|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 275, Issue 8, 5347-5354, February 25, 2000
From the Tumor associated gene-1/L amino acid
transporter-1 (TA1/LAT-1) was recently identified as a light chain of
the CD98 amino acid transporter and cellular activation marker. Our
previous studies with primary rat hepatocyte cultures demonstrated that TA1 RNA levels were responsive to media amino acid concentrations, suggesting adaptive regulation. High level TA1 expression associated with transformed cells also suggested a role in tumor progression. The
present study examined the relationship of TA1/CD98 expression, adaptive response, and associated amino acid transport to neoplastic transformation using a panel of well characterized rat hepatic cell
lines. We found 1) increased expression of TA1 in response to amino
acid depletion, specific for arginine but not glutamine; 2) loss of TA1
response to arginine in TA11 was cloned in our
laboratory on the basis of its differential expression between rat
hepatoma cells and normal adult rat liver and encodes a predicted
integral membrane protein with several membrane-spanning domains (1).
TA1 is identical to the C terminus (amino acids 272-512) of LAT-1,
which is a 512-amino acid protein with 12 transmembrane domains (2).
E16, cloned as a lymphocyte activation antigen, is the human homolog of
TA1 and is 95% identical at the amino acid level (3). TA1/LAT-1 is a
member of an emerging family of highly conserved molecules with
homologs in Xenopus, Schistosoma, mouse, and man.
More recently, TA1/LAT-1 and its homologs have been identified as the
light chain of the CD98 molecule (2, 4-8). The CD98 complex consists
of an 80-kDa heavy chain (4F2) and a 40-45-kDa light chain (9). CD98
has been implicated in a variety of functions including amino acid
transport, cell survival, integrin activation, and cell fusion
(10-13). Additional light chains have been identified and designated
y+LAT-1 (14-16), y+LAT-2 (14, 17), and LAT-2
(18, 19). The CD98 complex can mediate System L or System
y+L amino acid transport in Xenopus oocytes
depending on which CD98 light chain (CD98lc) is associated with the
CD98 heavy chain (CD98hc) (2, 6-8, 14). Co-injection of cRNA for
TA1/LAT-1 and 4F2 has been shown to mediate System L transport of large
neutral amino acids with branched or aromatic side chains in
Xenopus oocytes (2, 6). LAT-2 also mediates System L
transport in Xenopus oocytes upon co-injection with 4F2hc;
however, LAT-2 transports both large and small neutral amino acids with
a lower affinity (Km) than LAT-1 (18, 19). TA1/LAT-1
is not only expressed in rat hepatomas but also in a variety of human
cancers including human colon and breast carcinoma (20). Whereas TA1
expression is not seen in normal adult rat liver, it can be induced
transiently in liver and with the same kinetics as c-myc
after acute carbon tetrachloride injury, suggesting a role in
normal injury response in this organ (21). In primary hepatocyte
cultures, we also observed that changes in amino acid concentrations
resulted in altered levels of TA1 expression, suggesting adaptive
regulation (22).
We are interested in examining the regulation and role of TA1/E16 and
CD98-related molecules in hepatocarcinogenesis. We have utilized a
panel of rat hepatic WB cell lines that differ in their transformed and
tumorigenic properties. We have assessed how basal TA1/CD98lc and
4F2/CD98hc expression in response to an environmental factor, arginine
availability, are associated with transformation and tumorigenicity in
this system. We have found regulation at the level of TA1/CD98lc. The
possible physiological significance of this regulation is discussed.
4F2/CD98hc RNA levels remained fairly constant regardless of arginine
availability, transformation, or tumorigenicity. TA1/CD98lc RNA levels,
but not the total pool of immunologically reactive protein, were found
to be modulated by arginine availability in nontransformed and
GGT-negative transformed WB cells. TA1/CD98lc RNA levels were
constitutively high in GGT-positive transformed cells and hepatomas,
and TA1/CD98lc protein levels were elevated relative to nontransformed
cells. System L amino acid transport was significantly higher in
GGT-positive transformed, tumorigenic cells than nontransformed cells
and responded to availability of arginine, a nonsystem L substrate.
Cell Culture--
A brief description of all cell lines used in
this study including data on transformation and tumorigenicity is
presented in Table I. The normal diploid
hepatic epithelial line WB-F344 (WB) was isolated from an adult male
Fischer 344 rat (23). The derivative cell lines were produced by 11 brief repeated exposures of WB-F344 cells to 5 µg/ml of
N-methyl-N'-nitro-N-nitrosoguanidine, and from this heterogeneous, tumorigenic cell population, 18 clonal subpopulations were isolated based on their expression of the oncofetal
marker enzyme GGT (24). Cloned cell lines were determined to be either
GGT-negative (i.e. GN6) or GGT-positive (i.e.
GP6). Cell lines were also re-established from subcutaneous tumors
derived from GGT-positive (i.e. GP6TB, GP7TB) and
GGT-negative cells (i.e. GN6TF). 1683 cells are rat
transplantable hepatocellular carcinoma cells derived from primary
carcinomas induced by a choline-deficient, ethionine diet (25).
WB, GN6, GN6TF, and GP7TB cell lines were routinely cultured in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% FBS, whereas GP6 and GP6TB were routinely cultured in
Dulbecco's modified Eagle's medium /Ham's F-12 (1:1) (Life Technologies, Inc.) containing 10% FBS and supplemented with insulin, transferrin, selenous acid, bovine serum albumin, and linoleic acid at
1 ml/100 ml medium (Becton Dickinson). 1683 cells were routinely
cultured in Waymouth's medium containing 10% FBS. To better simulate
the normal hepatic milieu, a custom formulation of Chee's essential
medium (CEM) demonstrated to maintain long term differentiated hepatic
cell function in vitro was kindly provided by Dr. Hugo
Jauregui (Department of Pathology, Rhode Island Hospital) and prepared
as described previously (26). Arginine and glutamine were not present
in this custom formulation and were added from stock solutions. It was
not feasable to selectively delete other amino acids with this medium
because custom batches missing other amino acids were only available in
100-liter batches. For experiments in which gene expression was assayed
as a function of arginine availability, cells were seeded into T-75
flasks with CEM containing 5% FBS dialyzed or undialyzed with arginine
or without arginine. All experiments with all the cell lines were repeated with dialyzed serum to remove serum as a source of arginine with similar results to nondialyzed serum. Medium was changed every
day. Viability was at least 90% in all cases.
