From the
A gene has been described, Down Regulated in Adenoma (dra), which is expressed in normal colon but is absent
in the majority of colon adenomas and adenocarcinomas. However, the
function of this protein is unknown. Because of sequence similarity to
a recently cloned membrane sulfate transporter in rat liver, the
transport function of Dra was examined. We established that dra encodes for a Na
Marked progress has been made in understanding the molecular
pathogenesis of colorectal cancer by exploring hereditary and acquired
genetic alterations
(1) . Another approach to understanding the
development of the malignant phenotype is to identify genes that are
differentially expressed in normal and neoplastic tissue. In this
regard, a gene was recently described that is expressed in normal
colonic tissue but is significantly decreased or absent in adenomas and
adenocarcinomas
(2) . Because of this pattern of expression, the
cDNA was named Down Regulated in Adenoma, or
dra. In the course of examining the expression of dra in normal human tissues, dra mRNA was found to be limited
to the colon, although small intestinal epithelial cells were not
tested
(2) . It is unclear whether the unique pattern of
expression of dra in normal and neoplastic colonic tissue is
related to the development or progression of neoplasia. Furthermore,
there has been no function assigned to the Dra protein. The original
analysis of the protein structure of Dra revealed three potential
nuclear-targeting sites, a potential homeodomain motif, and a possible
acidic transcriptional activation domain
(2) . This analysis
suggested that the Dra protein may be a transcription factor or a
protein that interacts with transcription factors.
The aim of the
present study was to determine the function of the protein encoded by
the dra cDNA. The achievement of this goal was aided by the
comparison of the sequence for Dra with nucleotide and protein data
bases. A computer analysis of the amino acid sequences of Dra with
these data bases revealed varying homology with a soybean nodulin clone
(GMAK170), Neospora crassa sulfate permease II (Cys-14)
(3) and sulfate anion transporter-1 (Sat-1) of rat
hepatocytes
(4) . These proteins represent membrane-spanning
transport molecules, and comparison of the putative hydrophobic
membrane-spanning domains suggested that Dra may be in a similar class
of molecule. Furthermore, Cys-14 and Sat-1 are known transporters of
sulfate ions
(3, 4) , which narrowed the possibilities
for the function of Dra. Recently, the gene responsible for diastrophic
dysplasia (called DTDST) was found to have homology to these sulfate
transporters as well
(5) . The aligned amino acid sequences for
Sat-1, Dra, and DTDST are illustrated in that publication
(5) .
To examine whether dra encodes a transport protein, we
utilized the Xenopus laevis oocyte expression system. These
functional studies demonstrated that dra does encode for a
Na
Sulfate transport has been well characterized in
the rat liver. In rat liver canalicular membrane vesicles, Meier et
al.(13) characterized a bicarbonate-sulfate exchanger
that, given the bicarbonate gradient that exists at the canalicular
lumen, would mediate the excretion of sulfate and other substrates.
Substrate specificity experiments demonstrated that thiosulfate and
oxalate and, to a lesser degree, succinate could substitute for
sulfate, but chloride, nitrate, phosphate, acetate, lactate, glutamate,
aspartate, bile acids, and reduced and oxidized glutathione were not
substrates for this transporter. Since furosemide and the disulfonic
derivatives SITS and DIDS also inhibited transport, sulfonated drugs as
well as sulfate-conjugated metabolites might represent substrates for
this transport process, although the latter were not examined. A
similar transport process was described in rat and rabbit renal
basolateral membrane vesicles
(16, 18) . Subsequently,
the rat liver canalicular sulfate carrier (sulfate anion transporter-1
or Sat-1) was functionally expressed
(19) and cloned
(4) using Xenopus oocytes. Northern hybridization
(4) and hybrid depletion experiments using antisense
oligonucleotides derived from the Sat-1 cDNA sequence
(20) demonstrated that rat renal basolateral
Na
Less is known
about sulfate transport in the small intestine, but both basolateral
and apical transport processes have been partially characterized.
Sulfate transport in rat and rabbit small intestinal basolateral
membranes, which is different from that in renal basolateral membranes,
has been described
(21, 22) . Although this basolateral
transport was DIDS- and oxalate-sensitive, chloride, and not
bicarbonate, exchanges for sulfate in this transport
process
(21, 22) . In addition to this transporter, a
sulfate:bicarbonate exchanger that also transports oxalate has been
identified on the basolateral membrane of rabbit ileal villus
enterocytes, but not crypt cells
(23) . Apical sulfate transport
(absorption) by both the kidney and the intestine was thought to be
mediated by an entirely different transport process,
Na
In
contrast to hepatic, renal, and ileal sulfate transport, colonic
transport of sulfate has not been examined in any systematic manner.
Accumulation of sulfate by mouse intestine is maximal in the area
adjacent to the ileocecal valve, and during development there is a
continuous increase in accumulation in the terminal ileum, whereas
accumulation diminishes in the remainder of the gut, including colon
(28). The colon, however, has been shown to be an important site for
oxalate absorption and is required for enteric hyperoxaluria to
occur
(29) .
Where might the Dra sulfate transporter fit in
the physiology of the intestinal and colonic ion transport? From the
present data, it is impossible to determine whether Dra is the apical
sulfate:OH exchanger, basolateral Cl
dra was
discovered because of the intriguing property of loss of expression in
neoplastic colon cells, which led to the speculation that it might
represent a tumor suppressor gene involved in colon
carcinogenesis
(2) . With the discovery that dra encodes
for a sulfate transporter, it is possible that its expression
represents a phenotypic characteristic of a mature colonocyte. In a
neoplastic cell, expression of this gene may be lost as the cell
reverts to a more immature phenotype. It is well described in colon
carcinomas that genes expressed in fetal colonocytes are re-expressed
in the cancer
cells
(30, 31, 32, 33, 34, 35) .
The developmental expression of dra that we have shown in mice
would be consistent with the hypothesis that a phenotype more close to
a fetal colonocyte might lack dra expression. In this
scenario, the dra gene would not necessarily have a functional
role in the acquisition of the malignant phenotype.
It is not
immediately obvious how the loss of a sulfate transporter protein would
be important in the development of a malignant cells. It is interesting
to note that sulfate transport activity in the HTC hepatoma cell line
is also markedly reduced
(36) . The identification of tumor
suppressor genes that are integral to the process of colon
carcinogenesis such as APC
(37) , which interacts with
catenins
(38) , and DCC, which is a member of the cell adhesion
molecule family of proteins
(39) , has emphasized the potential
importance of interactions of the cell with the extracellular
environment. The importance of a sulfate transporter in a
osteochondrodysplasia syndrome (diastrophic dysplasia) has shown that
alteration of sulfate transport may change the sulfation of
proteoglycans, which results in abnormal morphogenesis of
joints
(5) . In addition, it is known that proteoglycans in liver
and kidney tumors have decreased sulfation
(40, 41) . It
is possible that abnormal synthesis of sulfated proteoglycans may play
a role in colon cell functions as proteoglycans bind many growth
factors
(42) . Sulfation is also decreased in mucins associated
with colorectal cancer (43-45) and in the rectum of inflammatory
states such as ulcerative colitis
(46) . In lung fibroblasts
carrier-mediated sulfate transport at the plasma membrane is
rate-limiting for macromolecular sulfation
(47) . Further
investigation is required to determine the function of Dra in the colon
and whether loss of function of the sulfate transporter may be involved
in the development of the malignant phenotype or simply a lost function
that is of little consequence to the tumor cell.
-independent transporter for both
sulfate and oxalate using microinjected Xenopus oocytes as an
assay system. Sulfate transport was sensitive to the anion exchange
inhibitor DIDS (4,4`-diisothiocyano-2,2`disulfonic acid stilbene).
Using an RNase protection assay, we found that dra mRNA
expression is limited to the small intestine and colon in mouse,
therefore identifying Dra as an intestine-specific sulfate transporter.
dra also had a unique pattern of expression during intestinal
development. Northern blot analysis revealed a low level of expression
in colon at birth with a marked increase in the first 2 postnatal
weeks. In contrast, there was a lower, constant level of expression in
small intestine in the postnatal period. Caco-2 cells, a colon
carcinoma cell line that differentiates over time in culture,
demonstrated a marked induction of dra mRNA as cells
progressed from the preconfluent (undifferentiated) to the
postconfluent (differentiated) state. These results show that Dra is an
intestine-specific Na
-independent sulfate transporter
that has differential expression during colonic development. This
functional characterization provides the foundation for investigation
of the role of Dra in intestinal sulfate transport and in the malignant
phenotype.
-independent sulfate transporter with similarities
to the Sat-1 liver transporter. In addition, this study describes the
tissue distribution of dra mRNA and its unique developmental
expression in the mouse, as well as the differential expression of
dra in preconfluent and postconfluent Caco-2 cells. Our
results suggest that Dra is a functional transport protein that is
likely a characteristic of fully developed colon and that loss of
expression in colonic neoplasia may represent reversion to an earlier
developmental phenotype, although a role in the development of the
malignant phenotype remains a possibility.
RNA Isolation
Total RNA was extracted from mouse
tissues using a modification of the technique of Chirgwin et al. (6). Small amounts of tissue were homogenized, in a solution
containing 4.2 M guanidinium thiocyanate, 25 mM
sodium citrate, 17 mM sodium lauryl sarcosine, and 0.7%
2-mercaptoethanol, using a Brinkmann homogenizer. The homogenate was
layered on top of 4 ml of a 5.7 M cesium chloride cushion
buffered with 25 mM sodium acetate, and RNA was separated by
ultracentrifugation (28,000 rpm, 20 °C, 18 h). The RNA pellet was
desolved in 0.3 M sodium acetate (pH 5) and
ethanol-precipitated twice, then resuspended in water treated with
diethyl pyrocarbonate (DEPC).(
)
In some
experiments RNA, from tissues and cell lines, was extracted as
described by Chomczynski and Sacchi
(7) and as described
previously (8). RNA from CaCo-2 cells was extracted at days 4 and 18
after plating for preconfluent and postconfluent cells, respectively.
Cloning of the Human dra cDNA and Portion of the Mouse
cDNA
Total RNA isolated from human ileum was reverse transcribed
into complimentary DNA (cDNA) using oligo(dT) (0.5 µg) and MMLV
reverse transcriptase, as described previously
(9) with minor
modifications. The MMLV reverse transcriptase and RNA were incubated at
37 °C for 1 h. The cDNA obtained was amplified by PCR using Taq polymerase with specific primers to the 5` and 3` ends of the
total human cDNA
(2) containing a 5` eukaryotic start and 3`
stop translation site based on those described by Cavener and
Ray
(10) : primer 1,
5`-CGCGTTAAGCGGCCGCACCATGATTGAACCCTTTGGGAATCA-GTAT-3`; primer 2,
5`-CGCGTTAAGCGGCCGCGATTAGAATTTTGTTTCAACTGGCACC-3`. NotI
restriction enzyme sites were incorporated into the primers and the PCR
products were cloned into Bluescript KS (Stratagene)
after digestion of the vector and PCR products with NotI. The
resulting 2300-bp cDNA was sequenced as described by the manufacturer
(Sequenase 2.0, U. S. Biochemical Corp.), and this was compared to the
published sequence
(2) . Total RNA isolated from adult mouse
colon was reverse transcribed into complimentary DNA (cDNA) using
random hexamer primers (125 pmol) and MMLV reverse transcriptase. The
cDNA was amplified by PCR with specific primers to dra from
the human sequence +1427 to +1867 from the start of
translation
(2) , using Taq polymerase (primer 1:
5`-TCATCTTCACCATTGTCCTGGG-3`; primer 2, 5`-TGGTATTGATTGGCTGGTCCAG-3`).
The 441-bp PCR product was cloned into Bluescript KS
(Strategene) by TA cloning (the vector was prepared according to
the protocol of Marchuk et al.(11) ). The cDNA clone
was sequenced utilizing the T3 and T7 primers of Bluescript
(Sequenase).
In Vitro Transcription of dra cDNA
dra plasmid (5 mg) was linearized with XbaI at the 3` end.
After phenol/chloroform extraction and ethanol precipitation, capped
mRNA was synthesized using T7 RNA polymerase in the presence of the
capping analog mG(5`)ppp(5`)G using a Promega Ribomax large
scale RNA system T7. Unincorporated nucleotides were removed with
Chromaspin-30 columns (Clontech), and synthesized mRNA was recovered by
ethanol precipitation, followed by resuspension in DEPC/water for
oocyte injection at a concentration of 10 ng/ml.
RNase Protection Assay
The RNase protection assay
was performed essentially as described previously
(8) .
Single-stranded P-labeled probe for mouse dra was
synthesized from pKS-dra linearized with XbaI, using
T3 RNA polymerase (Promega) and [
P]CTP (3000
Ci/mol). This yielded a probe of 441 bp in length. After synthesis, the
P-labeled RNA probe was purified by digestion with
RNase-free DNase I, extracted with phenol and chloroform, and
precipitated with ethanol. Solution hybridization was performed for 16
h at 45 °C in a solution containing 10 µg of total RNA, 1
10
cpm of probe, 80% formamide, 40 mM
PIPES (pH 6.4), 0.4 M NaCl, and 1 mM EDTA. Samples
were then digested with RNase A and T1 for 60 min at 15 °C,
extracted with phenol:chloroform (1:1), and precipitated with ethanol.
The pellets were resuspended in loading buffer (80% formamide, 0.1%
bromphenol blue, 0.1% xylene cyanol, and 1 mM EDTA, pH 8.0)
and separated in a 6% denaturing polyacrylamide gel.
Northern Analysis
Samples of total RNA were
size-separated by electrophoresis in 2.2 M formaldehyde, 1%
agarose gels. The RNA was transferred to Hybond-N (Amersham) nylon
membranes, UV cross-linked, and hybridized. The cloned mouse dra fragment was labeled with Klenow enzyme (Boehringer Mannheim)
using [P]dGTP and hybridized following the
protocol of Virca et al.(12) .
Oocyte Isolation and Injection
Mature X.
laevis females were purchased from Xenopus I, Inc. (Ann Arbor,
MI). Frogs were anesthetized by immersion for 15 min in ice-cold water
containing 0.3% 3-aminobenzoic acid ethyl ester. Oocytes were removed
and incubated at room temperature for 3 h in a solution containing, in
mM, 98 NaCl, 2 KCl, 1 MgCl, and 5 HEPES/NaOH (pH
7.5) supplemented with 2 mg/ml collagenase (Life Technologies, Inc.).
Oocytes were then washed with frog Ringer's (in mM, 98
NaCl, 2 KCl, 1.8 CaCl
, 1 MgCl
, and 5 HEPES/NaOH
(pH 7.5)), and stage V and VI oocytes were selected. After overnight
incubation at 18 °C in frog Ringer's supplemented with
penicillin (500 units/ml) and streptomycin (100 µg/ml), healthy
oocytes were injected with 500 pg of mRNA derived from in vitro transcription of dra or DEPC-treated water (total volume:
50 nl). Subsequently, oocytes were cultured for 4 days at 18 °C
with a daily change of antibiotic-supplemented frog Ringer's.
Transport Assay
[S]Sulfate
(carrier-free) and [
C] oxalate were obtained
from DuPont NEN. For sulfate uptake studies, 12-25 oocytes were
washed once in a sodium-free and sulfate-free uptake solution (in
mM, 100 choline chloride, 2 KCl, 1 CaCl
, 1
MgCl
, 10 HEPES/Tris, pH 7.5). The oocytes were then
incubated in 500 µl of uptake solution containing 1 mM
[
S] sulfate (20 µCi/ml) for the designated
time interval. Uptake was stopped by washing the oocytes three times
with ice-cold uptake solution containing 5 mM
K
SO
. Individual oocytes were then transferred
to scintillation vials, dissolved in 0.5 ml of 10% sodium dodecyl
sulfate, and after addition of scintillation fluid, oocyte-associated
radioactivity determined in a Beckman LS 1801 liquid scintillation
counter. For oxalate uptake studies, uptake solution contained, in
mM, 100 choline chloride, 2 KCl, and 10 HEPES/Tris, pH 7.5;
oocytes were incubated in 500 µl of uptake solution containing 1
mM [
C] oxalate (2 µCi/ml); and
uptake was stopped by washing the oocytes three times with ice-cold
uptake solution containing 5 mM oxalate.
RESULTS
Cloning of the Human cDNA and a Portion of the Mouse
cDNA of dra
Initially, we cloned the human dra cDNA
from the known sequence and a portion of the mouse cDNA for use in
expression studies in mice. First, RNA isolated from normal human ileum
was reverse transcribed and the complete translated region of the cDNA
was cloned by PCR using amplification primers based on the published
sequence
(2) . To obtain a clone with an efficient eukaryotic
start and stop translation site, we modified the primers according to
those described by Cavener and Ray
(10) . After sequencing, the
cDNA had the identical sequence to the previously reported
clone
(2) . A 441-bp fragment of the mouse cDNA of dra was cloned from normal mouse colonic RNA using the reverse
transcription-polymerase chain reaction. The mouse dra cDNA
corresponded to bases +1427 to +1867 of the human cDNA
(numbered from the translational start site) and contained the 12th and
last putative membrane-spanning domain
(3) . The murine and human
cDNAs, in this region, had 88% identity in the amino acid sequence.
Function of dra in Xenopus Oocytes
On the basis of
significant homology between the Dra sequence and the recently cloned
rat canalicular sulfate transporter Sat-1
(4) , RNA derived from
in vitro transcription of dra was injected into a
Xenopus oocyte expression system and sulfate transport
activity assayed. Na-independent
[
S] sulfate uptake into RNA-injected oocytes was
significantly greater than uptake in water-injected oocytes
(Fig. 1). Moreover, the expressed Na
-independent
[
S] sulfate uptake was sensitive to the anion
exchange inhibitor, 4,4`-diisothiocyano-2,2`-disulfonic acid stilbene
(DIDS) and oxalate (Fig. 2), exhibiting a cis-inhibition
pattern similar to that for Sat-1
(4) . Since oxalate appears to
be a substrate for hepatic
(13) ,
intestinal
(14, 15) , and renal sulfate transporters
(16), and showed cis-inhibition of sulfate transport
(Fig. 2), oxalate transport was also directly examined in
RNA-injected oocytes. Na
-independent
[
C]oxalate uptake into RNA-injected oocytes was
also significantly greater than uptake in water-injected oocytes
(Fig. 3).
Figure 1:
Time course of sulfate uptake in
Xenopus oocytes. Oocytes were injected with either 500 pg cRNA
() or DEPC-treated water (
) (total volume: 50 nl). Four days
after injection, uptake of 1 mM [
S]
sulfate was determined at 25 °C over 2 h. Uptake values represent
the means ± S.E. of four separate oocyte preparations using
8-12 oocytes for each determination. **, p <
0.005.
Figure 2:
cis-Inhibition of sulfate uptake
in Xenopus oocytes. Oocytes were injected with either 500 pg
cRNA () or DEPC-treated water (
) (total volume: 50 nl). Four
days after injection, 2-h uptake of 1 mM
[
S] sulfate was determined at 25 °C in the
presence or absence of 1 mM DIDS or 5 mM oxalate.
Uptake values represent the means ± S.E. of 13-28
determinations from two separate oocyte preparations. *, p < 0.05.
Figure 3:
Time course of oxalate uptake in
Xenopus oocytes. Oocytes were injected with either 500 pg cRNA
() or DEPC-treated water (
) (total volume: 50 nl). Four days
after injection, uptake of 1 mM [
C]
oxalate was determined at 25 °C over 3 h. Uptake values represent
the means ± S.E. of three separate oocyte preparations using
8-12 oocytes for each determination. **, p <
0.005.
Tissue-specific Expression of dra in the Mouse
In
the initial report of the cloning of dra, expressed mRNA was
found only in the colon when examined in several human
tissues
(2) . To examine the distribution of expression more
completely and to directly compare the small intestine to the colon, an
RNase protection assay of RNA isolated from different mouse tissues was
performed using the 441-bp mouse cDNA as a probe. The expression of
dra was limited to the small intestine and colon
(Fig. 4). In the small intestine, there was low expression of
dra along the entire length of the intestine. The most intense
expression was found in the cecum with a proximal to distal gradient in
the colon. Importantly, there was no expression in multiple other mouse
tissues.
Figure 4:
Tissue-specific expression of dra in the mouse. RNA extracted from tissues of adult mice were
analyzed by RNase protection assay. The arrow reflects the
probe binding to dra. Only intestinal tissues express DRA with
the highest level of expression seen in the
cecum.
Expression of dra in Developing Normal Mouse
Tissue
Because of its differential expression in normal and
neoplastic colonic tissue, we examined dra expression during
intestinal development in the mouse. Since many important developmental
events in the mouse small intestine and colon occur after birth, RNA
was isolated from the small intestine and colon of normal mice at
postnatal day 0, day 9, and adult. A Northern blot was prepared from
the RNA and probed with the 441-bp cDNA fragment of dra. In
the colon, a low level of dra mRNA was present at birth, with
a marked increase observed during postnatal development (Fig. 5).
In contrast, dra mRNA remained at a low constant level
throughout postnatal development in the small intestine. Quantification
of the increase in colonic expression revealed that there was a
134-fold increase in expression in the adult colon as compared to the
newborn mouse.
Figure 5:
Expression of DRA in developing
normal mouse tissue. RNA extracted from postnatal day 0, day 9, and day
40 colon and small intestine were characterized by Northern blot
analysis. The membrane was hybridized with a 441-bp mouse cDNA probe.
The probe identified an mRNA, at the expected size for dra, of
3.2 kilobases. dra expression is minimal at birth in the colon
and increases with development. Small intestine expression of dra is present at birth and continues to
adulthood.
Differential Expression of dra in Preconfluent and
Postconfluent Caco2 Cells and Other Tumor Cell
Lines
Postconfluent Caco-2 cells are a model for differentiated
enterocytes
(17) and as such are a potential system to evaluate
differentiation in tissue culture. Northern analysis of Caco-2 cell RNA
revealed a band of 2.8 kilobases, the predicted size of dra mRNA, in the normal human ileum control and only in the
postconfluent cells. There was also a smaller unidentified band in the
postconfluent lane (Fig. 6). In addition, the Colo DM cells,
another human colon cancer cell line, do not express dra. Hep
G2 cells, derived from a hepatoma, do not express dra at the
expected size, but a smaller transcript is identified.
Figure 6:
Differential expression of DRA in
preconfluent and postconfluent Caco-2 cells and other tumor cell lines.
RNA was extracted from Caco-2 cells at day 4 (preconfluent) and day 18
(postconfluent) from plating, HepG2 cells, Colo DM cells, and human
small intestine. The RNA was characterized by Northern blot analysis
with a 441-bp cDNA probe from mouse dra. The postconfluent
Caco-2 cells express dra with an identical size transcript
(3.2 kilobases) as the human small intestine. There is also a small
transcript of unknown identity, which also appears to be present in
HepG2 cells. The Colo DM cells do not express
dra.
DISCUSSION
We demonstrate that dra encodes for a protein that
mediates Na-independent, DIDS- and oxalate-sensitive
sulfate uptake and conclude that Dra is an intestine-specific sulfate
(and oxalate) transporter which is expressed predominantly in the
colon. Thus, the similarity in amino acid sequence shared by Dra and
Sat-1
(4, 5) is also reflected in a functional
similarity for transported molecules. These findings make it highly
likely that the recently described gene that is responsible for
diastrophic dystrophy, which also has sequence similarity to Sat-1 and
Dra, is a functional sulfate transporter
(5) . In fact, sulfate
transport is defective in fibroblasts from patients with this inherited
disease. The mRNA for the putative sulfate transporter that is mutated
in diastrophic dystrophy is expressed in many cells and
tissues
(5) . Therefore, it appears that there will be multiple
sulfate transporters, some of which are highly restricted to specific
tissues and others that serve a more generalized cellular function.
What is the potential role of dra in the intestine, and what
is the significance of its pattern of expression in colonic neoplasia
and development?
-independent sulfate transport activity is closely
related to Sat-1. No hybridization signal was detected in duodenum,
ileum, proximal colon, or distal colon
(4) .
:sulfate cotransport, which has also been cloned
recently
(20, 24) . However, demonstration that
sodium-stimulated sulfate uptake in the intestine exhibited sensitivity
to pH
(25, 26) led to the characterization of a
sulfate:OH exchanger in rabbit ileal brush-border membrane
vesicles
(14, 27) . Sulfate:OH exchange and
Na
:sulfate cotransport were shown to be separate
carriers on the basis of inhibition studies
(14) . The substrate
specificity of intestinal brush-border sulfate:OH exchange is similar
to that described for canalicular bicarbonate-sulfate exchange in that
probenecid, DIDS, and oxalate cis-inhibit sulfate uptake (15);
however, superimposition of a bicarbonate gradient does not further
stimulate sulfate uptake over a pH gradient alone
(27) .
:sulfate, or
basolateral sulfate:HCO
exchanger.
Moreover, since these transporters were characterized in the ileum, it
is possible that Dra represents a transport process that has not as yet
been physiologically characterized. Since all the characterized
transport processes have similar kinetic features and substrate
specificities, determination of the potential role of Dra will have to
await development of specific antibodies and immunolocalization
studies. Once the protein is localized to either the apical or
basolateral membrane, the Caco-2 cell line may prove a useful model to
examine its function. Furthermore, expression in eukaryotic cells will
be necessary to study its transport properties.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.