proteins such as LNX
enables them to act as scaffolding proteins interacting with multiple
targets simultaneously. Elucidation of the composition and
intracellular localization of possible LNX multiprotein complexes will
shed light upon the biological function of CAR in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mCARic (DBD/CAR) (bait)
harbors the Gal4 DNA binding domain fused to the complete intracellular
tail of mouse CAR-1 and was constructed by PCR amplification of
pBKCMVmCAR using primers 104 and 103. The resulting fragment was
digested with EcoRI/BfaI and cloned into
EcoRI/SmaI in pGBT9. pHA/tLNX is the
original mouse cDNA rescued in the yeast two-hybrid screen and
contains a truncated LNX cDNA (nucleotides 334-2144 in LNXp70,
accession number AF034746) in pACT-2. pHA/Wt p80 contains a double
HA-tag and was constructed by transfer of an HA-Wt p80 fragment from
the corresponding mammalian expression vector by
EcoRI/Klenow/XhoI treatment into pACT-2 vector treated likewise. pHA/Wt p70 was constructed by transfer of a Wt p70
fragment from the mammalian expression vector pWt p70 treated with
EcoRI/mungbean nuclease/XhoI into pACT-2 treated
likewise. pRXR is a pACT-2-derived plasmid expressing the activation
domain of the transcription factor RXR.
TVV, and pGST·CAR
C-term
were constructed by standard PCR amplification using pBKCMVmCAR as
template, a common 5'-primer, No. 125, and 3'-primers Nos. 126, 130, or
129, respectively. The PCR fragments were digested with
EcoRI/SmaI and cloned into pGEX-2T in the
corresponding sites.
mCARic
construct and the library were transformed sequentially into the yeast
strain AH109 (Clontech), and the transformation mixture was plated on medium-stringency selection plates that lacked
amino acids His, Leu, and Trp. In total, 3 × 106
clones were screened. Emerging colonies were replica-plated on high-stringency selection plates lacking the amino acids Ade, His, Leu,
and Trp. A colony lift assay to test
-galactosidase expression was then performed. Rescue of library-derived plasmids was
done in the bacterial strain KC8 (Clontech) that
has a defect in leuB that can be complemented by yeast LEU2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Gal was only apparent in the presence of
DBD/CAR. Yeast transformed with HA/tLNX and DBD alone or with HA/tLNX
only were not able to grow. Transformation per se did not
affect growth of the yeast host because all three types of
transformants were capable of growing on rich, nonselective yeast
peptone dextrose (YPD) medium (Fig. 2A,
left panel) as well as on plates selecting for the presence
of bait and/or cDNA plasmids (data not shown).
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Fig. 1.
Schematic representation of DNA constructs
and the unique C-terminal sequences of CAR-1 and CAR-2.
A, mouse LNX constructs with color-coded protein domains.
Blue boxes represent PDZ domains 1-4. The RING
finger domain is shown in yellow, the HA-tag in
red, and the LDNPAY sequence is marked as a black
line. B, GST fusion constructs. GST·CAR expresses a
fusion protein between GST and the complete intracellular tail of mouse
CAR-1. GST·CAR TVV and GST·CAR
C-term are C-terminal deletions
of GST·CAR. Numbers indicate the amino acids of CAR present in the
construct. C, amino acid sequence of the two different C
termini of CAR-1 and CAR-2. Gray box represents the part of
CAR common to both isoforms.
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Fig. 2.
Growth of yeast transformants on YPD and
high-stringency selection plates. One colony of each of the
indicated yeast transformants was resuspended in 0.5 ml of water and
streaked onto rich yeast peptone dextrose (YPD) medium (left
panel) or high-stringency selection plates containing X- -Gal
(right panel). Yeast cells were transformed with the
following plasmids: pHA/tLNX alone (tLNX), pHA/tLNX and
pGBT9 (tLNX+DBD), or pHA/tLNX and pGal4DBD-mCARic
(tLNX+DBD/CAR) (A); pHA/Wt p80 alone
(p80), pHA/Wt p80 and pGBT9 (p80 + DBD), or
pHA/Wt p80 and pGal4DBD-mCARic (p80 + DBD/CAR)
(B); pHA/Wt p70 alone (p70), pHA/Wt p70 and pGBT9
(p70 + DBD), or pHA/Wt p70 and pGal4DBD-mCARic (p70 + DBD/CAR) (C); pRXR alone (RXR), pRXR and
pGBT9 (RXR+DBD); or pRXR and pGal4DBD-mCARic
(RXR+DBD/CAR) (D).
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Fig. 3.
Analysis of the region in LNX required for
binding to CAR. Protein extracts from 293 cells transfected with
the indicated HA/LNX plasmids were incubated with GST or GST·CAR.