RNA Preparation and Northern Blot Analysis--
Total RNA was
isolated using the guanidinium isothiocyanate/cesium chloride method
(27) for normal adult rat liver and a modification of that method for
cultured cell lines (Totally RNA KitTM; Ambion, Austin, TX). RNA was
isolated from cultured cells after subculture in CEM at the appropriate
time points. Aliquots (12 µg) of total RNA were size fractionated on
1% agarose/formaldehyde gels as described previously (20). After
electrophoresis, gels were equilibrated in 1 M ammonium
acetate, and RNA was transferred to Nytran nylon membranes (Schleicher
& Schuell). Blots were baked for 2 h at 80 °C and hybridized at
65 °C according to Church and Gilbert (28). Rat TA1 p900,
a fragment corresponding to nucleotides 816-1536 of the full-length
rat LAT-1, inserted into Bluescript SK vector was labeled with
[32P]dCTP (NEN Life Science Products; 3000 Ci/mmol) by
random primed labeling (Roche Molecular Biochemicals) for use as a
probe. Blots wrapped in plastic wrap were exposed to x-ray film
(Eastman Kodak Co.) at Amino Acid Transport Assays--
The transport of radiolabeled
amino acids by cell monolayers was performed using a modification of
the cluster tray method developed by Gazzola et al. (31),
and described by Kilberg (32). All 3H-labeled amino acids
were purchased from NEN Life Science Products, and unlabeled amino
acids were purchased from Sigma. Cells were seeded in 24-well trays
such that the cells would be near confluent for the transport assay.
Before the transport assays, cells were rinsed with warm
Na+-free Krebs-Ringer phosphate buffer (cholKRP), in which
the sodium containing salts were iso-osmotically replaced with choline,
to remove extracellular Na+ and amino acids. Cells were
incubated in warm cholKRP for 10 min to deplete intracellular amino
acids. The uptake of radiolabeled amino acids (5 µCi of
3H-amino acid/ml) at 100 µmol/liter in either 200 µl of
cholKRP or NaKRP was measured for 30 s at 37 °C. Preliminary
experiments indicated that uptake of each 3H-labeled amino
acid was linearly dependent on incubation time up to at least 3 min;
therefore, uptake was measured for 30 s (data not shown). In
inhibition experiments, excess cold amino acid was added to the uptake
buffer at 10 mM final concentration. Uptake was terminated
by washing the cells rapidly four times with 1 ml/well of ice-cold
cholKRP. After the trays were allowed to dry, the cells were incubated
for 1 h with 0.2 ml/well of 0.2% (w/v) SDS plus 0.2 N
NaOH to release intracellular radioactivity. A 0.1-ml aliquot from each
well was neutralized with 0.1 ml of 0.2 N HCl and
quantified in a Beckman LS 6000SC liquid scintillation counter. The
remaining 0.1 ml was analyzed for protein content using the BCA protein
assay reagent (Pierce). Transport velocities were calculated from
radioactive counts, specific activities of uptake mixes, and protein
absorbance values and expressed as pmol amino acid transported per
milligram of protein per minute (averages ± S.E. of at least
three separate determinations). Data comparing two experimental results
were analyzed statistically by Student's t test using the
InStat Macintosh statistics program. Each experiment was repeated at
least twice to show qualitatively the same results.
Immunoblot Analysis--
Detergent extracts were prepared from
WB, GP6, and GP7TB cells cultured under the same conditions as for RNA
preparation and analyzed by SDS-polyacrylamide gel electrophoresis as
described previously (20). Rabbit antibodies prepared to synthetic
peptides corresponding to amino acids 211-221 of TA1/E16 (482-492 of
LAT-1) deduced sequence were used together with chemiluminescence
detection as described previously (20). Two rabbits (K and G) were
immunized with the identical preparation of keyhole limpet
hemocyamin-conjugated peptide. A recombinant fusion protein of TA1 with
six histidines produced in baculovirus and purified by nickel affinity
chromatography as previously reported (4) served as positive control.
As a negative control, blots were reacted with nonimmune rabbit serum at the same dilution.
TA1 mRNA Is Induced Following Arginine Deprivation of WB Cells
While 4F2hc mRNA Levels Remain Invariant--
In previous studies
with primary hepatocyte cultures, we found that TA1 mRNA levels
could be either induced or suppressed depending on the culture medium
and its amino acid concentrations (22). We extended these studies to
examine whether the same response to amino acid availability was found
in a normal rat liver epithelial cell line, WB, and whether deprivation
of arginine and/or glutamine, the two amino acids manipulable in our
culture system, could increase TA1 expression in these cells. WB is a diploid cell line isolated from the liver of an adult male Fischer-344 rat and has been used extensively as both an in vitro and an
in vivo model system for the liver (23, 33). WB cells were
cultured in CEM, CEM without arginine, CEM without glutamine, or CEM
without arginine or glutamine for 48, 72, and 96 h. Using
densitometry and normalizing to GAPDH, steady state levels of TA1 RNA
were 8-10-fold higher in WB cells cultured in the absence of arginine than in the presence of arginine (Fig.
1). Thus, the availability of arginine
was found to modulate TA1 expression, whereas glutamine availability
had no effect. The apparent increase in TA1 observed after WB cells are
cultured for 48 h without glutamine was found to be negligible
after standardization to ethidium staining and GAPDH hybridization. In
stark contrast to TA1, 4F2 mRNA levels did not vary in response to
arginine availability. Similarly, glutamine availability did not affect
4F2 expression. Other amino acids were not examined because of
constraints of medium composition. Since arginine availability was
found to modulate TA1 mRNA levels in these nontransformed
nontumorigenic cells, we used arginine availability subsequently as a
tool to examine the regulation of TA1 and 4F2 in a panel of rat hepatic
cell lines differing in stages of transformation and
tumorigenicity.
Early Response of TA1 and 4F2 mRNA to Arginine
Availability--
To determine the time course of the observed
response, we examined TA1 and 4F2 steady state message levels at
additional time points in WB cells cultured with and without arginine.
WB cells were cultured in Dulbecco's modified Eagle's medium,
trypsinized and then seeded and cultured in CEM with or without
arginine for 4, 8, 24, and 48 h. The results are presented in Fig.
