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J Biol Chem, Vol. 274, Issue 28, 19738-19744, July 9, 1999
From the Departament de Bioquímica i Biologia Molecular and
** Departament de Fisiologia, We have identified a new
human cDNA, L-amino acid transporter-2 (LAT-2), that induces a
system L transport activity with 4F2hc (the heavy chain of the surface
antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth
member of the family of amino acid transporters that are subunits of
4F2hc. The amino acid transport activity induced by the co-expression
of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs
(e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly
trans-stimulated, suggesting an exchanger mechanism of
transport. Expression of tagged N-myc-LAT-2 alone in
oocytes did not induce amino acid transport, and the protein had an
intracellular location. Co-expression of N-myc-LAT-2 and
4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These
data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L
transport activity with broad specificity for zwitterionic amino acids.
Human LAT-2 mRNA is expressed in kidney >>>
placenta Last year, three amino acid transporter cDNAs (LAT-1,
y+LAT-1, and
y+LAT-2)1 were
identified as subunits of the heavy chain of the cell surface antigen
4F2 (4F2hc, also named CD98) (1-3). These subunits co-express amino
acid transport activity with 4F2hc in oocytes (i.e. system L
for LAT-1, and system y+L for y+LAT-1 and
y+LAT-2) (1-4). The role of this family of proteins in
amino acid transport has recently been demonstrated by the fact that
mutations in the y+LAT-1 gene cause
lysinuric protein intolerance, an inherited amino aciduria due to a
defective renal reabsorption mechanism of dibasic amino acids (5, 6).
The structural and functional similarities between 4F2hc and its
homologous protein rBAT suggest that a member of this family of
subunits might be the subunit of rBAT needed to fully express the amino
acid transport system bo,+ activity (reviewed in Refs. 7
and 8). After the identification of rBAT as the Type I
cystinuria gene (9), this subunit is a good candidate for non-Type I
cystinuria (7). A search throughout gene data bases suggests that there
may be as many as four new human members of the family of subunits of
4F2hc and rBAT.
Kanai and co-workers (1) identified rat LAT-1 (also known as TA1) by
co-expression cloning with 4F2hc in oocytes. The co-expressed transport
activity shows clear characteristics of the amino acid transport system
L: high affinity (Km in the low µM
range), sodium-independent, and trans-stimulated transport
for large zwitterionic amino acids. Some of these characteristics have
also been demonstrated for the human (E16, Ref. 2) and Xenopus
laevis orthologs of LAT-1 (ASUR4, Ref. 2; IU12, Ref. 3). System L
is almost ubiquitous (10), and variants of system L have been described (11, 12). The expression of rat LAT-1 is not ubiquitous, and it is not
present in tissues such as kidney and liver (1), which suggests that
homologs of LAT-1 might encode system L amino acid transporter variants.
In this study we have identified the fourth human member (LAT-2) of
this family of amino acid transporters. LAT-2 does not induce transport
of amino acids in oocytes when it is injected alone, but a variant of
system L transport activity (i.e. with broad specificity for
small and large zwitterionic amino acids) is co-expressed when LAT-2 is
injected with 4F2hc. We demonstrate here that co-expression of LAT-2
with 4F2hc brings the former to the oocyte plasma membrane. Its
expression in the epithelial cells of the proximal tubule suggests a
role of LAT-2/4F2hc in the renal reabsorption of neutral amino acids.
PCR Amplification, Sequencing, and LAT-2 cDNA
Construction--
For PCR amplification, first-strand cDNA was
synthesized from 5 µg of total RNA purified from opossum kidney (13)
cells using SuperScript II kit (Life Technologies, Inc.). Two
degenerate forward and reverse primers were designed based on two
highly conserved regions among the first known members of this family of amino acid transporters. PCR amplification, subcloning into pGEM-T
easy vector (Promega), and sequencing were carried out as described
elsewhere (3).
The open reading frame (ORF) of LAT-2 was obtained from two partial
human LAT-2 cDNA clones (IMAGE No. 322502 and No. 267204). Clone
322502 was cut with NotI and AvrII to create the
5'-end fragment of LAT-2 (from nt 1 to 1152 of the LAT-2 cDNA).
Clone 267204 was digested with EcoRI and AvrII to
create the 3'-end fragment of the LAT-2 coding sequence (nt 1152 to
3'-end of the LAT-2 cDNA) ligated to the pT7T3D vector. Both
fragments were ligated to create a LAT-2 cDNA fragment covering the
ORF (5'-end to nt 2050; see Fig. 1) in pT7T3D vector. To improve
expression in oocytes, an SspI-NotI fragment of
LAT-2 was cloned into pNKS2-myc NotI vector (a
gift from G. Schmalzing; Ref. 14). To create an N-myc-tagged
LAT-2 cDNA, pT7T3D-LAT-2 was PCR-amplified with primers M13 forward
(16-mer) and 5'-ACGTCTAGTCGACATGGAAGAAGGAGCCAGGCAC-3' (containing a
SalI site and the first 21 nt of the ORF of LAT-2). The PCR
product was digested with SalI and NotI. The
resulting fragment of LAT-2 was cloned into pNKS2-myc NotI.
