From the Departments of Immunology and Cell Biology
and ¶ Physiology, Graduate School of Medicine, Kyoto University,
Kyoto 606-8501, Japan
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ABSTRACT |
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4F2, also termed CD98, is an integral membrane
protein consisting of a heavy chain linked to a light chain by
disulfide bond. We have generated a monoclonal antibody to the mouse
4F2 light chain and cloned the cDNA. It encodes a mouse counterpart
of rat L-type amino acid transporter-1, and induces system L amino acid transport in Xenopus oocytes in the presence of 4F2 heavy
chain. Transfection studies in mammalian cells have indicated that the 4F2 heavy chain is expressed on the plasma membrane on its own, whereas
the 4F2 light chain can be transported to the surface only in the
presence of 4F2 heavy chain. 4F2 heavy chain is expressed diffusely on
the surface of fibroblastic L cells, whereas it is localized
selectively to the cell-cell adhesion sites in L cells expressing
cadherins. These results indicate that the 4F2 heavy chain is
associated covalently with an amino acid transporter and controls the
cell surface expression as well as the membrane topology of the 4F2
heterodimer. Although 4F2 heavy and light chains are expressed
coordinately in most tissues, the light chain is barely detected by the
antibody in kidney and intestine, despite the presence of heavy chain
in a complex form. The results predict the presence of multiple 4F2
light chains.
4F2 antigen, also called CD98, has been originally identified as
an activation antigen of lymphocytes (1). It is known to be rather
ubiquitously expressed in many types of cells and notably in almost all
tumor cell lines (2). Early biochemical studies have revealed that 4F2
antigen is a heterodimer consisting of a type 2 glycosylated integral
membrane protein of around 80 kDa (heavy chain
(H-chain))1 and a protein
with apparent molecular mass of 37 kDa (light chain (L-chain)) linked
by disulfide-bond (2, 3). Although 4F2 H-chain cDNA has been
previously cloned (4, 5), 4F2 L-chain remained unidentified. 4F2
H-chain shares some 30% homology with a broad specificity amino acid
transporter (BAT), which activates system b0,+-like amino
acid transport (6, 7), and is shown to induce system y+L
amino acid transport when expressed in Xenopus oocytes (8, 9). Since 4F2 H-chain has been indicated subsequently to induce multiple amino acid transport systems (10), there has been speculation that 4F2 H-chain is a specific activator of the transport systems rather than a carrier (11). Besides its relation to amino acid transport, a variety of functional implications have been made on 4F2.
For instance, anti-4F2 H-chain antibody has been reported to inhibit
growth of some tumor cells (12) and hematopoietic progenitor cells
(13). Also, it was indicated to be involved in the virus-induced
syncytium formation as well as cell fusion of normal monocytes in the
absence of virus infection (14, 15). More recently, 4F2 H-chain has
been shown to reverse the dominant negative effect of overexpressed
cytoplasmic domain of In the present study, we first have generated a monoclonal antibody to
mouse 4F2 L-chain and isolated the 4F2 L-chain cDNA by expression
cloning using it. 4F2 L-chain consists of 512 residues and is predicted
to be a very hydrophobic protein with 11 or possibly 12 membrane-spanning regions. GenBankTM search has revealed
that the cDNA is a mouse counterpart of the most recently reported
rat gene termed L-type amino acid transporter-1, LAT1. LAT1
cRNA could induce system L amino acid transport in Xenopus
oocytes in the presence of rat 4F2 H-chain and has been suggested to be
a 4F2 L-chain (17). We have confirmed that the mouse 4F2 L-chain
induces high affinity amino acid transport with features of system L in
the presence of mouse 4F2 H-chain and have proved that it is associated
covalently with 4F2 H-chain by a disulfide bond via cysteine at
position 103 of the latter. The 4F2 H-chain has been indicated to be
expressed on the cell surface as a monomer on its own, whereas 4F2
L-chain is transported to the plasma membrane only in the presence of
4F2 H-chain. 4F2 H-chain is expressed on the epithelial cell surface of
most embryonic tissues in vivo, and the analysis on cultured
cells has indicated further that 4F2 H-chain is expressed selectively
at cell-cell adhesion sites generated by cadherins. The present results
thus reveal a critical role of 4F2 H-chain in the control of
intracellular trafficking as well as the cell surface topology of the
4F2 heterodimer and provide a new clue to delineate the mechanisms for
its involvement in diverse cellular functions. We also present the
results predicting the presence of additional 4F2 L-chains that is
distinct from that of LAT1 in some normal epithelial tissues such
as kidney and intestine.