Bound proteins were analyzed by immunoblotting using an HA-specific
antibody. Input: 5% of the protein extract used in the
binding reactions. Arrowheads indicate the position of the
HA/LNX proteins. Proteins in lanes 1-15 and
16-21 were separated on 10 and 15% SDS-PAGE gels,
respectively. Positions of protein molecular weight markers are shown
to the left.
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Fig. 4.
Analysis of the region in CAR required for
binding to LNX. A, protein extract from 293 cells
transfected with pHA/tLNX was incubated with the indicated GST fusion
proteins. Bound proteins were separated on a 10% SDS-PAGE gel and
analyzed by immunoblotting using an HA-specific antibody. B,
GST fusion proteins were separated on a 10% SDS-PAGE gel and stained
with Coomassie Brilliant Blue. Positions of protein molecular weight
markers are shown to the left.
TVV, indicating that the last three amino acids of CAR are
indeed important for binding (Fig. 4A, compare lanes 2 and 3). Surprisingly, GST·CAR
Cterm, which
expresses a fusion protein lacking the last 13 amino acids of CAR,
could still bind HA/tLNX despite the absence of the C-terminal TVV
residues (Fig. 4A, lane 4). This result suggested
that other sequences besides the TVV terminus are involved in binding
to LNX. The fact that an affinity column harboring a peptide
encompassing the last 13 amino acids of CAR-1 was not able to retain
HA/tLNX from cell extracts further argued for this conclusion (data not
shown). In GST·CAR
Cterm the part of the CAR intracellular tail
expressed is identical in both splice variants of CAR, suggesting that
LNX might also bind to CAR-2 (Fig. 1C). Indeed, a fusion
construct between GST and the complete intracellular tail of the human
CAR-2 homologue interacted with HA/tLNX in a GST pull-down assay (data not shown). The interaction between CAR and HA/tLNX was specific, because no binding to GST alone was seen (Fig. 4A,
lane 1). All GST fusion proteins were used in equal amounts
(Fig. 4B, upper bands). Together these
experiments indicated that binding to LNX may require both an intact
TVV C terminus in mCAR-1 and a region of CAR present in both CAR splice variants.
TVV compared with GST·CAR (compare lanes 3 and
4). Binding was retained when binding was instead done with
GST·CAR
Cterm (lane 5). These results were in
agreement with the binding obtained using extract from
HA/tLNX-transfected cells (Fig. 4A), thus supporting the
conclusion that there might be more than one region in CAR-1 involved
in the interaction with LNX.
View larger version (29K):
[in a new window]
Fig. 5.
Binding of untagged full-length LNXp70 and
LNXp80 to CAR. Wt p70 (A) and Wt p80 (B)
were translated in vitro in the presence of
[35S]Met and incubated with the indicated GST fusions.
Bound proteins were separated on a 10% SDS-PAGE gel. The gel was
dried, and labeled proteins were analyzed by autoradiography.
Input: 10% of the labeled extract used in the binding
reactions.
View larger version (24K):
[in a new window]
Fig. 6.
Colocalization of CAR-1 and LNX in 293 cells. pBKCMVmCAR was cotransfected with pHA/tLNX (A),
pHA/Wt p70 (B), or pHA/Wt p80 (C) in 293 cells.
24 h post-transfection, CAR-1 and LNX were localized by indirect
double immunofluorescence. CAR-1 was detected with a CAR-1-specific
polyclonal antibody and a fluorescein isothiocyanate-conjugated
anti-rabbit secondary antibody. HA-tagged proteins were detected with
an HA-specific monoclonal antibody and Alexa Flour 546 anti-mouse
secondary antibody. Right panels show merged
images.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TVV protein.
2
in the C terminus of the ligand and in other cases by extracellular
signals to a transmembrane ligand (19).
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ACKNOWLEDGEMENT |
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We thank Anita Bergström for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by the Swedish Cancer Society.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.
§ To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, Stockholm Branch, Karolinska Intitutet, Box 240, SE-17177 Stockholm, Sweden. Tel.: 468-7287009; Fax: 468-332812; E-mail: kerstin.sollerbrant@licr.ki.se.
¶ Both authors contributed equally to this work.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M205927200
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ABBREVIATIONS |
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The abbreviations used are: CAR, Coxsackie virus and adenovirus receptor; LNX, Ligand-of-Numb protein-X; tLNX, truncated LNX; Wt, wild-type; HA, hemagglutinin; RT, reverse transcriptase; DBD, DNA binding domain; GST, glutathione S-transferase; RXR, retinoid X receptor.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Tomko, R. P., Xu, R.,
and Philipson, L.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3352-3356 |
2. |
Bergelson, J. M.,
Cunningham, J. A.,
Droguett, G.,
Kurt-Jones, E. A.,
Krithivas, A.,
Hong, J. S.,
Horwitz, M. S.,
Crowell, R. L.,
and Finberg, R. W.