2. TA1 steady state mRNA levels were
responsive to arginine availability in WB cells within four hours after
culture in CEM without arginine. After densitometric normalization to
GAPDH steady state mRNA levels, TA1 mRNA levels were
approximately ten-fold greater in cells cultured without arginine for
four hours versus with arginine. Normalized 4F2 mRNA
levels at this time point were approximately 2.5 fold higher in WB
cells cultured without arginine versus with arginine. For
subsequent experiments, we chose to examine differences between response of normal and transformed, tumorigenic cell lines at time
points from 24 to 72 h, since cell attachment and spreading of
this integrin-associated molecule may confound data interpretation.
TA1 and 4F2 RNA Levels Differentially Respond to Arginine
Availability in GGT-negative Transformed WB Cells versus GGT-positive
Transformed WB Cells--
Loss of response to positive/negative
signals is one of the major events marking the transition from a normal
to a malignant cell. We examined TA1 and 4F2 response to arginine
availability in chemically transformed GGT-negative (GN6) and
GGT-positive (GP6) WB cells, and tumor derived WB cell lines which were
both GGT-negative (GN6TF) and GGT-positive (GP6TB, GP7TB) to determine if TA1 and/or 4F2 response would correlate with transformation, GGT
status, or tumorigenicity. Normally, GGT is expressed in fetal rat
hepatocytes, adult bile duct cells, and kidney, but during hepatocarcinogenesis it becomes up-regulated as an early event in
neoplastic conversion. Hanigan has elegantly demonstrated that GGT-negative and GGT-positive mouse hepatoma (Hepa 1-6) cells grow
similarly in cysteine-rich medium (>100 µM cysteine),
but at physiological concentrations of cysteine (<100 µM
cysteine), GGT expression confers a selective growth advantage on
GGT-positive mouse hepatoma cells (34). Cells were cultured in CEM with
or without arginine for 24, 48, and 72 h prior to RNA extraction. Northern analysis was performed and the results are shown in Figs. 3 and 4.
Although 4F2 expression remained fairly constant in all cells,
conditions, and time points, TA1 expression varied considerably. In the
GGT-negative WB lines, TA1 RNA levels increased 8 to 10 fold in the
absence of arginine for 72 h. In contrast, expression increased
only about 2 to 4 fold in GGT-positive transformed cells largely
because of higher basal level expression in the presence of arginine.
Similar results were also obtained at the earlier time points, although
the fold differences were lower.
Despite the transformation status of these cell lines, in all cases TA1
RNA was expressed at a high level in cells cultured without arginine.
Interestingly, although basal 4F2 expression was about 2-fold greater
in transformed cells, it remained fairly constant for each cell type
whether or not arginine was present in the medium. At early time points
after culture in CEM, GGT-negative transformed cells express high
levels of TA1 regardless of arginine availability, similar to
GGT-positive cells at all time points. After 72 h of culture in
the presence of arginine, reduced TA1 levels were observed in
GGT-negative transformed cells similar to those seen in nontransformed
WB cells cultured in CEM with arginine. These results imply the loss of
regulation of TA1 at different stages of progression in these rat
hepatic lines.
TA1 and 4F2 Are Constitutively Expressed in a Non-WB Rat Hepatoma
Cell Line--
We also examined the response of TA1 and 4F2 in another
rat hepatoma cell line separate from the WB tumor-derived cell lines. In contrast to WB lines that were transformed by carcinogen treatment in vitro, 1683 cells are rat transplantable hepatocellular
carcinoma cells derived from primary carcinomas induced by feeding rats a choline-deficient, ethionine diet (25). 1683 cells were cultured in
CEM with or without arginine for 24, 48, and 72 h. Neither TA1 nor
4F2 expression significantly changed whether or not arginine was
included in the medium (Fig. 5). Steady
state levels of both messages were high all the time, with only slight
increases of TA1 expression (2-3-fold) at 72 h after arginine
deprivation. These cells constitutively express high levels of
TA1/CD98lc and 4F2/CD98hc message. Thus loss of arginine response does
not appear related to the type of carcinogen involved in
transformation.
Relative TA1 and 4F2 mRNA Levels in All Cell Lines
Examined--
RNA samples from each cell line were included on the
same Northern blot so that comparisons could be made between all the cell lines. These results are presented in a graph in Fig.
6. Each bar represents the average
densitometric value determined from three independent blots. 4F2/CD98hc
RNA levels are fairly constant in every cell line, and every time point
examined had no greater than a 2-fold difference between cells cultured
with or without arginine. WB cells have the lowest TA1 and 4F2 RNA levels in all conditions and time points, although TA1 RNA levels do
increase 8-10-fold when cells are cultured without arginine. When GN6
and GN6TF cells were cultured in medium with arginine, TA1 RNA levels
showed a large decrease from 24 to 72 h relative to cells
maintained without arginine. It is also interesting to compare the TA1
response to arginine availability by in vitro transformed
cells (GP6 cells) versus cell cultures derived from solid
tumors of these cells (GP6TB). In both GP6 and GP6TB, TA1 RNA levels
differ only about 2-4-fold during the course of culture with or
without arginine. GP6TB cells, however, express higher basal levels of
both TA1 and 4F2 than GP6 cells and also induce higher levels of TA1
when cells are cultured without arginine, suggesting an alteration
associated with tumor progression.
System L Transport Is Differentially Regulated in Nontransformed
Versus GGT-positive Transformed and Tumorigenic Rat Hepatic
Cells--
Because TA1/LAT-1 functions as the light chain of CD98 and
has been demonstrated to mediate system L transport in
Xenopus oocytes, we hypothesized that the altered expression
of TA1 was likely to be associated with differences in transport
activity. We thus examined whether arginine availability affected
system L amino acid transport in WB cells and whether there were
differences between nontransformed and transformed GGT-positive
tumorigenic lines. As shown in Fig.
7a, WB cells cultured without
arginine for 48 h transported approximately 3-fold more leucine
(significant at p < 0.05) than WB cells cultured in
the presence of arginine regardless of the presence of sodium. As
expected, addition of excess cold tyrosine inhibited leucine transport,
consistent with system L activity. Arginine transport (system
y+) was not significantly different in WB cells cultured
with or without arginine (Fig. 7b). As shown in Fig.
7c, GP7TB cells cultured without arginine for 48 h also
transported significantly more leucine (about 2.5-fold) than GP7TB
cells cultured with arginine. Arginine transport was not significantly
different in GP7TB cells under the two culture conditions (Fig.