Antibodies and Cell Lines--
Anti-4F2 H-chain monoclonal
antibody (mAb), 14.37, has been reported previously (3). To raise
anti-4F2 mAbs for multiple purposes, Armenian hamsters were immunized
with pooled SDS-PAGE gel slices corresponding to the 80-kDa 4F2 H-chain
or 37-kDa L-chain from the P3U1 cell lysates that had been
immunoprecipitated with 14.37 mAb. Hybridoma supernatants were screened
by two independent assays: immunoprecipitation of
125I-labeled P3U1 cell lysates, and immunoblotting of the
cell lysates immunoprecipitated with 14.37 mAb. By these procedures,
two additional anti-4F2 H-chain mAbs were obtained: 10.10 mAb, which is
capable of efficiently immunoprecipitating the 4F2 heterodimer from
P3U1 cells as well as the 4F2 H-chain expressed by cDNA
transfection, and 10.4 mAb, which is effective for the detection of
80-kDa 4F2 H-chain by immunoblotting. The 10.10 mAb could be used for
immunoprecipitation, immunostaining, and immunohistochemistry, whereas
the 10.4 mAb could be used for immunoblotting. Another mAb, 10.7, was
also capable of immunoprecipitating the 4F2 heterodimer from P3U1
cells. It specifically reacted to the 37-kDa band by the immunoblotting and was indicated to recognize the 4F2 L-chain (see also text). The
10.7 mAb has been shown to stain the cells only after the permeabilization, suggesting that its epitope is in the cytoplasmic region. Anti-E-cadherin (ECCD-2) was purchased from Takara Co.Ltd, Kyoto, Japan, and anti-N-cadherin (NCD-2) was provided by Dr. Takeichi,
Kyoto University, Kyoto, Japan. Anti-Myc antibody (9E10) was purified
in our laboratory. L cells and those stably transfected with E-cadherin
(EL) and N-cadherin cDNA (NL) were also provided by Dr. Takeichi.
All cell lines were maintained in Dulbecco's modified minimal
essential medium supplemented with 10% fetal calf serum.
cDNA Transfection and Expression Cloning--
cDNA
library of BAL 17.2 mouse B lymphoma cells was constructed in the
expression vector pPISC,2 and
COS cells were transfected with the cDNA library (10 µg/5 × 106 cells) by electroporation. Cells were harvested by
trypsinization 3 days after the transfection and fixed and
permeabilized by Fix and Parm (Caltag, S. San Francisco, CA), as
instructed by manufacturer. Cells were stained with 10.7 mAb followed
by FITC-conjugated anti-hamster IgG (Caltag, S. San Francisco, CA).
Positively stained cells were collected by cell sorting with
fluorescence-activated cell sorter Vantage (Becton Dickinson, Mountain
View, CA). Episomal plasmids were directly recovered from such
collected cells by the method described by Davis et al.
(18). Briefly, the sorted cells were treated with buffer containing 100 mM EDTA, 10 mM Tris-Cl, pH 8.0, 0.1% SDS, and
100 µg/ml of proteinase K at 55 °C overnight followed by
phenol/chloroform extraction and ethanol precipitation. Samples were
suspended in 2 µl of water and transformed into bacteria. Plasmids
were purified from liquid culture of bacteria and again transfected
into COS cells. After three cycles of the procedure, a single plasmid
clone, p10.7, was isolated and sequenced. The cDNA transfection
into COS and HeLa cells was done using electroporation and
CaPO4 method, respectively.
Plasmid Construction--
4F2 L-chain cDNA tagged with Myc
epitope at the C terminus was constructed by subcloning synthetic
oligonucleotides encoding the epitope tag and a part of cDNA into
the 3'-end of the cDNA. Single residue mutants of 4F2 H-chain
(cysteine at position 103 being substituted for serine, C103S, and
cysteine at 325 for serine, C325S) were constructed by the two-step PCR
and confirmed by DNA sequencing. For cRNA synthesis, cDNAs of both
4F2 H-chain (3) and 4F2 L-chain were subcloned into pSP73 vector
(Promega, Madison, WI). After linearizing the plasmids with
XhoI, cRNAs were synthesized by using mMASSAGE, mMACHINE SP6
kit (Ambion, Austin, TX), as instructed by the manufacturer.