(1997)
Science
275,
1320-1323 |
3. | Philipson, L., and Pettersson, R. F.(2003) Curr. Top. Microbiol. Immunol., in press |
4. | Andersson, B., Tomko, R. P., Edwards, K., Mirza, M., Darban, H., Öncü, D., Sonnhammer, E., Sollerbrant, K., and Philipson, L. (2000) Gene Func. Dis. 2, 11-15 |
5. | Honda, T., Saitoh, H., Masuko, M., Katagiri-Abe, T., Tominaga, K., Kozakai, I., Kobayashi, K., Kumanishi, T., Watanabe, Y. G., Odani, S., and Kuwano, R. (2000) Mol. Brain Res. 77, 19-28[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Cohen, C. J.,
Shieh, J. T.,
Pickles, R. J.,
Okegawa, T.,
Hsieh, J. T.,
and Bergelson, J. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
15191-15196 |
7. |
Okegawa, T., Li, Y.,
Pong, R. C.,
Bergelson, J. M.,
Zhou, J.,
and Hsieh, J. T.
(2000)
Cancer Res.
60,
5031-5036 |
8. | Tomko, R. P., Johansson, C. B., Totrov, M., Abagyan, R., Frisen, J., and Philipson, L. (2000) Exp. Cell Res. 255, 47-55[CrossRef][Medline] [Order article via Infotrieve] |
9. | Fechner, H., Haack, A., Wang, H., Wang, X., Eizema, K., Pauschinger, M., Schoemaker, R., Veghel, R., Houtsmuller, A., Schultheiss, H. P., Lamers, J., and Poller, W. (1999) Gene Ther. 6, 1520-1535[CrossRef][Medline] [Order article via Infotrieve] |
10. | Nalbantoglu, J., Pari, G., Karpati, G., and Holland, P. C. (1999) Hum. Gene Ther. 10, 1009-1019[CrossRef][Medline] [Order article via Infotrieve] |
11. | Hutchin, M. E., Pickles, R. J., and Yarbrough, W. G. (2000) Hum. Gene Ther. 11, 2365-2375[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Rebel, V. I.,
Hartnett, S.,
Denham, J.,
Chan, M.,
Finberg, R.,
and Sieff, C. A.
(2000)
Stem Cells
18,
176-182 |
13. |
Carson, S. D.,
Hobbs, J. T.,
Tracy, S. M.,
and Chapman, N. M.
(1999)
J. Virol.
73,
7077-7079 |
14. |
Ito, M.,
Kodama, M.,
Masuko, M.,
Yamaura, M.,
Fuse, K.,
Uesugi, Y.,
Hirono, S.,
Okura, Y.,
Kato, K.,
Hotta, Y.,
Honda, T.,
Kuwano, R.,
and Aizawa, Y.
(2000)
Circ. Res.
86,
275-280 |
15. |
Songyang, Z.,
Fanning, A. S., Fu, C., Xu, J.,
Marfatia, S. M.,
Chishti, A. H.,
Crompton, A.,
Chan, A. C.,
Anderson, J. M.,
and Cantley, L. C.
(1997)
Science
275,
73-77 |
16. |
Dho, S. E.,
Jacob, S.,
Wolting, C. D.,
French, M. B.,
Rohrschneider, L. R.,
and McGlade, C. J.
(1998)
J. Biol. Chem.
273,
9179-9187 |
17. | Knoblich, J. A. (1997) Curr. Opin. Cell Biol. 9, 833-841[CrossRef][Medline] [Order article via Infotrieve] |
18. | Andersson, A. M., Melin, L., Bean, A., and Pettersson, R. F. (1997) J. Virol. 71, 4717-4727[Abstract] |
19. |
Hung, A. Y.,
and Sheng, M.
(2002)
J. Biol. Chem.
277,
5699-5702 |
20. | Uemura, T., Shepherd, S., Ackerman, L., Jan, L. Y., and Jan, Y. N. (1989) Cell 58, 349-360[Medline] [Order article via Infotrieve] |
21. | Rhyu, M. S., Jan, L. Y., and Jan, Y. N. (1994) Cell 76, 477-491[Medline] [Order article via Infotrieve] |
22. | Guo, M., Jan, L. Y., and Jan, Y. N. (1996) Neuron 17, 27-41[Medline] [Order article via Infotrieve] |
23. | Rice, D. S., Northcutt, G. M., and Kurschner, C. (2001) Mol. Cell Neurosci. 18, 525-540[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Nie, J.,
McGill, M. A.,
Dermer, M.,
Dho, S. E.,
Wolting, C. D.,
and McGlade, C. J.
(2002)
EMBO J.
21,
93-102 |
25. |
Bezprozvanny, I.,
and Maximov, A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
787-789 |