The N-myc-tagged LAT-2 cDNA was tested by sequencing.
All sequences carried out in this work were performed in both
directions with d-rhodamine dye terminator cycle sequencing ready
reaction kit (Perkin-Elmer). The sequence reactions were analyzed with
an Abi Prism 377 DNA sequencer.
Oocytes, Injections, and Uptake Measurements--
Oocyte origin,
management, and injections were as described elsewhere (15, 16).
Defolliculated stage VI X. laevis oocytes were injected with
10 ng/oocyte human 4F2hc, human LAT-2, N-myc-LAT-2, or
X. laevis IU12 cRNA. Synthesis of human 4F2hc cRNA (17) was as described (18). X. laevis LAT-1 (i.e. IU12)
was a gift from Y. B. Shi (19), and the cRNA was synthesized as
described elsewhere (3). Human LAT-2 cRNA was obtained by cutting the
cDNA with NotI and using T7 polymerase.
Influx rates of L-[3H]arginine,
L-[3H]leucine,
L-[3H]alanine, and
L-[3H]glutamine (Amersham Pharmacia Biotech)
were measured in 100 mM NaCl or 100 mM
CholineCl medium at the indicated number of days after injection and
under linear conditions as described (15). Amino acid transport rates
obtained with oocytes injected with water (50 nl) were similar to those
of noninjected oocytes (data not shown). For
L-[3H]isoleucine efflux measurements, groups
of five cRNA-injected or noninjected oocytes were incubated with 50 µM L-[3H]isoleucine (3 µCi/µl) for the
indicated period of time (see legend to Fig. 7). Efflux was measured as
described elsewhere (20).
Computer Analysis--
Amino acid or nucleotide sequence
homology search and the prediction of transmembrane segments of LAT-2
were performed as indicated elsewhere (3).
Northern Blot Analysis--
A human adult poly(A+)
membrane from CLONTECH (Palo Alto, CA) was used.
The insert of clone 267204 was separated from the pT7T3D vector by
NotI-EcoRI digestion. This ~1-kb DNA fragment
was purified, labeled with [ In Situ Hybridization--
Sense and antisense cRNA probes were
labeled with digoxigenin-11-UTP (Roche Molecular Biochemicals) by
transcription of a LAT2 fragment (1-310 nt of the contig shown in Fig.
1) contained in the pT7T3D vector. The transcription reactions were set
up at room temperature by mixing 7.5 µl of double-distilled water treated with diethyl pyrocarbonate, 1 µl of linearized template cDNA (1 µg), 4 µl of 5× transcription buffer (Promega), 2 µl
of NTPmix (10 mM ATP, CTP, GTP, 6.5 mM UTP, 3.5 mM digoxigenin-11-UTP, Roche Molecular Biochemicals), 1 µl of RNAsin (30.6 units/µl, Amersham Pharmacia Biotech), and 2 µl of RNA polymerase (T7 or T3, 15 units/µl, Promega). Labeling
reactions were performed at 37 °C for 2 h and stopped by
incubation with 2 µl of RNase-free DNase (10 units/µl, Stratagene)
for 15 min at 37 °C. cRNA fragments were precipitated overnight with
1/10 vol of 4 M LiCl and 2.5 volumes of ethanol at
Fresh human kidney was fixed in 4% paraformaldheyde, 0.1 M
phosphate buffer and kept at 4 °C before use. Thereafter, the
sections were washed in 0.1 M phosphate buffer (2 h, room
temperature) and dehydrated with 70, 90, and 100% alcohol,
alcohol/xylene (v/v), and xylene (2 h for each). Pieces were embedded
in paraffin. 5-µm sections were cut on a Leica RM 2135 microtome and
mounted on silenized slides (Perkin-Elmer). Sections were deparaffined
with xylene and hydrated with 100, 90, and 70% ethanol and
double-distilled water treated with diethyl pyrocarbonate,
permeabilized with proteinase K (Roche Molecular Biochemicals) (1 µg/ml) in Tris-EDTA buffer, pH 8 (3 min, 37 °C), 0.2 N HCl (20 min, room temperature), and washed twice in 2× SSC solution (30 min,
room temperature). The hybridization step was carried out with a
solution containing 50% formamide, 10% dextran sulfate, 2× SSC
solution, 1× Denhardt's solution, 400 ng/µl denatured salmon sperm
DNA, and denatured (4 min, 70 °C) sense or antisense probes (5 ng/µl) for 16 h at 42 °C in a moist chamber. The sections
were then washed in 4× SSC solution containing 45% formamide (2 min,
room temperature), 0.1× SSC (1 h, 37 °C), digested with 20 µg/ml
RNase A (Roche Molecular Biochemicals) (30 min, room temperature), and
washed in 0.1× SSC solution (5 min, room temperature). Sections were
rinsed twice in Tris-buffered saline (TBS), pH 7.5 (10 min, room
temperature), blocked with 1% bovine serum albumin in TBS (30 min,
room temperature), and incubated overnight with an alkaline
phosphatase-conjugated anti-digoxigenin antibody (1:500) (Biocell,
Cardiff, UK). They were then washed in TBS, pH 7.5 (10 min, room
temperature), and TBS, pH 9.5, containing 50 mM
MgCl2 (10 min, room temperature) and developed with
nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche
Molecular Biochemicals). Slides were examined on an Olympus microscope.