Immunoprecipitation, Immunoblotting, and Northern
Blotting--
Cells either unlabeled or surface labeled with biotin
using biotin-XX succinimidyl ester (Molecular Probes, Eugene, OR) were lysed with a lysis buffer (1% Nonidet P-40, 50 mM Tris-Cl,
pH 7.4, 0.15 M NaCl, 10 mM EDTA, PMSF,
leupeptin, antipain, chymostatin trypsin inhibitor), incubated with
antibodies (2-5 µg) at 4 °C for 3 h, and then precipitated
with protein A-Sepharose 4B (Amersham Pharmacia Biotech, Uppsala,
Sweden) at 4 °C for 30 min. Lysates were electrophoresed in
SDS-PAGE, blotted on polyvinylidine difluoride membranes, incubated
with antibodies followed by horseradish peroxidase-conjugated second
antibodies or with avidin-biotin complex (ABC) reagent (Vector,
Burlingame, CA) for biotinylated samples, and developed using a
Supersignal Western blotting detection system (Pierce, Rockford, IL).
Northern blotting was done as described previously (3).
Immunofluorescence Staining and Immunohistochemistry--
Cells
were cultured on the coverslips, rinsed with TBS+
(Tris-buffered saline, pH7.4, supplemented with 10 mM
CaCl2), fixed with 3% formaldehyde in TBS+,
and blocked with 2% bovine serum albumin in TBS+. For
double staining, the cells were incubated with anti-4F2 H-chain mAb
(10.10) and anti-E-cadherin or anti-Myc for 1 h at room
temperature and then with biotin-conjugated goat anti-hamster IgG
(Caltag) and FITC-conjugated rabbit anti-rat IgG or anti-mouse IgG
(Caltag) for 1 h at room temperature followed by Texas red-avidin (Biomeda, Foster City, CA). The samples were dried, mounted in ProLong
antifade kit (Molecular Probes, Inc., Eugene, OR), and analyzed with a
confocal laser microscopy (Olympus, Osaka, Japan). Immunohistochemistry
was performed as described before (19). Briefly, whole embryos (E14)
were fixed with 4% paraformaldehyde at 4 °C for 30 min. Frozen
sections at 10-16-mm thickness were preblocked, incubated with 10.10 mAb, and then with biotin-conjugated goat anti-hamster IgG followed by
ABC kit.
Measurement of Amino Acid Uptake in Xenopus Oocytes--
Amino
acid uptake was measured as described (6) with slight modifications.
Briefly, five to seven Xenopus oocytes per condition were
washed twice in amino acid-free uptake solution (100 mM
choline chloride, 2 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, and 10 mM Hepes, pH 7.5). Oocytes injected with cRNAs or water as
a control were incubated with 200 µl of uptake solution containing 50 µM radiolabeled amino acids at 370 KBq/ml for 30 min at
25 °C. Amino acid competition experiments were performed by adding 5 mM inhibitors to the uptake solutions. After incubation,
oocytes were washed five times with 1 ml of ice-cold wash solution (80 µM choline chloride, 20 mM L-arginine, 20 mM L-leucine, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM Hepes, pH 7.5).
Each oocyte was then transferred to a vial, dissolved with 200 µl of
10% SDS, followed by addition of 3 ml of scintillation solution for
scintillation counting.
4F2 Heavy Chain Is Associated Covalently with a System L Amino Acid
Transporter by Disulfide Bond--
An anti-mouse 4F2 mAb, 10.7, was
produced, that was capable of immunoprecipitating a band at 120 kDa in
nonreducing condition and two bands at 80 and 37 kDa positions in
reducing condition from the surface-labeled P3U1 cell lysates (data not
shown). When the immunoprecipitate of P3U1 lysate with either anti-4F2
H-chain (14.37) or 10.7 mAb was blotted with 10.7, a 37-kDa band was
detected (Fig. 1a,
left), and conversely, immunoprecipitation with 10.7 resulted in the coprecipitation of 80 kDa 4F2 H-chain (Fig.