7d).
GP7TB cells transported approximately 50% more leucine than WB cells
whether the cells were cultured with arginine or without arginine
(significant at p < 0.05). These data correlate well with TA1 mRNA levels in GP7TB and WB cells cultured with and
without arginine for 48 h (compare Figs. 1 and 4) and are
consistent with its role as a system L transporter. The increase in
transport seen in these cells is less than the increase in mRNA
levels, suggesting that transcriptional mechanisms alone are unlikely to account for regulation.
TA1 Immunoreactive Protein Pools Are Elevated in Transformed and
Tumorigenic Cell Lines--
To determine whether levels of TA1 protein
in transformed lines derived from the WB line were increased relative
to normal hepatic cells and how these levels compared with transport
activity, immunoblot analysis was performed with anti-TA1 peptide
antibodies (Fig. 8). Lanes were
loaded with equivalent amounts of total protein per cell extract
(15 µg), as confirmed by Ponceau S staining after gel transfer (not
shown). Relative to nontransformed WB cells, K anti-peptide antibody
detected multiple bands in cell extracts from transformed GP6 and
tumorigenic GP7TB cells. The size range of these bands is similar to
what has been reported for in vitro translated 512-amino
acid LAT-1 (2). Although antibodies from both rabbits reacted with the
recombinant TA1, for reasons that are not known, rabbit serum G reacted
only with a band of approximately 35 kDa present in GP7TB cells. In
contrast to the increase observed in message levels in cells cultured
without arginine, no increase was observed in any immunoreactive bands
under these culture conditions. None of the bands shown were present
after blots were incubated with the same dilution of nonimmune rabbit
serum (data not shown). Whether the multiple bands detected with
antibody K may correspond to proteolytic fragments or TA1/LAT-1
cross-reactive molecules is not known.
We have examined the response of TA1/CD98lc and 4F2/CD98hc
expression and amino acid transport to arginine availability in a panel
of rat hepatic epithelial cell lines differing in GGT-expression, transformation, and tumorigenicity. We have focused our initial studies
primarily at the RNA level to assess changes associated with tumor
progression. The CD98 light chain has been implicated by others in
amino acid transport specificity (2, 6-8, 14). Our results show that
steady state levels of TA1/CD98lc RNA can be up-regulated 10-fold in
nontransformed cells in response to arginine but not glutamine
deprivation, whereas 4F2hc levels only vary by 2-fold under identical
conditions. To our knowledge this is the first demonstration that the
light and heavy chains of the CD98 complex respond differentially to an
environmental cue. Using this medium formulation, we were not able to
deplete other nutrients, and thus it is not known if the response of
CD98lc is specific to the stress of this amino acid limitation.
However, we have not observed up-regulation after rat hepatic cells are cultured in conditions of heat shock, hypoxia, or anoxia (data not
shown). This study was not intended to be a complete study of nutrient
response, and further studies are needed to determine the specificity,
threshold, and reversibility of the response.
TA1/CD98lc is constitutively expressed in GGT-positive transformed WB
(both in vitro transformed and tumor-derived) cells and 1683 cells. When comparing the tumor-derived cell lines, it is interesting
to note that GGT-negative GN6TF cells, which can modulate TA1 RNA
levels in response to amino acid availability, do not form tumors in
the liver, whereas the GGT-positive tumor-derived cells (GP6TB and
GP7TB), which do not modulate TA1/CD98lc RNA levels, do form tumors
rapidly in the liver (33). 4F2 mRNA levels do not vary
significantly in these cells, although in general the levels are much
higher in transformed versus nontransformed cells (Fig.
6b and compare 4F2 mRNA levels in WB versus
GN6, GN6TF, GP6TB, GP7TB, and 1683). System L activity was also found
to respond to arginine availability and correlated with TA1 mRNA
levels in the cells. Tumorigenic rat hepatic cells express higher basal and inducible system L activity than nontumorigenic cells. We have also
shown that the loss of response to arginine can occur at various points
in hepatic cell transformation/progression. By the time the cells have
attained tumorigenic capacity, the regulation is lost.
The correlation between alteration in TA1 regulation and GGT expression
is interesting because hepatic GGT is up-regulated during
carcinogenesis as an early event in neoplastic conversion (35-37).
Hanigan and Ricketts (38) have found that under conditions of low
cysteine, such as those found in the tumor microenvironment, GGT
converts glutathione to a source of cysteine. Furthermore, GGT
expression confers a selective growth advantage on GGT-positive mouse
hepatoma cells at physiological concentrations of cysteine (34).
Similarly, we hypothesize that TA1 expression may give cells a
selective growth or survival advantage particularly under conditions of
enhanced requirements for amino acids as is found in the tumor
microenvironment (39).
Perhaps not surprisingly, our immunoblot analysis failed to show an
alteration in the total pool of immunoreactive TA1/LAT-1 comparable
with the observed changes in RNA levels or transport activity, although
we did see elevated TA1/LAT-1 protein in transformed and tumorigenic
cells relative to nontransformed cells. These studies are complicated
by a number of factors including assessment of cell surface
versus cytoplasmic pools of protein, free light chain
versus that in complex with heavy chain and whether
antibodies cross-react with closely related members of the LAT-1
family, including LAT-2, which may be expressed in these cells.
Furthermore, as was recently demonstrated for the arginine transporter,
cat-1, translation of specific pools of messenger RNAs may
be compromised under conditions in which amino acids are limiting (40).
Because our light chain anti-peptide antibodies were not able to
immunoprecipitate the CD98 complex, further studies involving specific
heavy chain antibodies and cell localization or subfractionation will
be necessary to fully assess regulation of this molecule.
Hepatic amino acid transport and transporters are known to change
dramatically during the process of hepatocarcinogenesis such that
transporters up-regulated in neoplastic cells are often different from
their counterparts in normal cells (41). There are at least two System
L activities in liver: L1 is a high affinity transporter
found in fetal hepatocytes and transformed cells, and L2 is
a low affinity transporter located in freshly isolated hepatocytes (42,
43). LAT-1/TA1 may correspond to the L1 transporter, whereas LAT-2 or an unidentified light chain may correspond to the
L2 transporter. The present data do not exclude the
possibility that TA1 is a partial cDNA of LAT-1 or alternatively
that TA1 is a 6-transmembrane variant of the 12-transmembrane form. The detection of an immunoreactive protein of a smaller size than the
40-kDa major band in GP7TB cell extracts by two distinct anti-peptide antibodies is noteworthy in this regard.