Chromosome Mapping--
Chromosome mapping was done using the
Stanford Human Genome Center G3 radiation hybrid panel (medium
resolution). DNA samples of this panel, along with total genomic DNA
and pT7T3-249835 (used as a positive control), were PCR screened for
the presence of the genomic sequences flanked by the primers 12D
(5'-GGCATCTCTCTTCCTAATG-3') and 7R (5'-GCCAATGCTCTCCTCAGT-3'), which
are located in the 3'-untranslated region of the cDNA. PCR
amplifications were carried out in a Perkin-Elmer 9600 thermocycler as
described elsewhere (3). Amplification conditions were as follows: 35 cycles of denaturing (94 °C, 30 s), annealing (58 °C,
40 s), and extension (74 °C, 30 s). PCR results were
transformed into zeros (for no amplification) and ones (for positive
amplification) and submitted to the radiation hybrid mapping e-mail
server at the Stanford Human Genome Center (SHGC). The resulting
chromosomal location, referred to a SHGC marker, was obtained
automatically via e-mail from this server.
Localization of LAT-2 Expression by Confocal
Microscopy--
Groups of five oocytes were prepared for
immunofluorescence 2 days after injection with 10 ng/oocyte human 4F2hc
or N-myc-LAT-2 cRNA, alone or in combination. Oocytes were
placed in 500 mm3 cryomolds (Tissue-Tek, Miles Inc.,
Elkhart, IN), sliced, fixed, and permeabilized as described elsewhere
(18). Slices were incubated with monoclonal antibody 9E10
anti-myc (ATCC, Manassas, VA), diluted 1/500 in 10%
phosphate-buffered saline, at room temperature for 1 h. Slices
were washed three times in phosphate-buffered saline, incubated with
7.5 µg/ml Texas red-conjugated goat anti-mouse (Molecular Probes,
Leiden, The Netherlands) at room temperature for 1 h, washed three
times in phosphate-buffered saline, and mounted in Immunofluore (ICN,
Madrid, Spain).
Our goal was to identify any new member of the amino acid
transporter-related family expressed in the kidney and potentially involved in reabsorption of amino acids. For this purpose, reverse transcription-PCR amplification of total RNA from opossum kidney cells
was performed with degenerated primers as described for the
identification of y+LAT-1 (3). Electrophoretic analysis of
the PCR reaction showed one band of 286 bp, which was subcloned into
pGEMT-easy vector and amplified in Escherichia coli. The
deduced amino acid sequence of one clone (b2c2) showed a significant
degree of identity to the amino acid transporter-related proteins: 46, 45, 43, 41, and 43% with human y+LAT-1 and
y+LAT-2 and Xenopus, rat, and human LAT-1,
respectively. This homology is compatible with the assumption that b2c2
is part of a cDNA corresponding to a new member of this family. By
using the same computer approach (BLAST and EST Cluster Assembly
Machine) as we recently used for the identification of
y+LAT-1 (3), a human EST (W39098, IMAGE clone 322502) that shows homology with the b2c2 fragment (92% identity in the amino acid
sequence) was identified. Subsequently, EST W39098 was used to identify
two other ESTs from the same cluster (N23973 and H84042, from IMAGE
clones 267204 and 249835, respectively). Sequences of these overlapping
EST clones revealed a 3'-polyadenylated cDNA contig (LAT-2) of 3733 bp (Fig. 1). The first ATG codon lies within a good consensus initiation sequence (5'-GAAGG) (21). The ORF
continues to the first stop codon (TGA) at base 1827 and codes for a
protein of 535 amino acid residues with a predicted molecular mass of
58,577 Da. The nucleotide sequences of EST clones 322502, 267204, and
249835 and LAT-2 cDNA have been deposited in the GenBankTM/EBI
data base (accession numbers AF135828, AF135829, AF135830, and
AF135831, respectively).
A multiple sequence alignment of the predicted amino acid sequence of
human LAT-2, LAT-1, y+LAT-1, and y+LAT-2 is
shown in Fig. 2. Human LAT-2 shows an
amino acid sequence identity of 50, 44, and 45% to human LAT-1,
y+LAT-1 and y+LAT-2, respectively.
Hydrophobicity studies show 12 transmembrane domains with both C- and
N-terminal segments intracytoplasmatic, which is the same protein
structure suggested for the other members of this family (1-3, 19).
Only the consensus for the position of the transmembrane segment III
can vary for the proteins presented in Fig. 2. There is only one
putative N-glycosylation site (Fig. 2, boxed) between the
putative transmembrane segments VIII and IX. In our predicted model
this segment is cytoplasmic and cannot be glycosylated. This finding is
in full agreement with previous expression studies with rat and human
LAT-1 (1-2). 4F2hc is associated with its subunits in a disulfide
bond-dependent manner (2-4, 22) through cysteine residue
109 of human 4F2hc (18) and cysteine residue 164 of Xenopus
LAT-1 (23). This cysteine residue is conserved in all human 4F2 light
chains including LAT-2 (cysteine residue 154) (Fig. 2).