1a, right). The results indicate that 10.7 mAb
recognizes the light chain of 4F2 heterodimer. We then isolated a 4F2
L-chain cDNA, p10.7, by expression cloning using the mAb as
described under "Experimental Procedures." When COS cells were
transfected with either 4F2 H-chain (p14.37, Ref. 3) or p10.7 cDNA,
an 80- or 37-kDa band, respectively, was immunoprecipitated only with
the corresponding mAb as expected. On the other hand, when COS cells were cotransfected with both cDNAs, anti-4F2 H-chain mAb could immunoprecipitate the 37-kDa band reactive to 10.7 mAb in addition to
the 80-kDa 4F2 H-chain band (Fig. 1b). Because 4F2 H-chain has only two cysteines at positions 103 and 325 in the extracellular region (5), a single residue mutation for each cysteine to serine was
introduced, C103S and C325S, and cotransfected with p10.7 cDNA. As
also shown in Fig. 1b, the C103S mutation of 4F2 H-chain
abrogated the coprecipitation of the 37-kDa band by anti-4F2 H-chain
mAb, whereas the C325S mutation did not. These results have proven that
the cDNA indeed encodes the 4F2 L-chain.
The p10.7 cDNA consists of 3456 bp and contains an open reading
frame (nucleotides 27-1565) encoding 512 residues
(GenBankTM/DDBJ accession number AB17189). The deduced
amino acid sequence is highly homologous (98% identity) to the
recently reported rat LAT1 (17). The hydrophobicity profile shown in
Fig. 2a predicts at least 11 and possibly 12 helical transmembrane domains. As shown in Fig.
2b, injection of 4F2 L-chain cRNA alone into
Xenopus oocytes induced negligible
Na+-independent uptake of Leu or Arg, whereas 4F2 H-chain
cRNA induced Arg uptake as reported previously (8, 9). When both cRNAs were coinjected, potent Na+-independent Leu uptake was
induced, whereas Arg-uptake tended to be suppressed as compared with
that induced by 4F2 H-chain cRNA alone. The Leu uptake in the double
cRNA transfectants was almost completely inhibited by Ile, Val, His,
and Phe as well as by 2-( 4F2 H-chain Guides the 4F2 L-chain to Plasma Membrane That Is
Independent of Disulfide Linkage--
We then examined the
intracellular trafficking of each protein. COS cells transfected with
either 4F2 L- or H-chain cDNA alone, or with both, were
surface-labeled with biotin, lysed, and immunoprecipitated with
anti-4F2 H- or L-chain mAb followed by the detection of biotinylated proteins with ABC system. As controls, aliquots of the same cell lysates (one-fourth) were immunoprecipitated similarly and blotted with
the corresponding mAbs. The level of biotinylated 4F2 L-chain was found
to be marginal as compared with the total 4F2 L-chain in the L-chain
single transfectants, whereas the vast majority of 4F2 L-chain was
estimated to be expressed on the cell surface in the H-chain/ L-chain
double transfectants (Fig. 3a,
left panel). In contrast, comparable levels of biotinylated
4F2 H-chain were detected in both H-chain single and H-chain/L-chain
double transfectants (Fig. 3a, right panel).
Biotinylated 4F2 H-chain in the former was detected as a monomer
without covalently associated molecule as 4F2 H-chain (C103S) (Fig.
3b), eliminating the possibility that mouse 4F2 H-chain was
associated with the endogenous 4F2 L-chain in COS cells and expressed
on the cell surface. 4F2 H-chain (C103S), that failed to form
disulfide-linkage with 4F2 L-chain, however, was capable of inducing
cell surface expression of 4F2 L-chain as efficiently as wild type 4F2
H-chain (Fig. 3a). These results were confirmed by immunofluorescence staining. When 4F2 H- or
L-chain cDNA was singly transfected into HeLa cells, 4F2 H-chain
was expressed on the cell surface, whereas 4F2 L-chain was remained
mostly in the cytosol particularly in the Golgi area (Fig. 4,
a versus b). With the cotransfection
of 4F2 H- and L-chain cDNAs, on the other hand, 4F2 L-chain was
expressed on the cell surface with the same pattern as 4F2 H-chain
(Fig. 4, c versus d). An essentially
similar effect was obtained by the cotransfection with 4F2 H-chain
(C103S) cDNA as well (Fig. 4, e versus
f). These results indicate that 4F2 H-chain functions as a
"guidance molecule" for 4F2 L-chain to the plasma membrane, for
which the covalent linkage by a disulfide bond is not essential.
4F2 H- and 4F2 L-chains Are Coordinately Induced in Normal
Lymphocytes following Activation--
In normal mouse lymphocytes, the
4F2 L-chain transcript is induced rapidly following the mitogenic
stimulation in vitro with concanavalin A in a coordinated
manner as with that of 4F2 H-chain (Fig.