Amino acid availability may be one of the signals that regulates the
expression of transport proteins, and loss of response to amino acid
availability may contribute to the neoplastic process. Some examples of
mammalian mRNAs or proteins whose synthesis is enhanced in response
to amino acid deprivation include: cat-1 (40), asparagine
synthase (44), ornithine decarboxylase (45, 46), insulin-like growth
factor binding protein-1 (47), System A (48) and System L (49) amino
acid transport, and c-jun and c-myc (46, 50).
Although modulation of a System L amino acid transporter by a System
y+ substrate may appear contradictory, there are numerous
examples in which deprivation of amino acids can lead to increases in
expression of genes even though the deprived amino acid is not directly
synthesized/transported by the gene product. Examples include
asparagine synthetase (44), jun, fos, and
myc (46), cat-1, System A, and L17 (50, 51). Kilberg et al. (51) has hypothesized that mammalian
cells may have a general response to amino acid deprivation similar to
the response seen in yeast such that deprivation of any single amino acid up-regulates various activities.
In our current working model, 4F2/CD98hc is proposed to associate with
a low affinity transporter such as LAT-2 or an as yet unidentified
member of the LAT family in the normal adult hepatocyte. Under
conditions of transient nutrient stress, the high affinity transporter
TA1/LAT-1 would be up-regulated transiently and serve as the major
System L activity associated with 4F2. During hepatic cell
transformation/progression to a tumorigenic cell, this regulation would
be lost such that high affinity System L transport becomes constitutive.
CD98 has been associated with many functions in different cell types.
Up-regulation of TA1 in hepatic cells may conceivably block or uncouple
other (e.g. nontransport) functions including CD98
clustering and thereby contribute to neoplastic transformation. Recently, Hara et al. (52) have shown that overexpression of human 4F2/CD98hc in murine NIH3T3 cells resulted in malignant transformation of these cells. Whether levels of the light chain or
heavy to light chain ratios were affected was not examined in this
study. Although it is not yet known whether 4F2/CD98hc overexpression
would have similar effects in epithelial cells, our studies suggest a
potential mechanism whereby CD98lc may contribute to malignant
transformation of hepatic cells by providing neoplastic cells with a
selective growth advantage, particularly under conditions of nutrient
stress. Experiments are underway to test this hypothesis directly.
We thank Dr. H. Jauregui and S. Naik for
generous gifts of media and Dr. D. C. Hixson for valuable discussions.
*
This work was supported by NIEHS, National Institutes of
Health Training Grant T32ESO7272, National Institutes of Health Grant CA73611, and funds from the Rhode Island Hospital George Oncology Foundation.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 abbreviations used are:
TA1, tumor
associated gene-1;
LAT, L amino acid transporter;
4F2, heavy chain of
CD98 cell surface antigen;
GGT,
TA1/LAT-1/CD98 Light Chain and System L Activity, but Not
4F2/CD98 Heavy Chain, Respond to Arginine Availability in Rat Hepatic
Cells
LOSS OF RESPONSE IN TUMOR CELLS*
,,,
Division of Medical Oncology and
§ Department of Surgery, Rhode Island Hospital, Brown
University School of Medicine and Graduate Program in Pathobiology,
Providence, Rhode Island 02903 and the ¶ Department of Pathology,
University of North Carolina, Chapel Hill, North Carolina 27599
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyl transpeptidase-positive transformed and tumorigenic cells; 3) no appreciable response of
4F2/CD98 heavy chain to arginine levels; and 4) correlation of system L
amino acid transport activity in response to arginine with changes in
TA1/LAT-1 mRNA but not total immunoreacting protein. Our results
suggest this CD98 light chain may act as an environmental sensor,
responding to amino acid availability and that its regulation is
complex. We hypothesize that altered TA1 expression is an early event
in hepatocarcinogenesis giving neoplastic cells a growth or survival
advantage, particularly under conditions of limited amino acid availability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Characteristics of cell lines used in this study
70 °C in the presence of intensifying
screens. Blots were stripped and rehybridized to an 1800-bp
EcoRI fragment of human 4F2hc (provided by Dr.
Martin Hemler) (29). Blots were also stripped and hybridized to an
800-bp fragment of the 5' end of LAT-1 derived through reverse
transcription-polymerase chain reaction of the 1600-bp coding region of
LAT-1 using rat placenta as an RNA source followed by polymerase chain
reaction of the 5' end of LAT-1. A 1.25-kbp PstI fragment of
human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used in
combination with ethidium bromide staining to evaluate RNA loading
variations (30). Densitometry using the Quantity OneTM IBM software
package was used to quantify differences in RNA levels with
normalization to 18 S ribosomal RNA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (41K):
[in a new window]
Fig. 1.
CD98lc/TA1 and CD98hc/4F2 response to amino
acid availability in WB cells. Shown is total RNA from WB cells
cultured in CEM without arginine ( R), without arginine and
glutamine (R/Q), without glutamine (Q), or
CEM with arginine and glutamine (+R/+Q) for 48, 72, and
96 h. Aliquots (12 µg) of total RNA were analyzed by sequential
Northern blot hybridization to a 900-bp TA1 probe (top
panel), a 1.8-kbp 4F2 probe (middle panel), and a
1.25-kbp GAPDH probe (bottom panel). RNA from 1683 rat
hepatoma cells and normal adult rat liver were included for comparison.
Ethidium bromide staining was used as a loading control between lanes.
Approximate transcript size is indicated to the right.
Arginine, but not glutamine, availability modulated TA1 RNA levels,
whereas neither arginine nor glutamine availability changed 4F2 RNA
levels.
View larger version (47K):
[in a new window]
Fig. 2.