The human LAT-2 gene was chromosome-mapped by
using a radiation hybrid panel (see "Experimental Procedures") with
primers corresponding to the 3'-untranslated region of the LAT-2
cDNA. From this screening we obtained 16 positive and 66 negative
results. Chromosome mapping results, obtained from the SGHC server,
linked Lat-2, with a logarithem odds score of 12.6, to a distance of 13 cR (286 kb) from the marker SHGC-13507 (D14S1349). The nearest centromeric marker to this one, marker SHGC-6999 (X52889),
is located at chromosome 14q11.2-13.
cRNA from LAT-2 was injected into oocytes alone or in combination with
an equimolar quantity of human 4F2hc cRNA and tested for amino acid
transport (Fig. 3). 4F2hc alone induced,
as previously reported (16, 18, 24-27), y+L amino acid
transport activity (i.e. sodium-independent
L-arginine transport and sodium-dependent
L-leucine transport). LAT-2 alone induced weakly
sodium-independent L-leucine transport. Interestingly, when
4F2hc and LAT-2 were co-injected, the induction of
L-arginine transport was lower than that induced by 4F2hc
alone, whereas the induction of sodium-independent
L-leucine transport increased dramatically (Fig. 3). From
four independent experiments the average co-expression of
L-leucine transport relative to the induction of
4F2hc alone was 30-fold (ranging from 6- to 100-fold). The co-expression of leucine transport by 4F2hc and LAT-2 is
sodium-independent, suggesting induction of a system L-type amino acid
transport activity (Fig. 3). Kinetic analysis revealed an apparent
Km of 221 ± 54 µM for the
transport of L-leucine induced by 4F2hc/LAT-2 (data not
shown).
Identification of a Membrane Protein, LAT-2, That
Co-expresses with 4F2 Heavy Chain, an L-type Amino Acid Transport
Activity with Broad Specificity for Small and Large Zwitterionic
Amino Acids*
§,¶,
,
Serveis Científico-Tècnics, Universitat
de Barcelona, Avda. Diagonal 645, 08028 Barcelona, Spain
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
brain, liver > spleen, skeletal muscle, heart,
small intestine, and lung. Human LAT-2 gene localizes at
chromosome 14q11.2-13 (13 cR or ~286 kb from marker D14S1349). The
high expression of LAT-2 mRNA in epithelial cells of proximal
tubules, the basolateral location of 4F2hc in these cells, and the
amino acid transport activity of LAT-2 suggest that this transporter
contributes to the renal reabsorption of neutral amino acids in the
basolateral domain of epithelial proximal tubule cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (Amersham
Pharmacia Biotech) using a random oligonucleotide-priming labeling kit
(Amersham Pharmacia Biotech), and used as a probe. Hybridization and
washing conditions were as recommended by CLONTECH. In these conditions, y+LAT-1 and y+LAT-2 cRNAs were not detected (data
not shown).
80 °C. The precipitated cRNA was recovered in 10 µl of
double-distilled water treated with diethyl pyrocarbonate.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequence of
LAT-2 cDNA. The size of the cDNA contig is 3733 bp,
containing a 5'-untranslated region of 221 bp followed by an ORF of 535 amino acids and a 3'-untranslated region of 1904 bp that contains a
23-bp long poly(A) tail. The stop codon (TGA) is indicated by a
star. The possible polyadenylation signal is
underlined. The sequences of the overlapping segments were
identical in all three clones except for nt 1422. At this position,
clone 267204 has a G, as in the ESTs N32639 and N31874, and the
corresponding amino acid residue is Val401. In contrast,
clone 249835 has an A, and the corresponding amino acid is
Ile401. This is probably a polymorphism. In agreement with
the majority of available EST sequences, the contig LAT-2 shown in this
figure and the cRNA injected in this study has G at position 1422 (i.e. Val401).
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Fig. 2.
Amino acid sequence comparison of the four
human members of the family of amino acid transporters that are
subunits of 4F2hc. Multi-alignment was done using the program
CLUSTALW Sequence Alignment from the Baylor College of Medicine. The
thin horizontal lines indicate the 12 putative transmembrane
domains determined by computer analysis (see "Experimental
Procedures"). The amino acid residues identical to LAT-2 sequence are
indicated in gray boxes. The solid frame box
indicates a potential N-glycosylation site, but according to our
membrane topology prediction, this site is intracellular and cannot be
glycosylated. The conserved cysteine residue is indicated with a
star.
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Fig. 3.
Co-expressed amino acid transport activity by
4F2hc and LAT-2. Oocytes were injected with LAT-2 cRNA alone or in
combination with human 4F2hc cRNA. Three days after the injection, the
uptake of 50 µM L-[3H]arginine
(Arg) and 50 µM L-[3H]leucine
(Leu) in the presence (+, slashed bars) or
absence ( , open or closed bars) of 100 mM NaCl was determined for 5 min. Transport of
L-[3H]leucine in the absence of sodium is
highlighted in the closed bars. Amino acid uptake
rates (pmol/5 min per oocyte) were calculated by subtracting the uptake
of the noninjected group from that of the cRNA-injected groups. The
amino acid uptake activity of uninjected oocytes was as follows:
L-[3H]arginine uptake, 3.0 ± 0.2 (choline medium) and 4.0 ± 0.7 (sodium medium);
L-[3H]leucine uptake, 1.8 ± 0.2 (choline medium) and 3.3 ± 0.4 (sodium medium). Data (mean ± S.E.) correspond to a representative experiment with 7-8 oocytes
per group.