5a). Also, both transcripts
are expressed in all leukemic cell lines examined and with similar
relative intensities (data not shown). In Fig. 5b,
expression profiles of 4F2 H- and 4F2 L-chain transcripts in normal
adult organs are shown. Although both mRNAs are expressed rather
ubiquitously, the level of 4F2 L-chain mRNA appears to be
disproportionally low as compared with that of 4F2 H-chain in kidney,
small intestine, and liver (see below).
4F2 H-chain Is Sorted Specifically to the Cell-Cell Adhesion Sites
Generated by Cadherins--
The expression pattern of 4F2 on the cells
was then investigated. In OTF9 embryonic carcinoma cells, endogenous
4F2 H-chain was found to be located selectively at the cell-cell
adhesion sites (Fig. 6b).
Because the distribution was found to be nearly identical to that of
E-cadherin (Fig. 6a), we intended to examine directly the
effect of cadherins on 4F2 H-chain distribution at the cell surface
using fibroblastic L cells and those stably transfected with E-cadherin
(EL) or N-cadherin (NL). In L cells, 4F2 H-chain was stained diffusely
on the surface and E-cadherin was undetectable (Fig. 6, c
and d). In EL and NL cells, which exhibited significant cell-cell adhesion, 4F2 H-chain was concentrated at the cell-cell adhesion sites and colocalized with the cadherins (Fig. 6,
e, f and g, h).
Immunoprecipitation analysis revealed that 4F2 H-chain was associated
with 4F2 L-chain in all these cells (data not shown). These results
thus suggest that 4F2 H-chain is sorted specifically to the cell-cell
adherent membrane sites, most likely together with 4F2 L-chain, once
the stable cell adhesion is generated by cadherins.
Expression of 4F2 H-chain in Various Normal Tissues, Localization
at Cell-Cell Adhesion Sites in Polarized Epithelial Cells and
Implication for Multiple 4F2 L-chain(s)--
Finally, we examined the
cellular localization of 4F2 H-chain in various normal tissues.
Immunohistochemistry of mouse embryos has indicated that 4F2 H-chain is
expressed on the surface of epithelial cells of most tissues, including
epidermis (Fig. 7a), the
choroid plexus in the brain (b), retina (c), as
well as intestinal (d), renal (e), and thymic
epithelium (f). In polarized epithelial cells such as in
intestine and kidney, 4F2 H-chain expression is restricted apparently
at the lateral adhesion sites (Fig. 7, d and e),
consistent with the colocalized expression with E-cadherin in cell
lines. We wished to know whether 4F2 H-chain on the cell surface of
these tissues is associated with the 4F2 L-chain. Thus far, the only
available anti-4F2 L-chain antibody, 10.7 mAb, worked poorly in
immunohistochemistry. Therefore, immunoblotting analysis was performed.
The 4F2 H-chain is expressed in all embryonic and adult tissues
examined (Fig. 8A). The 4F2
L-chain, however, is detected barely in the kidney, intestine, and
adult liver, whereas it is expressed strongly in others including
brain, testis, and spleen as well as fetal liver (Fig. 8a).
Nonetheless, the 80-kDa 4F2 H-chain in kidney and intestine is detected
still as a 120-kDa complex in nonreducing condition (Fig.
8b), strongly implying that the 4F2 H-chain forms complex
with 4F2 L-chain(s) that is distinct from the LAT1 in these polarized
epithelial tissues.
In the present study, we have generated a monoclonal antibody to
the mouse 4F2 L-chain and cloned its cDNA. The deduced amino acid
sequence has revealed that 4F2 L-chain consists of 512 residues with at
least 11 or possibly 12 helical transmembrane domains, calculated
molecular mass being 56 kDa.
Results3 indicate that both
N- and C-terminal ends of the coding region are retained in the 4F2
L-chain, and thus much faster migration of 4F2 L-chain in SDS-PAGE at
37-kDa position appears to be because of the intrinsic structural
features. 4F2 L-chain is highly homologous (98% identity) to the very
recently reported rat LAT1 (17) and thus considered to be its mouse
counterpart. Rat LAT1 has been shown to induce system L amino acid
transport in Xenopus oocytes in the presence of 4F2 H-chain.
Based on the functional dependence on 4F2 H-chain, LAT1 is suggested to
be a 4F2 H-chain. Our present results have confirmed that the mouse 4F2
L-chain can mediate amino acid transport with typical features of high
affinity system L when expressed in Xenopus oocytes together
with the 4F2 H-chain. We have indicated further that the 4F2 L-chain is
associated covalently with 4F2 H-chain in the cells by a disulfide bond
via cysteine at position 103, which is conserved in the 4F2 H-chain of
mouse, rat, and human (4, 5). Thus, it has been proved that 4F2 H-chain
is associated covalently with a system L amino acid transporter.