Time course of CD98lc/TA1 and CD98hc/4F2
response to arginine availability in WB cells. Shown is total RNA
from WB cells cultured with arginine (+R) or without
arginine ( R) for 4, 8, 24, and 48 h. Aliquots (12 µg) of total RNA were analyzed by sequential Northern blot
hybridization to a 900-bp TA1 probe (top panel), a 1.8-kbp
4F2 probe (middle panel), and a 1.25-kbp GAPDH probe
(bottom panel). Ethidium bromide staining was used as a
loading control between lanes. Approximate transcript size is indicated
to the right. Steady state TA1 mRNA levels increased
within four hours of culture without arginine versus with
arginine. Although TA1 mRNA was induced up to ten-fold in WB cells
cultured without arginine, 4F2 mRNA levels were induced only
2-3-fold in cells cultured without arginine after densitometric
analysis with normalization to GAPDH mRNA levels.
View larger version (50K):
[in a new window]
Fig. 3.
Expression of CD98lc/TA1 and CD98hc/4F2 in
cultured GN6 and GN6TF cells in response to arginine availability.
Shown is total RNA from GN6 and GN6TF cells cultured in CEM with or
without arginine for 24, 48, and 72 h. Aliquots (12 µg) of total
RNA were analyzed by Northern blot hybridization to a 900-bp TA1 probe
(top panel), stripped and then rehybridized with a 1.8-kbp
4F2hc probe (middle panel), or stripped and then
rehybridized with a 1.25-kbp GAPDH probe (bottom panel).
Ethidium bromide staining was used as a loading control between lanes.
Approximate transcript sizes are indicated to the right.
Steady state TA1 RNA levels were high in cells cultured without
arginine but were gradually lowered in cells cultured with arginine.
4F2 RNA levels were constitutively high.
View larger version (64K):
[in a new window]
Fig. 4.
Expression of CD98lc/TA1 and CD98hc/4F2 in
cultured GP6, GP6TB, and GP7TB cells in response to arginine
availability. Shown is total RNA from GP6, GP6TB, and GP7TB cells
cultured in CEM with or without arginine for 24, 48, and 72 h
(Note: RNA for GP6 cells at 24 h was not available). Northern blot
hybridization of total RNA (12 µg) was analyzed by sequential
hybridization to probes for TA1 (top panel), 4F2hc
(middle panel), and GAPDH (bottom panel).
Ethidium bromide staining was used as a loading control between lanes.
Approximate transcript sizes are indicated to the right.
Neither TA1 nor 4F2 RNA levels varied significantly in any cell line
(more than 2-3-fold) under these culture conditions.
View larger version (26K):
[in a new window]
Fig. 5.
Expression of CD98lc/TA1 and CD98hc/4F2 in
cultured 1683 hepatoma cells in response to arginine availability.
Total RNA from 1683 cells cultured in CEM with or without arginine for
24, 48, and 72 h. Northern blot hybridization of total RNA (12 µg) was analyzed by sequential hybridization to probes for TA1
(top panel), 4F2hc (middle panel), and GAPDH
(bottom panel). Ethidium bromide staining was used as a
loading control between lanes. Approximate transcript sizes are
indicated to the right. Both TA1 and 4F2 were constitutively
expressed at high levels in these tumorigenic cells throughout the time
course of the experiment.
View larger version (25K):
[in a new window]
Fig. 6.
Graphs of relative CD98lc/TA1 and CD98hc/4F2
mRNA levels in the various rat hepatic cell lines. RNA samples
from each cell line were included on the same Northern blot to yield
these comparisons. Each bar represents the average
densitometric value determined from three independent blots.
a, relative units were plotted for TA1 mRNA levels in
the various cell lines cultured in the presence (open bars)
or absence (closed bars) of arginine for 4, 8, 24, 48, or
72 h. b, relative units were plotted for 4F2 mRNA
levels in the various cell lines cultured in the presence (open
bars) or absence (closed bars) of arginine for 72 h. RNA levels at 72 h are representative of levels at earlier
times because there was little change in 4F2 RNA levels.
View larger version (10K):
[in a new window]
Fig. 7.
Amino acid transport activity of leucine and
arginine in WB and GP7TB cells. The uptake of 100 µM
radiolabeled leucine and arginine was measured in both WB and GP7TB cells cultured in
CEM without arginine (closed bars) or with arginine
(open bars) for 48 h. All transport values are
expressed as pmol/mg of protein/min. Each experiment was repeated at
least twice with similar results. Values are the means ± S.E. of
three measurements. Values from a representative experiment are shown.
a, leucine transport was measured in WB cells. Excess cold
tyrosine inhibited leucine transport. b, arginine transport
was measured in WB cells. c, leucine transport was measured
in GP7TB cells. d, arginine transport was measured in GP7TB
cells. Leucine transport was as follows: GP7TB-R > WB-R > GP7TB + R > WB + R. These results correlated well with TA1/LAT-1
mRNA levels. Arginine transport did not vary in cells cultured with
or without arginine; however, the tumorigenic GP7TB cell line
transported more arginine than immortalized but not transformed WB
cells.
View larger version (33K):
[in a new window]
Fig. 8.
Immunoblot analysis of CD98lc/TA1 in WB,
GP7TB, and GP6 cells. Detergent extracts from WB, GP7TB, and GP6
cells cultured in CEM with or without arginine for 72 h and
analyzed by SDS-polyacrylamide gel electrophoresis under reducing
conditions. Rabbit antibodies (K and G) prepared to synthetic peptides
of CD98lc/TA1 (20) were used together with chemiluminescence detection.
A recombinant six-histidine fusion protein of TA1 (rTA1)
produced in baculovirus (3) served as a positive control. Relative to
nontransformed WB cells, K anti-peptide antibody specifically detected
multiple bands in cell extracts from transformed GP6 and tumorigenic
GP7TB cells. The size range of these bands is similar to what has been
reported for in vitro translated 512-amino acid LAT-1 (2).
The 35-kDa band was detected only in GP7TB cells, and this band was the
only band detected by G anti-peptide antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Medical Oncology,
Rhode Island Hospital, 593 Eddy St., Providence, RI 02903. Tel.: 401-444-8860; E-mail: Nancy_Thompson@brown.edu.
ABBREVIATIONS
-glutamyl transpeptidase;
KRP, Krebs-Ringer phosphate buffer;
FBS, fetal bovine serum;
CEM, Chee's
essential medium;
bp, base pair(s);
kbp, kilobase pair(s);
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Sang, J.,
Lim, Y.-P.,
Panzica, M.,
Finch, P.,
and Thompson, N. L.
(1995)
Cancer Res.
55,
1152-1159 2.
Kanai, Y.,
Segawa, H.,
Miyamoto, K.,
Uchino, H.,
Takeda, E.,
and Endou, H.