To further characterize the uptake activity co-expressed by LAT-2 and
4F2hc, we measured the inhibition of sodium-independent leucine uptake
by different amino acids at a 100-fold excess concentration (5 mM). Fig. 4 shows the
inhibition pattern for the transport activity induced by LAT-2 and
4F2hc compared with that induced by X. laevis LAT-1 and
4F2hc. These results showed clearly that the transport activity induced
by 4F2hc/LAT-2 and 4F2hc/LAT-1 is restricted to zwitterionic amino
acids. The pattern of inhibition in the case of X. laevis
LAT-1, restricted to large zwitterionic amino acids and analogs
(i.e. BCH), is in full agreement with the pattern described
for rat LAT-1 (1). In contrast, 4F2hc/LAT-2-induced transport activity
was also practically abolished by small zwitterionic amino acids
(i.e. glycine, alanine, serine, threonine, and cysteine), and it is clearly inhibited by glutamine and asparagine. To demonstrate transport of small zwitterionic amino acids via this variant of system
L, the uptake of 50 µM L-[3H]alanine was
determined in oocytes expressing 4F2hc/LAT-2 or 4F2hc/LAT-1.
Interestingly, co-expression of 4F2hc/LAT-2 in oocytes, but not of
4F2hc/LAT-1, resulted in the induction of L-alanine transport above background (i.e. noninjected or
4F2hc-injected oocytes) (Fig.
5a). Kinetic analysis of this
transport revealed an apparent Km of 978 ± 142 µM (Fig. 5b). Similarly to alanine,
4F2hc/LAT-2 induced sodium-independent L-glutamine
transport. Two days after injection, the induced uptake of 200 µM L-[3H]glutamine was 0.3 ± 0.4 and
34.6 ± 4.0 pmol/5 min per oocyte for 4F2hc- and
4F2hc/LAT-2-injected oocytes, respectively. All of the above suggests
that 4F2hc/LAT-2 represents a broad specificity variant of system L
transporter for small and large zwitterionic amino acids.
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0.001).
In agreement with previous reports for the transport activity induced
by 4F2hc in oocytes (18, 20) by LAT-1/4F2hc (1-2) and
y+LAT-1/4F2hc (4), LAT-2/4F2hc showed a high level of
trans-stimulation. Fig. 6
shows that the efflux of L-[3H]isoleucine in
oocytes expressing LAT-2/4F2hc is dependent on the presence of a
substrate in the medium (e.g. leucine), but it is not
trans-stimulated by amino acids that are not substrates (e.g. proline) of LAT-2/4F2hc amino acid transporter.
The level of efflux in trans0 conditions (no
amino acid substrates in the medium) in oocytes expressing
LAT-2/4F2hc is identical to that of noninjected oocytes or oocytes
expressing 4F2hc alone (Fig. 6). This result suggests a high level of
exchanger coupling via LAT-2/4F2hc.
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) or
proline (Pro, 
) or no amino acids (none, 
).
Data (mean ± S.E.) correspond to a representative experiment with
three groups of 5 oocytes per data point. The efflux rates in
4F2hc-injected and noninjected oocytes in the presence of 1 mM
L-leucine in the medium were 1630 ± 200 and 1350 ± 100 × 103 cpm/5 oocytes per min, respectively. These
efflux rates are indistinguishable from those of 4F2hc/LAT-2-injected
oocytes in medium containing 1 mM proline or no amino
acids.
Recently, it has been shown that y+LAT-1, LAT-1, and SPRM1
form a disulfide bond heterodimeric complex with 4F2hc (2-4, 22, 23).
Moreover, Verrey and co-workers (2) have shown that 4F2hc brings SPRM1
to the oocyte plasma membrane. Fig. 7
shows that 4F2hc also brings LAT-2 to the oocyte plasma membrane. To follow the expression of LAT-2, a tagged LAT-2 cRNA
(N-myc-LAT-2) was expressed in oocytes.
N-myc-LAT-2 co-expresses with 4F2hc L-transport
activity, but N-myc-LAT-2 alone does not induce amino acid
transport activity (see legend to Fig. 7). Confocal immunofluorescence detected N-myc-LAT-2 at the oocyte plasma membrane when
co-expressed with 4F2hc, but its expression was intracellular when
expressed alone in oocytes (Fig. 7).
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The tissue expression of the mRNA corresponding to LAT-2 was
examined by Northern blot analysis at high stringency conditions (Fig.
8). mRNA species of ~5 and ~3.7
kb hybridize with the LAT-2 cDNA; the size of the shorter
transcript corresponds to that of the LAT-2 cDNA identified here.
Both transcripts are expressed most conspicuously in the kidney.
Placenta brain, liver > skeletal muscle, and heart also
express these transcripts. The last two also showed a very faint band
of ~7 kb. Long exposures also revealed the 5- and 3.7-kb transcripts
in the small intestine and the lung. In situ hybridization
studies specifically localized the renal expression of LAT-2 mRNA
to the epithelial cells of proximal tubules, most probably in the
convoluted part (Fig. 9). No other
components of the nephron, including distal tubules and glomeruli, were
reactive with LAT-2 cRNA probe. A similar pattern of expression has
been shown on inmunolocalization of 4F2hc protein on kidney cortex (Ref. 28, and data not shown).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In this study we have identified a new member (LAT-2) of the family of amino acid transporters, which are subunits of 4F2hc and in humans are composed also of LAT-1, y+LAT-1, and y+LAT-2. We report here on the human LAT-2 cDNA sequence, chromosomal location, and pattern of expression of its mRNA. Moreover, we show that 4F2hc brings LAT-2 to the oocyte plasma membrane, which induces a system L amino acid transport activity with broad specificity. Therefore, LAT-2 is a putative new light subunit of the surface antigen 4F2.
Before the identification of the 4F2hc subunits, functional expression
experiments in oocytes revealed that 4F2hc induced both system
y+L (16, 24-27) and system L (29) transport activities. In
agreement with this finding, the 4F2hc subunits y+LAT-1,
y+LAT-2, LAT-1, and LAT-2 are isoforms of systems
y+L and L, respectively. Before our cloning of system
y+L, there were no reports of variants of system
y+L in the literature (30). y+LAT-1 is
defective in lysinuric protein intolerance (5, 6), and
y+LAT-2 might be responsible for system y+L in
the cell types in which this transport activity is not defective in
lysinuric protein intolerance (e.g. erythrocytes (31)). In contrast, system L variants L1 (substrate affinity in the micromolar range) and L2 (substrate affinity in the millimolar range) have been
described previously (10). These subtypes are expressed in hepatoma
cell lines and hepatocytes, respectively (11). An L3 subtype was
described in fibroblasts with an affinity between that of the L1 and L2
subtypes (12). LAT-1 fits the transport characteristics and the tissue
distribution of subtype L1 (Ref. 1 and present study). LAT-2 shows
characteristic features of system L (i.e. sodium-independent
transport of zwitterionic amino acids inhibitable by the analog BCH),
but it also shows features that are dissimilar to both system L
subtypes. Thus, LAT-2 is expressed in the liver and has a substrate
affinity in the micromolar range for L-leucine
(Km 
220 µM). This is a lower
affinity than has been described for rat (18 µM) and
Xenopus (32 µM) LAT-1 (1, 2). Moreover, in
contrast to the hepatic system L, LAT-2 also transports small
zwitterionic amino acids (e.g. L-alanine with an apparent Km 1 mM). A
transport system with similar characteristics
(sodium-independent, trans-stimulated transport
for large and small zwitterionic amino acids and a similar apparent
Km for L-alanine) to that of LAT-2 has
been described in the basolateral membrane of the intestinal enterocyte (32, 33) and the placental syncytiotrophoblast (34).
What is the physiological role of a system L transporter with broad
specificity for zwitterionic amino acids, including the small ones?
Christensen (10) hypothesized that system L serves the exchange (efflux
and influx) of zwitterionic amino acids through the plasma membrane to
fulfill the inter-organ fluxes of these amino acids (10). The transport
activity induced by 4F2hc/LAT-2 is highly trans-stimulated
in oocytes. Indeed, LAT-2 behaves as an exchanger, because the efflux
via this transporter in oocytes is totally dependent on the presence of
a substrate in the medium. The kidney showed the highest LAT-2 mRNA
expression (present study), whereas LAT-1, the other system L isoform
transporter, is not expressed in the kidney (1). The involvement of
rBAT and y+LAT-1 in Type I cystinuria
and lysinuric protein intolerance, respectively (5, 6, 9), demonstrates
the role of these transporters in the apical reabsorption of cystine
and dibasic amino acids (rBAT) and in the basolateral efflux of dibasic
amino acids (4F2hc/y+LAT-1). LAT-2 mRNA is restricted
to the epithelial cells of the proximal tubule of the human kidney
(present study). In these cells 4F2hc has a basolateral location (28).
This result suggests that 4F2hc/LAT-2 might mediate the efflux of
zwitterionic amino acids, including those with a short side chain,
through the basolateral plasma membrane. Moreover, the fact that
cysteine, the main intracellular form of cystine (35, 36), strongly
inhibits 4F2hc/LAT-2 transport activity suggests the role of this
transporter in the renal reabsorption of cystine. The expression of
LAT-2 mRNA in the small intestine suggests a similar role in the
intestinal absorption of zwitterionic amino acids. The expression of
LAT-2 mRNA in the placenta also implicates it in the transfer of
zwitterionic amino acids from the placenta to the fetus. This
hypothesis needs confirmation by the localization of LAT-2 at the
basolateral plasma membrane in the epithelial cells of the proximal
tubule and in the placental trophoblast. The expression of LAT-2
mRNA in the brain, liver, spleen, and skeletal muscle suggests a
role in the release of glutamine and short zwitterionic amino acids
from these tissues as follows: (i) glial cells release glutamine to
neurons as a substrate for glutamate and 
-aminobutyric acid
synthesis (37); (ii) hepatocytes in the perivenous zone release
glutamine as an ammonium detoxification pathway (38); (iii) the
skeletal muscle releases most of the aminic nitrogen as glutamine and
alanine (39); and (iv) spleen and small intestinal enterocytes
metabolize glutamine and produce alanine and other small zwitterionic
amino acids (e.g. serine and glycine) (40). Identification
of the cells expressing LAT-2 in these tissues will help us to
understand the role of this system L amino acid transporter with broad specificity.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Judith Garcia for technical assistance, Belen Peral for helping with and making possible the radiation hybrid analysis, and Núria Reig for help in reviewing this manuscript. We thank the Serveis Científico-Tècnics de la Universitat de Barcelona for sequencing and for help with immunochemical and in situ hybridization analysis. We thank the Fundació Institut d'Urologia, Nefrologia i Andrologia-Puigvert for donating human tissue and also thank Robin Rycroft for editorial help.
| |
FOOTNOTES |
|---|
* This work was supported in part by Dirección General de Investigación Científica y Técnica Research Grant PM96/0060 and by Grant 1995 SGR 537 from the Generalitat de Catalunya (Spain).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) AF135828, AF135829, AF135830, and AF135831.
These two authors contributed equally to this study.
§ Recipients of a grant from the Comissió Interdepartamental de Recerca i Innovació Tecnològica, Catalonia, Spain.
¶ Recipients of a grant from the Ministerio de Educación y Cultura, Spain.
To whom correspondence should be addressed. Tel.:
34-93-4021543; Fax: 34-93-4021559; E-mail: mpalacin@porthos.
bio.ub.es.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: LAT, L-amino acid transporter; y+LAT, y+ L-amino acid transporter; 4F2hc, heavy chain of the cell surface antigen 4F2; BCH, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid; rBAT, related to bo,+ amino acid transporter; PCR, polymerase chain reaction; ORF, open reading frame; nt, nucleotide; bp, base pair(s); kb, kilobase or kilobase pair(s); TBS, Tris-buffered saline; SHGC, Stanford Human Genome Center; EST, expressed sequence tag.
| |
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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]
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D. Merlin, S. Sitaraman, X. Liu, K. Eastburn, J. Sun, T. Kucharzik, B. Lewis, and J. L. Madara CD98-mediated Links between Amino Acid Transport and beta 1 Integrin Distribution in Polarized Columnar Epithelia J. Biol. Chem., October 12, 2001; 276(42): 39282 - 39289. [Abstract] [Full Text] [PDF]
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 E. C. H. Friesema, R. Docter, E. P. C. M. Moerings, F. Verrey, E. P. Krenning, G. Hennemann, and T. J. Visser Thyroid Hormone Transport by the Heterodimeric Human System L Amino Acid Transporter Endocrinology, October 1, 2001; 142(10): 4339 - 4348. [Abstract] [Full Text] [PDF]
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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]
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 B. P. Bode Recent Molecular Advances in Mammalian Glutamine Transport J. Nutr., September 1, 2001; 131(9): 2475S - 2485. [Abstract] [Full Text] [PDF]
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 G. Hennemann, R. Docter, E. C. H. Friesema, M. de Jong, E. P. Krenning, and T. J. Visser Plasma Membrane Transport of Thyroid Hormones and Its Role in Thyroid Hormone Metabolism and Bioavailability Endocr. Rev., August 1, 2001; 22(4): 451 - 476. [Abstract] [Full Text] [PDF]
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 V. T. Cannon, R. K. Zalups, and D. W. Barfuss Amino Acid Transporters Involved in Luminal Transport of Mercuric Conjugates of Cysteine in Rabbit Proximal Tubule J. Pharmacol. Exp. Ther., August 1, 2001; 298(2): 780 - 789. [Abstract] [Full Text] [PDF]
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Y. Kudo and C A R Boyd Characterisation of L-tryptophan transporters in human placenta: a comparison of brush border and basal membrane vesicles J. Physiol., March 1, 2001; 531(2): 405 - 416. [Abstract] [Full Text] [PDF]
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Y. Kudo and C A R Boyd The role of L-tryptophan transport in L-tryptophan degradation by indoleamine 2,3-dioxygenase in human placental explants J. Physiol., March 1, 2001; 531(2): 417 - 423. [Abstract] [Full Text] [PDF]
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 M. Font, L. Feliubadalo, X. Estivill, V. Nunes, E. Golomb, Y. Kreiss, E. Pras, L. Bisceglia, A. P. d'Adamo, L. Zelante, et al. Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria Hum. Mol. Genet., February 1, 2001; 10(4): 305 - 316. [Abstract] [Full Text] [PDF]
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C. A Wagner, A. Broer, A. Albers, N. Gamper, F. Lang, and S. Broer The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1 J. Physiol., July 1, 2000; 526(1): 35 - 46. [Abstract] [Full Text] [PDF]
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R. J. Reimer, F. A. Chaudhry, A. T. Gray, and R. H. Edwards Amino acid transport System A resembles System N in sequence but differs in mechanism PNAS, June 14, 2000; (2000) 140152797. [Abstract] [Full Text]
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D. P. Rajan, W. Huang, R. Kekuda, R. L. George, J. Wang, S. J. Conway, L. D. Devoe, F. H. Leibach, P. D. Prasad, and V. Ganapathy Differential Influence of the 4F2 Heavy Chain and the Protein Related to b0,+ Amino Acid Transport on Substrate Affinity of the Heteromeric b0,+ Amino Acid Transporter J. Biol. Chem., May 5, 2000; 275(19): 14331 - 14335. [Abstract] [Full Text] [PDF]
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Y. Fukasawa, H. Segawa, J. Y. Kim, A. Chairoungdua, D. K. Kim, H. Matsuo, S. H. Cha, H. Endou, and Y. Kanai Identification and Characterization of a Na+-independent Neutral Amino Acid Transporter That Associates with the 4F2 Heavy Chain and Exhibits Substrate Selectivity for Small Neutral D- and L-Amino Acids J. Biol. Chem., March 24, 2000; 275(13): 9690 - 9698. [Abstract] [Full Text] [PDF]
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W. A. Campbell, D. E. Sah, M. M. Medina, J. E. Albina, W. B. Coleman, and N. L. Thompson 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 J. Biol. Chem., February 25, 2000; 275(8): 5347 - 5354. [Abstract] [Full Text] [PDF]
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Y. Kudo and C A R Boyd Heterodimeric amino acid transporters: expression of heavy but not light chains of CD98 correlates with induction of amino acid transport systems in human placental trophoblast J. Physiol., February 15, 2000; 523(1): 13 - 18. [Abstract] [Full Text] [PDF]
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J. Mykkanen, D. Torrents, M. Pineda, M. Camps, M. E. Yoldi, N. Horelli-Kuitunen, K. Huoponen, M. Heinonen, J. Oksanen, O. Simell, et al. Functional analysis of novel mutations in y+LAT-1 amino acid transporter gene causing lysinuric protein intolerance (LPI) Hum. Mol. Genet., February 12, 2000; 9(3): 431 - 438. [Abstract] [Full Text] [PDF]
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H. Varoqui, H. Zhu, D. Yao, H. Ming, and J. D. Erickson Cloning and Functional Identification of a Neuronal Glutamine Transporter J. Biol. Chem., February 11, 2000; 275(6): 4049 - 4054. [Abstract] [Full Text] [PDF]
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R. J. Boado, J. Y. Li, M. Nagaya, C. Zhang, and W. M. Pardridge Selective expression of the large neutral amino acid transporter at the blood-brain barrier PNAS, October 12, 1999; 96(21): 12079 - 12084. [Abstract] [Full Text] [PDF]
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A. Chairoungdua, H. Segawa, J. Y. Kim, K.-i. Miyamoto, H. Haga, Y. Fukui, K.'i. Mizoguchi, H. Ito, E. Takeda, H. Endou, et al. Identification of an Amino Acid Transporter Associated with the Cystinuria-related Type II Membrane Glycoprotein J. Biol. Chem., October 8, 1999; 274(41): 28845 - 28848. [Abstract] [Full Text] [PDF]
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D. P. Rajan, R. Kekuda, W. Huang, H. Wang, L. D. Devoe, F. H. Leibach, P. D. Prasad, and V. Ganapathy Cloning and Expression of a b0,+-like Amino Acid Transporter Functioning as a Heterodimer with 4F2hc Instead of rBAT. A NEW CANDIDATE GENE FOR CYSTINURIA J. Biol. Chem., October 8, 1999; 274(41): 29005 - 29010. [Abstract] [Full Text] [PDF]
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Y. Kanai, Y. Fukasawa, S. H. Cha, H. Segawa, A. Chairoungdua, D. K. Kim, H. Matsuo, J. Y. Kim, K.-i. Miyamoto, E. Takeda, et al. Transport Properties of a System y+L Neutral and Basic Amino Acid Transporter. INSIGHTS INTO THE MECHANISMS OF SUBSTRATE RECOGNITION J. Biol. Chem., June 30, 2000; 275(27): 20787 - 20793. [Abstract] [Full Text] [PDF]
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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]
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D. K. Kim, Y. Kanai, A. Chairoungdua, H. Matsuo, S. H. Cha, and H. Endou Expression Cloning of a Na+-independent Aromatic Amino Acid Transporter with Structural Similarity to H+/Monocarboxylate Transporters J. Biol. Chem., May 11, 2001; 276(20): 17221 - 17228. [Abstract] [Full Text] [PDF]
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A. Chairoungdua, Y. Kanai, H. Matsuo, J. Inatomi, D. K. Kim, and H. Endou Identification and Characterization of a Novel Member of the Heterodimeric Amino Acid Transporter Family Presumed to be Associated with an Unknown Heavy Chain J. Biol. Chem., December 21, 2001; 276(52): 49390 - 49399. [Abstract] [Full Text] [PDF]
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R. J. Reimer, F. A. Chaudhry, A. T. Gray, and R. H. Edwards Amino acid transport System A resembles System N in sequence but differs in mechanism PNAS, July 5, 2000; 97(14): 7715 - 7720. [Abstract] [Full Text] [PDF]
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