We then addressed the molecular basis for the functional dependence of
4F2 L-chain on 4F2 H-chain. Present results have indicated that 4F2
H-chain alone is expressed efficiently on the cell surface as monomer.
In contrast, 4F2 L-chain is expressed minimally at the plasma membrane
in mammalian cells, remaining mostly in the Golgi area, and requires
4F2 H-chain to be sorted to the cell surface. The results thus indicate
that one of the functions of 4F2 H-chain is to guide 4F2 L-chain to the
plasma membrane. Rather unexpectedly, the guidance effect is
independent of disulfide-linkage, implying the involvement of
noncovalent steric association. A similar mechanism has been proposed
for the heterodimeric P-type cation-exchange ATPases, in which a
Amino acid permeases in lower eukaryocytes are expressed usually as
monomeric proteins (21). Membrane expression of permeases in yeast,
however, is shown to be controlled by a unique ER-resident protein,
SHR3, without which the transport of permeases from ER to plasma
membrane is impaired selectively (22). In this aspect, it is noted that
a mutation of BAT (M467T), most commonly detected in the patients of
type I cysteinuria, results in the defective expression of BAT protein
on the cell surface (23). The BAT is reported also to be associated
with an as yet undefined 50-kDa protein in the kidney cells (24). It
thus seems possible that 4F2 H-chain/BAT family, often called
"transport-related" proteins, represents specific "guidance
molecules" for selected amino acid transporters to the plasma
membrane in mammalian cells. At present, it remains to be seen whether
4F2 H-chain has additional functions as an integral part of the
transport carrier.
In normal mouse embryos, 4F2 H-chain has been shown to be expressed
prominently in the epithelial cells of most tissue, in addition to the
vascular and lymphohematopoietic cells. In the polarized epithelial
cells such as in kidney and intestine, 4F2 H-chain expression appears
to be restricted at the lateral sites, which primarily depended on
cadherins (25). Indeed, immunofluorescence analysis of OTF9 embryonic
carcinoma cells in culture has indicated clearly that the 4F2 H-chain
is expressed selectively at the cell-cell adhesion sites and
colocalizes with E-cadherin. Furthermore, in L cells stably transfected
with E- or N-cadherin cDNA, 4F2 H-chain is expressed at the
cell-cell adhesion sites, whereas it is expressed diffusely on the
surface of L cells without cadherin, indicating that the membrane
topology of 4F2 H-chain is regulated by cadherins. Results4 have indicated that E-cadherin is
coimmunoprecipitated with 4F2 heterodimer from OTF9 cells lysed with
mild detergents, suggesting that 4F2 complex is included in the
membrane domain generated by E-cadherin. Similar cell-cell
adhesion-dependent restriction of the cell surface topology
has been reported for Na+/K+-ATPase and
Cl Our present results have also implicated the presence of multiple 4F2
L-chain(s) that are associated covalently with 4F2 H-chain. In cells of
most tissues including lymphoid cells and most cancer cells, both 4F2
H- and L-chain are detected at comparable levels. In the intestine and
kidney, however, 4F2 L-chain is detected barely by the 10.7 mAb.
Nonetheless, the 4F2 H-chain is present mostly as a 120-kDa complex
rather than an 80-kDa monomer form also in these tissues, suggesting
the presence of distinct 4F2 L-chain(s). Molecular heterogeneity in the
system L transport activity has been previously demonstrated (28).
Because 4F2 H-chain in these polarized epithelial cells is localized
selectively on the lateral, but not apical, surface, the yet undefined
4F2 L-chain(s) may be expected to exhibit unique functions, directional solute transports for instance.
The 4F2 antigen has been suggested to be involved in a wide variety of
cellular functions, including cellular growth (12, 13), virus-induced
cell aggregation, and fusion (14, 15), and affinity regulation of
INTRODUCTION
Top
Abstract
Introduction
References
1 integrin on the ligand affinity
of integrin (16). Although these results imply the involvement of 4F2
antigen in diverse cellular activities, exact mechanisms underlying
them remain unknown.
EXPERIMENTAL PROCEDURES
RESULTS
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Fig. 1.