(1998)
J. Biol. Chem.
273,
23629-23632 3.
Gaugitsch, H. W.,
Preischl, E. E.,
Kalthoff, F.,
Huber, N. E.,
and Baumruker, T.
(1992)
J. Biol. Chem.
267,
11267-11273 4.
Mannion, B. A.,
Kolesnikova, T. V.,
Lin, S. H.,
Wang, S.,
Thompson, N. L.,
and Hemler, M. E.
(1998)
J. Biol. Chem.
273,
33127-33129 5.
Tsurudome, M.,
Ito, M.,
Takebayashi, S.,
Okumura, K.,
Nishio, M.,
Kawano, M.,
Kusagawa, S.,
Komada, H.,
and Ito, Y.
(1999)
J. Immunol.
162,
2462-2466 6.
Nakamura, E.,
Sato, M.,
Yang, H.,
Miyagawa, F.,
Harasaki, M.,
Tomita, K.,
Matsuoka, S.,
Noma, A.,
Iwai, K.,
and Minato, N.
(1999)
J. Biol. Chem.
274,
3009-3016 7.
Prasad, P. D.,
Wang, H.,
Huang, W.,
Kekuda, R.,
Rajan, D. P.,
Leibach, F. H.,
and Ganapathy, V.
(1999)
Biochem. Biophys. Res. Commun.
255,
283-288[CrossRef][Medline]
[Order article via Infotrieve]
8.
Mastroberardino, L.,
Spindler, B.,
Pfeiffer, R.,
Skelly, P. J.,
Loffing, J.,
Shoemaker, C. B.,
and Verrey, F.
(1998)
Nature
395,
288-291[CrossRef][Medline]
[Order article via Infotrieve]
9.
Hemler, M. E.,
and Strominger, J. L.
(1982)
J. Immunol.
129,
623-628[Abstract]
10.
Broer, S.,
Broer, A.,
and Hamprecht, B.
(1997)
Biochem. J.
324,
535-541
11.
Warren, A. P.,
Patel, K.,
McConkey, D. J.,
and Palacios, R.
(1996)
Blood
87,
3676-3587 12.
Fenczik, C. A.,
Sethl, T.,
Ramos, J. W.,
Hughes, P. E.,
and Ginsberg, M. H.
(1997)
Nature
390,
81-85[CrossRef][Medline]
[Order article via Infotrieve]
13.
Ohgimoto, S.,
Tabata, N.,
Suga, S.,
Nishio, M.,
Ohta, H.,
Tsurudoma, M.,
Komada, H.,
Kawano, M.,
Watanabe, N.,
and Ito, Y.
(1995)
J Immunol.
155,
3585-3592[Abstract]
14.
Torrents, D.,
Estevez, R.,
Pineda, M.,
Fernandez, E.,
Lloberas, J.,
Shi, Y.-B.,
Zorzano, A.,
and Palacin, M.
(1998)
J. Biol. Chem.
273,
32437-32445 15.
Torrents, D.,
Mykkanen, J.,
Pineda, M.,
Feliubadalo, L.,
Estevez, R.,
de Cid, R.,
Sanjurjo, P.,
Zorzano, A.,
Nunes, V.,
Huoponen, K.,
Reinikainen, A.,
Simell, O.,
Savontaus, M.-L.,
Aula, P.,
and Palacin, M.
(1999)
Nat. Genet.
21,
293-296[CrossRef][Medline]
[Order article via Infotrieve]
16.
Borsani, G.,
Bassi, M. T.,
Sperandeo, M. P.,
DeGrandi, A.,
Buoninconti, A.,
Riboni, M.,
Manzoni, M.,
Incerti, B.,
Pepe, A.,
Andria, G.,
Ballabio, A.,
and Sebastio, G.
(1999)
Nat. Genet.
21,
297-301[CrossRef][Medline]
[Order article via Infotrieve]
17.
Nagase, T.,
Seki, N.,
Ishikawa, K.,
Ohara, O.,
and Nomura, N.
(1996)
DNA Res.
3,
321-329[Abstract]
18.
Pineda, M.,
Fernandez, E.,
Torrents, D.,
Estevez, R.,
Lopez, C.,
Camps, M.,
Lloberas, J.,
Zorzano, A.,
and Palacin, M.
(1999)
J. Biol. Chem.
274,
19738-19744 19.
Segawa, H.,
Fukasawa, Y.,
Miyamoto, K.,
Takeda, E.,
Endou, H.,
and Kanai, Y.
(1999)
J. Biol. Chem.
274,
19745-19751 20.
Wolf, D. A.,
Wang, S.,
Panzica, M. A.,
Bassily, N. H.,
and Thompson, N. L.
(1996)
Cancer Res.
56,
5012-5022 21.
Shultz, V. D.,
Degli Esposti, S.,
Panzica, M. S.,
Abraham, A.,
Finch, P.,
and Thompson, N. L.
(1997)
Pathobiology
65,
14-25[Medline]
[Order article via Infotrieve]
22.
Shultz, V. D.,
Campbell, W.,
Karr, S.,
Hixson, D. C.,
and Thompson, N. L.
(1999)
Toxicol. Appl. Pharmacol.
154,
84-96[CrossRef][Medline]
[Order article via Infotrieve]
23.
Tsao, M.-S.,
Grisham, J. W.,
Nelson, K. G.,
and Smith, J. D.
(1984)
Exp. Cell. Res.
154,
38-52[CrossRef][Medline]
[Order article via Infotrieve]
24.
Tsao, M.-S.,
Grisham, J. W.,
Nelson, K. G.,
and Smith, J. D.
(1985)
Am. J. Pathol.
118,
306-315[Abstract]
25.
Hixson, D. C.,
McEntire, K. D.,
and Obrink, B.
(1985)
Cancer Res.
45,
3742-3749 26.
Waxman, D. J.,
Morrissey, J.,
Naik, S.,
and Jauregui, H.
(1990)
Biochemistry
271,
113-119
27.
Chirgwin, J. M.,
Przbyla, A. E.,
MacDonald, R. J.,
and Rutter, W. J.
(1979)
Biochemistry
18,
5294-5299[CrossRef][Medline]
[Order article via Infotrieve]
28.
Church, G.,
and Gilbert, W.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
1991-1995 29.