A monoclonal antibody (10.7) specific to 4F2
L-chain. a, P3U1 cell lysate was immunoprecipitated
with control hamster IgG, anti-4F2 H-chain (10.10), or 10.7 mAb,
electrophoresed in SDS-PAGE, and immunoblotted with either anti-4F2
H-chain (10.4) or with 10.7 mAb. b, COS cells were
transfected with p10.7, 4F2 H-chain cDNA (p14.37), or with p10.7
combined with p14.37, p14.37 (C103S), or p14.37 (C325S) in a pSR
expression vector by electroporation. The cells were harvested 3 days
after the transfection, lysed in a lysis buffer containing 1% Nonidet
P-40, and immunoprecipitated followed by immunoblotting at the
indicated combinations of mAbs.
)-endoamino-bicycloheptane-2-carboxylic acid
(BCH), a specific inhibitor of system L transport. A kinetic study
revealed that the Na+-independent Leu uptake was saturable
and high affinity, with the Km calculated to be
around 25 µM (Fig. 2b).
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Fig. 2.
4F2 L-chain is a multimembrane-spanning
protein and mediates system L amino acid transport in the presence of
4F2 H-chain in Xenopus oocytes. a,
hydrophobicity profile of the deduced amino acid sequence of 4F2
L-chain cDNA (Kyte-Doolittle program). b, (left
panel) oocytes were injected with cRNA (2.5 ng/egg) of 4F2 L-chain
(lc), 4F2 H-chain (hc), or both (lc + hc), and assayed for the uptake of indicated amino acids 3 days after the injection. Control oocytes ( ) received water. The
uptake of amino acids was measured by incubating oocytes with 50 µM indicated radiolabeled amino acids for 30 min at
25 °C in the uptake solution containing 100 mM choline
chloride in place of NaCl. Means ± S.D. of five to seven oocytes
are indicated. Center panel, the Na+-independent
L-leucine uptake was determined as above in the presence of
5 mM indicated inhibitors using the oocytes that had been
injected with both 4F2 L-chain and 4F2 H-chain cRNAs (2.5 ng of each) 3 days before. Means ± S.D. of five to seven oocytes are indicated.
Right panel, oocytes that had been injected with both 4F2 L-
and 4F2 H-chain cRNAs (2.5 ng of each) 3 days before were incubated
with varying concentrations of L-leucine for 30 min, and
the uptake was measured as above. The mean base-line uptake of control
oocytes was subtracted from that of cRNA-injected oocytes at each
concentration. Inset, Eadie-Hofstee plot of the data.
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Fig. 3.
4F2 H-chain guides the intracellular
trafficking of the 4F2 L-chain to the plasma membrane independently of
a disulfide-linkage. a, COS cells were transfected with
4F2 L-chain cDNA, 4F2 H-chain cDNA, or with 4F2 L-chain
together with 4F2 H-chain or 4F2 H-chain (C103S) cDNA by
electroporation. The cells were harvested 3 days later, surface
biotinylated, lysed in a lysis buffer, immunoprecipitated with anti-4F2
L-chain or H-chain mAb, and electrophoresed in SDS-PAGE. The
biotinylated proteins were detected with ABC kit. One-fourth of each
sample was similarly electrophoresed and immunoblotted with
corresponding mAbs to estimate the total amounts of expressed proteins.
b, COS cells were transfected as above. After the surface
biotinylation, the lysates were immunoprecipitated with anti-4F2
H-chain, and electrophoresed in SDS-PAGE at either reducing (left
panel) or nonreducing (right panel) condition. The
biotinylated proteins were detected with ABC kit. Left
panel: open arrow head, 4F2 H-chain; closed arrow
head, coprecipitated 4F2 L-chain; right panel:
open arrow head, 4F2 H-chain in complex form; closed
arrow head, 4F2 H-chain monomer.
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Fig. 4.
4F2 H-chain guides the intracellular
trafficking of 4F2 L-chain to the plasma membrane independently of
disulfide-linkage immunostaining analysis. HeLa cells
were transfected with 4F2 H-chain cDNA (a), 4F2 L-chain
cDNA tagged with Myc epitope at the C terminus (b), 4F2
H-chain and Myc-4F2 L-chain cDNAs (c and d),
or with 4F2 H-chain (C103S) and Myc-4F2 L-chain cDNAs (e
and f) by CaPO4 method. Three days later, the
cells were fixed and stained with biotin-conjugated anti-4F2 H-chain
followed by Texas Red-avidin (a), or anti-Myc mAb followed
by FITC-anti-mouse IgG (b). For the double transfectants,
the cells were double-stained with anti-4F2 H-chain (c and
e) and anti-Myc (d and f) as above.
Panels c and d, as well as
e and f, represent the same fields,
respectively.
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Fig. 5.
Comparable expression profiles of the 4F2
H-chain and 4F2 L-chain transcripts in normal tissues.
a, normal adult BALB/c spleen cells were cultured in the
presence of concanavalin A (2 µg/ml) for varying periods. Total RNA
was extracted from the cells and blotted using 32P-labeled
4F2 H-chain and 4F2 L-chain cDNA probes. b, filters of
poly(A)+ RNAs from various murine adult organs (OriGene,
Rockville, MD) were blotted sequentially with 32P-labeled
cDNA probes of 4F2 H-chain, 4F2 L-chain, and -actin.
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Fig. 6.
4F2 H-chain is expressed selectively at the
cell-cell adherent sites and colocalizes with cadherins. OTF9
cells (a and b), L cells (c and
d), EL cells (e and f), and NL cells
(g and h) were cultured on coverslips, fixed, and
double-stained with anti-E-cadherin (a, c, and
e) or anti-N-cadherin (g) and biotin-conjugated
anti-4F2 H-chain (b, d, f, and
h) followed by FITC-conjugated anti-rat IgG and Texas
Red-avidin. The stained cells were analyzed with a confocal laser
microscopy.
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Fig. 7.
Expression of 4F2 H-chain in normal embryonic
tissues. Sections of fixed whole embryos (E14) of BALB/c mice were
stained with anti-4F2 H-chain mAb (10.10) followed by biotin-conjugated
anti-hamster IgG and detected with an ABC kit. a, skin (× 200) b, choroid plexus in brain; c, retina;
d, intestine; e, kidney; f, thymus (× 400).
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Fig. 8.
Dissociated expression of the 4F2 H-chain and
4F2 L-chain (LAT1) in kidney, intestine, and liver implication
for the multiple 4F2 L-chain(s). a, various organs from
E18 embryos (E18) and adult (A) mice were
homogenized, extracted in a lysis buffer, electrophoresed in SDS-PAGE
in reducing condition, and immunoblotted with anti-4F2 H-chain (10.4)
and anti-4F2 L-chain (10.7) mAbs. b, tissue extracts of
indicated organs from adult mice were electrophoresed in SDS-PAGE in
either nonreducing or reducing condition and immunoblotted with
anti-4F2 H-chain mAb.
DISCUSSION
-subunit is responsible for the correct intracellular trafficking of
/
heterodimeric holoenzymes from ER to the cell surface (20).
/HCO3
channel (26, 27).
1-integrins (16). It has been reported also that anti-4F2 H-chain
antibodies affect the Ca2+-influx in sarcolemmal vesicles
(29). Although system L transport of the 4F2 heterodimer should play
certainly important roles in proliferative cells, including tumor
cells, to meet critical nutritional requirements, the relation of it to
many other suspected functions remains obscure. Further studies on the
guidance mechanisms of 4F2 H-chain for 4F2 L-chain(s) and possibly
other proteins, with or without covalent linkage, to the cell surface
as well as the analysis on the mechanisms for regulation of membrane
topology of 4F2 heterodimer by cell adhesion molecules might provide
new clues to delineate the multiple functions of 4F2 antigen in various cell types.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. M. Takeichi and Y. Minami for valuable discussion and providing cells, and Dr. M. Maeda for the technical assistance. Proofreading of the manuscript by Dr. L. Reid is also appreciated.
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FOOTNOTES |
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* This work was supported by grants from the Ministry of Education and Science, Japanese Government.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB17189.
§ The first two authors equally contributed to the work.
To whom correspondence should be addressed: Dept. of
Immunology and Cell Biology, Faculty of Medicine, Kyoto University,
Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4659; Fax:
81-75-753-4403; E-mail: minato{at}med.kyoto-u.ac.jp.
The abbreviations used are: H-chain, heavy chain; L-chain, light chain; ABC, avidin-biotin complex; BAT, broad specificity amino acid transporter; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; LAT1, L-type amino acid transporter 1; mAb, monoclonal antibody; EL, L cells stably transfected with E-cadherin; NL, L cells transfected with N-cadherin; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline.
2 K. Iwai, unpublished data.
3 H. Yang and N. Minato, unpublished observations.
4 K. Suga and N. Minato, unpublished observations.
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REFERENCES |
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