Quackenbush, E.,
Clabby, M.,
Gottesdiener, K. M.,
Barbosa, J.,
Jones, N. H.,
Strominger, J. L.,
Speck, S.,
and Leiden, J. M.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
6526-6530 30.
Fort, P.,
Marty, L.,
Piechaczyk, M.,
el Sabrouty, S.,
Dani, C.,
Jeanteur, P.,
and Blanchard, J. M.
(1985)
Nucleic Acids Res.
13,
1431-1442 31.
Gazzola, G. C.,
Dall'Asta, V.,
Franchi-Gazzola, R.,
and White, M. F.
(1981)
Anal. Biochem.
115,
368-374[CrossRef][Medline]
[Order article via Infotrieve]
32.
Kilberg, M. S.
(1989)
Methods Enzymol.
173,
564-575[Medline]
[Order article via Infotrieve]
33.
Coleman, W. B.,
McCullough, K. D.,
Esch, G. L.,
Faris, R. A.,
Hixson, D. C.,
Smith, G. J.,
and Grisham, J. W.
(1997)
Am. J. Pathol.
151,
353-359[Abstract]
34.
Hanigan, M. H.
(1995)
Carcinogen
16,
181-185 35.
Solt, D. B.,
Calderon-Solt, L.,
and Odajima, T.
(1985)
J. Natl. Cancer. Inst.
74,
437-445
36.
Ozono, S.,
Homma, Y.,
and Oyasu, R.
(1985)
Cancer Lett.
29,
49-57[CrossRef][Medline]
[Order article via Infotrieve]
37.
Pitot, H. C.
(1996)
J. Cancer Res. Clin. Oncol.
122,
257-265[CrossRef][Medline]
[Order article via Infotrieve]
38.
Hanigan, M. H.,
and Ricketts, W. A.
(1993)
Biochemistry
32,
6302-6306[CrossRef][Medline]
[Order article via Infotrieve]
39.
Inoue, Y.,
Bode, B. P.,
Copeland, E. M.,
and Souba, W. W.
(1995)
Cancer Res.
55,
3525-3530 40.
Aulak, K. S.,
Mishra, R.,
Zhou, L.,
Hyatt, S. L.,
de Jonge, W.,
Lamers, W.,
Snider, M.,
and Hatzoglou, M.
(1999)
J. Biol. Chem.
274,
30424-30432 41.
Bode, B. P.,
Kaminski, D. L.,
Souba, W. W.,
and Li, A. P.
(1995)
Hepatology
21,
511-520[CrossRef][Medline]
[Order article via Infotrieve]
42.
Weissbach, L.,
Handlogten, M. E.,
Christensen, H. N.,
and Kilberg, M. S.
(1982)
J. Biol. Chem.
257,
12006-11 43.
Novak, D. A.,
Kilberg, M. S.,
and Beveridge, M. J.
(1994)
Biochem. J.
301,
671-674
44.
Gong, S. S.,
Guerrini, L.,
and Basilico, C.
(1991)
Mol. Cell. Biol.
11,
6059-6066 45.
Chen, Z. P.,
and Chen, K. Y.
(1992)
J. Biol. Chem.
267,
6946-6951 46.
Pohjanpelto, P.,
and Holtta, E.
(1990)
Mol. Cell Biol.
10,
5814-5821 47.
Jousse, C.,
Bruhat, A.,
Ferrara, M.,
and Fafournoux, P.
(1998)
Biochem. J.
334,
147-153
48.
Bracy, D. S.,
Handlogten, M. E.,
Barber, E. F.,
Han, H.-P.,
and Kilberg, M. S.
(1986)
J. Biol. Chem.
261,
1514-1520 49.
Shotwell, M. A.,
Mattes, P. M.,
Jayme, D. W.,
and Oxender, D. L.
(1982)
J. Biol. Chem.
257,
2974-2980 50.
Hyatt, S. L.,
Aulak, K. S.,
Malandro, M.,
Kilberg, M. S.,
and Hatzoglou, M.
(1997)
J. Biol. Chem.
272,
19951-19957 51.
Kilberg, M. S.,
Hutson, R. G.,
and Laine, R. O.
(1994)
FASEB J.
8,
13-19[Abstract]
52.
Hara, K.,
Kudoh, H.,
Enomoto, T.,
Hashimoto, Y.,
and Masuko, T.
(1999)
Biochem. Biophys. Res. Commun.
262,
720-725[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Fernandez, D. Torrents, A. Zorzano, M. Palacin, and J. Chillaron Identification and Functional Characterization of a Novel Low Affinity Aromatic-preferring Amino Acid Transporter (arpAT): ONE OF THE FEW PROTEINS SILENCED DURING PRIMATE EVOLUTION J. Biol. Chem., May 13, 2005; 280(19): 19364 - 19372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Dave, N. Schulz, M. Zecevic, C. A. Wagner, and F. Verrey Expression of heteromeric amino acid transporters along the murine intestine J. Physiol., July 15, 2004; 558(2): 597 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Fanelli, E. F. Grollman, D. Wang, and N. J. Philp MCT1 and its accessory protein CD147 are differentially regulated by TSH in rat thyroid cells Am J Physiol Endocrinol Metab, December 1, 2003; 285(6): E1223 - E1229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Lahoutte, V. Caveliers, P. R. Franken, A. Bossuyt, J. Mertens, and H. Everaert Increased Tumor Uptake of 3-123I-Iodo-L-{alpha}-Methyltyrosine After Preloading with Amino Acids: An In Vivo Animal Imaging Study J. Nucl. Med., September 1, 2002; 43(9): 1201 - 1206. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chillaron, R. Roca, A. Valencia, A. Zorzano, and M. Palacin Heteromeric amino acid transporters: biochemistry, genetics, and physiology Am J Physiol Renal Physiol, December 1, 2001; 281(6): F995 - F1018. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Wagner, F. Lang, and S. Broer Function and structure of heterodimeric amino acid transporters Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1077 - C1093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. P. Bode Recent Molecular Advances in Mammalian Glutamine Transport J. Nutr., September 1, 2001; 131(9): 2475S - 2485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. A. Campbell and N. L. Thompson Overexpression of LAT1/CD98 Light Chain Is Sufficient to Increase System L-Amino Acid Transport Activity in Mouse Hepatocytes but Not Fibroblasts J. Biol. Chem., May 11, 2001; 276(20): 16877 - 16884. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |