From the
Department of Environmental Medicine,
University of Rochester School of Medicine, Rochester, New York 14642,
Mount Desert Island Biological Laboratory,
Salsbury Cove, Maine 04672, and the ¶Department
of Medicine and Liver Center, Yale University School of Medicine, New Haven,
Connecticut 06520
Received for publication, January 31, 2003 , and in revised form, April 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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Using a comparative approach, a novel type of organic solute and steroid
transporter was recently identified in the liver of an evolutionarily ancient
vertebrate, the little skate Raja erinacea
(8). In contrast to all other
organic anion carriers, this skate transporter is generated by co-expression
of two distinct and novel gene products, Ost and
Ost
.1 Substrates
for this multispecific transporter include estrone 3-sulfate, taurocholate,
digoxin, and prostaglandin E2. Interestingly, the overall predicted
membrane topology of skate Ost
-Ost
is similar to that of the
heterodimeric sensory rhodopsins, suggesting that Ost
-Ost
may
have evolved from an ancestral rhodopsin-like molecule but has acquired the
ability to transport steroids and eicosanoids, compounds that also function as
ligands for some G-protein-coupled receptors.
Initially, Ost and Ost
orthologues were not
identified in the human genome or in any other sequenced genomes, indicating
that these genes might be specific to marine elasmobranchs. However, sequences
for hypothetical human and mouse proteins have recently been entered into the
data bases that exhibit 2541% predicted amino acid sequence identity
with skate Ost
and Ost
(see "Results"). The present
study tested whether these mammalian genes are expressed, whether they encode
for orthologues of the skate gene products, and if so, whether they
functionally complement the transport activity of one another.
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EXPERIMENTAL PROCEDURES |
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Cloning StrategyA multistep RT-PCR-based strategy was
employed to obtain cDNAs for the putative open reading frames (ORFs) of
hypothetical proteins OST (GenBankTM/EBI accession number
CAC51162
[GenBank]
), OST
(GenBankTM/EBI accession number XP_058693
[GenBank]
), and
mOST
(GenBankTM/EBI accession number XP_134984/AY279396).
Oligonucleotide primers were designed to generate ORFs for each gene based on
the cDNA sequences listed on GenBankTM
(Table I). RT-PCR products of
predicted sizes were created for putative hOST
,
hOST
, and mOst
using either human or mouse liver
poly(A)+ RNA as a template (Clontech). The reaction products were
isolated via agarose gel electrophoresis and ligated into the pCR-II TOPO
vector (Invitrogen) utilizing Invitrogen's TOPO TA cloning kit. The resulting
plasmids were used to transform DH5
Escherichia coli, from
which positive clones were selected for ampicillin resistance and blue/white
staining. Upon isolation and identification, a single clone expressing the new
plasmid was used as a template in a PCR with primers designed to generate a
final product flanked by two unique restriction sites
(Table I). Following isolation
and enzymatic digestion, the cDNAs with sticky ends were directionally ligated
into the pSP64 poly(A) vector. These plasmids then were used to transform
DH5
E. coli, and positive clones were selected for ampicillin
resistance. Plasmid purifications were completed using Promega's DNA Wizard
kit.
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The cDNA for the hypothetical mOst (GenBankTM/EBI
accession numbers BC024441
[GenBank]
.1 and AAH25912
[GenBank]
) was obtained from the American Type
Culture Collection (ATCC #6994476). This clone was used as a template in a PCR
to generate the ORF for GenBankTM sequence BC024441
[GenBank]
.1. The primers were
designed to add a HindIII site to the 5' end and an
XbaI site to the 3' end of the PCR product
(Table I). The PCR product was
purified by agarose gel electrophoresis, and the band of expected size (1060
bp) was excised and enzymatically digested with HindIII and
XbaI. The resulting cDNA sequence was then directionally cloned into
the pSP64 poly(A) vector and used to transform DH5
E. coli. A
positive clone was selected, and the plasmid was isolated with Promega's DNA
Wizard miniprep kit.
Final clones for all four genes were sequenced in both directions with a series of specific oligonucleotide primers at the Mount Desert Island Biological Laboratory DNA Sequencing Core (Salsbury Cove, ME) to confirm sequence identity. The plasmids were then linearized with EcoRI and used to synthesize cRNA via Ambion's mMessage mMachine kit for injection into Xenopus oocytes.
Real Time Quantitative PCR to Determine Tissue Distribution and
Expression Levels of hOST and hOST
in 19 Human
Tissue cDNAsHuman tissue cDNAs were purchased from Origene (human
Sure-RACETM panel). Synthetic oligonucleotide primers were designed to
amplify portions of hOST
, hOST
, and human
-actin for use in quantitative real time PCR
(Table I). Reactions were
conducted and analyzed on a Roto-Gene 2000 real time light cycler from Corbett
Research (Phenix Corporation, Hayward, CA). Qiagen's QuantiTect Sybr Green
quantitative RT-PCR kit was used for PCR analysis. Expression levels are
reported as a ratio to
-actin within each tissue examined.
Xenopus Oocyte Preparation, Microinjection, and Transport Assays Isolation of Xenopus oocytes was performed as described by Goldin (9) and employed previously in our laboratory (1012). Stage V and VI defolliculated oocytes were selected and incubated at 18 °C in modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, and 20 mM HEPES-Tris, pH 7.5) supplemented with penicillin (100 units/ml) and streptomycin (100 µg/ml). The oocyte medium was changed daily, and healthy oocytes, defined as those with a clean brown animal pole and a distinct equator line, were selected for experiments. Transport studies were performed as described previously (10, 11).
DNA Sequence and Hydropathy AnalysisDouble-stranded cDNA clones were sequenced in both directions at the Mount Desert Island Biological Laboratory DNA Sequencing Core. The full-length sequences for all clones were obtained using synthetic oligonucleotide primers. Sequence analysis was performed with the DNA and protein sequence analysis program Lasergene from DNAStar Inc. (Madison, WI). Membrane topology and putative membrane-spanning domains were determined by hidden Markov model analysis (www.enzim.hu/hmmtop) and by Kyte Doolittle hydropathy analysis (13). The presence of possible signal peptides was evaluated with the SignalP V1.1 program (www.cbs.dtu.dk/services/).
Synthesis of FLAG Epitope-tagged Skate and Human OST
and of c-MYC-tagged Skate and Human OST
The FLAG
epitope (DYKDDDDK) was added in-frame to the 3' end of skate and human
OST
cDNA, and the c-MYC epitope (EQKLISEEDL) was added to the
3' end of skate and human OST
cDNA. Constructs were
created via PCR using skate and human OST
and
OST
cDNA as templates with oligonucleotide primers containing
the FLAG or c-MYC sequences flanked by unique restriction sites. PCR products
of predicted size were identified by agarose gel electrophoresis, excised, and
purified with Qiagen's agarose gel purification kit. The isolated fragments
were subcloned into the pSP64 poly(A) vector, and that vector was used to
transform DH5
E. coli. Positive clones were grown and plasmid
DNA isolated using Promega's DNA Wizard plasmid isolation kit. After
restriction digestion and sequencing to establish clone identity, cRNA was
prepared for each of the four constructs using Ambion's mMessage mMachine
kit.
Immunofluorescence Labeling of Intact OocytesIntact Xenopus oocytes on day 3 after cRNA injection were fixed in methanol:acetone (1:1) for 10 min on ice, followed by four washes of 5 min each at room temperature and an overnight wash at 4 °C in antibody dilution buffer (0.01 M PBS + 0.05% Tween 20, 1% bovine serum albumin, 1% normal goat serum, and 0.01% sodium azide). Oocytes were washed at room temperature with 1x PBS + 1% Tween 20 for 1 h with 10-min buffer changes followed by a 15-min incubation in blocking solution (3% bovine serum albumin in antibody dilution buffer). Oocytes were then washed with 1x PBS + 1% Tween 20 for another 1 h with 10-min buffer changes followed by an overnight wash at 4 °C. The oocytes were incubated with either anti-FLAG M2 monoclonal antibody (Sigma; 4.9 mg/ml, diluted 1:200 with antibody dilution buffer) or monoclonal anti-c-MYC Cy3-conjugated antibody (Sigma; 1.2 mg/ml, diluted 1:100 with antibody dilution buffer) for 1 h. To remove excess antibody, oocytes were washed in 1x PBS + 1% Tween 20 three times for 5 min each, two times for 10 min each, and then overnight. The oocytes labeled with the anti-FLAG M2 monoclonal antibody were incubated with an Alexa fluor 488 F(ab')2 fragment of goat anti-mouse antibody in the dark for 1 h (Molecular Probes; 2 mg/ml, diluted 1:200). Secondary antibody was removed by washing the oocytes with 1x PBS + 1% Tween 20 three times for 5 min each, two times for 10 min each, and then overnight. Cells were imaged using a x10 objective on a Leica TCS-SP laser-scanning confocal microscope.
Statistical AnalysisKinetic data from experiments measuring uptake of radiolabeled substrate were fit to the Michaelis-Menten equation by nonlinear least squares regression analysis. Vmax and Km values with standard errors were derived from these curves. Comparison of data measuring initial rates of uptake of radiolabeled substrates in the presence and absence of inhibitors was performed by unpaired Student's t test and correlated to p < 0.05.
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RESULTS |
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Likewise, possible human and mouse orthologues of skate Ost were
recently entered into the data bases, but these sequences exhibit only
2529% predicted amino acid identity with skate Ost
(Fig. 2). The hypothetical
human protein with GenBankTM sequence XP_058693
[GenBank]
and the hypothetical
mouse protein with GenBankTM sequence XP_134984/AY279396 exhibit 25 and
29% predicted amino acid identity with skate Ost
, respectively
(Fig. 2). These hypothetical
human and mouse proteins exhibit 62.5% amino acid identity with each other,
both encoding for proteins containing 128 amino acids with a putative single
TM domain (Fig. 2).
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An RT-PCR-based strategy was employed to obtain cDNAs for the predicted
ORFs of putative human OST and OST
using human
liver mRNA and of putative mouse Ost
using mouse liver mRNA.
The cDNA for the putative mouse Ost
was obtained from the
American Type Culture Collection (ATCC #6994476). Sequence analysis of the
synthesized cDNAs for the four genes indicated that both mouse clones were
identical to GenBankTM sequences, whereas the human sequences varied by a
single nucleotide in the reading frame of each gene. Both differences in the
human gene sequences result in amino acid substitutions (Figs.
1 and
2; GenBankTM/EBI accession
numbers AY194243
[GenBank]
and AY194242
[GenBank]
). It is unclear whether the observed differences
are due to polymorphisms or whether they result from mutations introduced
during PCR. They are unlikely to be sequence artifacts as both occur in
regions of strong sequence data. When compared with the published human
genomic DNA data base
(www.ncbi.nlm.hih.gov:80/BLAST/),
our human OST
sequence matches perfectly, whereas the
GenBankTM cDNA sequence for the protein with GenBankTM/EBI accession
number CAC51162
[GenBank]
contains an adenine at the base in question rather than a
guanine (position 604 of the ORF). This difference predicts a valine at
position 202 of human OST
(circled in
Fig. 1), whereas the
GenBankTM sequence CAC51162
[GenBank]
predicts an isoleucine. In contrast, our
human OST
sequence differs from the genomic DNA sequence at the
base in question, containing a cytosine at nucleotide 197 of the ORF instead
of an adenine. As a result, the sequence for human OST
obtained in the
present study predicts a threonine at position 66 (circled in
Fig. 2), whereas the
GenBankTM sequence XP_058693
[GenBank]
predicts a lysine. Because the human liver
poly(A)+ RNA used as a template for RT-PCR in the present study was
pooled from four different people, the reaction product containing the inserts
used for cloning human OST
was sequenced. Upon analysis it was
determined that an equal distribution of adenine and cytosine occurred at this
nucleotide position (data not shown), indicating that this may be a naturally
existing polymorphism.
A search of the human genomic data base
(www.ncbi.nlm.hih.gov:80/BLAST/)
revealed that human OST is located on chromosome 3 and is
coded by nine exons in the 3q29 region, whereas human OST
is on
chromosome 15 and is coded by four exons located in the 15q21 region. Mouse
Ost
is located on chromosome 16 and is coded by nine exons at
16B2, whereas mouse Ost
is found on chromosome 9 and is coded
by four exons located at 9C.
The Human and Mouse Proteins Function as Organic Solute
TransportersTo assess whether the human and mouse proteins
function as organic solute transporters, uptake of [3H]estrone
3-sulfate was measured in X. laevis oocytes injected with cRNA
synthesized from the human, mouse, or skate genes. As expected, co-expression
of skate Ost and Ost
was required to generate transport activity
(Fig. 3A)
(8). When the putative human
OST
and mouse OST
were expressed individually in oocytes, no
transport activity was detected (Fig.
3A); however, when these proteins were co-expressed with
skate Ost
, a strong transport signal was obtained
(Fig. 3A), indicating
that human OST
and mouse OST
can functionally complement the
corresponding skate protein. Likewise, human OST
and mouse OST
did
not induce transport activity when expressed individually in oocytes but
generated a functional transporter when co-expressed with skate Ost
(Fig. 3B). Thus, the
human and mouse proteins not only are functional orthologues of the skate
proteins but also are able to complement each other across species. Moreover,
co-expression of the two human (OST
-OST
) or the two mouse
(mOST
-mOST
) proteins generated a very strong transport signal, as
did the human-mouse
-
pairs
(Fig. 3C).
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Characteristics of Human OST-OST
- and
Mouse OST
-OST
-mediated
TransportOocytes injected with human OST
and
OST
cRNA (1 ng each) or with mouse Ost
and
Ost
cRNA (1 ng each) were able to transport taurocholate,
estrone 3-sulfate, digoxin, and prostaglandin E2 but not estradiol
17
-D-glucuronide or p-aminohippurate
(Fig. 4), indicating that this
transport system is multispecific and that it may participate in cellular
uptake of conjugated steroids and eicosanoids. This substrate profile is
similar to that of skate Ost
-Ost
(8).
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The skate, mouse, and human transporters shared a number of other features
as well. Transport was sodium-independent
(Fig. 5), saturable
(Fig. 6), and inhibited by bile
salts, steroids, and other organic anions
(Table II). Replacement of the
NaCl in the oocyte incubation medium with either choline chloride or lithium
chloride had no effect on estrone 3-sulfate uptake
(Fig. 5), indicating that
transport is not coupled to the sodium electrochemical gradient. Initial rates
of estrone 3-sulfate uptake into human OST-OST
- or mouse
OST
-OST
-expressing oocytes were saturable, although the apparent
Michaelis constants (Km) were relatively high
(320 ± 30 µM and 290 ± 24 µM,
respectively; Fig. 6). The
Km for estrone 3-sulfate uptake by skate
Ost
-Ost
is lower (85 µM)
(8). Uptake of
[3H]estrone 3-sulfate in hOST
-hOST
- and
mOST
-mOST
-expressing oocytes was inhibited by a variety of bile
salts, steroids, and other organic anions
(Table II). As reported
previously for skate Ost
-Ost
(8), [3H]estrone
3-sulfate uptake was inhibited by sulfated steroids, including lithocholic
acid sulfate and taurolithocholic acid sulfate
(Table II).
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Tissue Distribution of Human OST and OST
mRNA OST
and OST
mRNA levels were
measured in 19 human tissue cDNAs and were expressed relative to
-actin
mRNA levels using quantitative real time PCR analysis. The results revealed
that OST
and OST
are widely expressed in human
tissues (Fig. 7). Tissues that
had high levels of OST
mRNA generally also had high levels of
OST
mRNA, indicating coexpression of these genes. Relatively
high levels of both mRNAs were found in testis, colon, liver, small intestine,
kidney, ovary, and adrenal gland (Fig.
7A); and lower levels were measured in heart, lung,
brain, pituitary, thyroid gland, uterus, prostate, mammary gland, and fat
(Fig. 7B). The mRNA
for OST
and OST
was below our limit of
detection in skeletal muscle and peripheral blood leukocytes (data not
shown).
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Trafficking of OST and OST
to the Plasma
MembraneTo gain insight into the mechanism by which OST
and
OST
interact to generate a functional transporter, epitope-tagged
constructs were synthesized, and these constructs were tested for functional
activity (Fig. 8) and cellular
localization (Fig. 9) in
Xenopus oocytes. The FLAG epitope was added in-frame to the 3'
end of skate and human OST
cDNA, and the c-MYC epitope was
added to the 3' end of skate and human OST
cDNA. As
expected, there was no transport activity when the individual epitope-tagged
proteins were expressed in oocytes; however, when human or skate
-FLAG
and
-c-MYC were co-expressed, there was strong estrone 3-sulfate
transport activity (Fig. 8).
Interestingly, immunofluorescence analysis revealed that OST
-FLAG and
OST
-c-MYC were able to reach the plasma membrane when expressed either
individually or in pairs (Fig.
9). Thus, the inability of the individual proteins to generate
transport activity does not appear to be due to impaired trafficking to the
cell surface.
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DISCUSSION |
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Identification of OST and OST
was made
possible by the recent cloning of orthologous genes from the liver of an
evolutionarily ancient marine vertebrate, the little skate
(8). Surprisingly, when skate
Ost
and Ost
genes were identified in 2001,
comparable genes were not present in any of the sequenced genomes, including
the human genome, suggesting that they may be unique to elasmobranchs
(8). However, because the human
genome remains in draft form, many genes have not yet been discovered.
Hypothetical human and mouse genes that recently were entered into the data
bases are predicted to encode proteins exhibiting 2541% amino acid
identity with skate Ost and Ost
(Figs.
1 and
2). Although this level of
amino acid identity is low, it is not insignificant given an evolutionary
distance of 200 million years between skates and humans. Thus, despite the
modest level of amino acid identity, these skate and mammalian proteins may be
carrying out the same biological functions. To test this possibility, the
present study assessed whether these hypothetical human and mouse genes are
expressed in human and mouse liver, and if so, whether they function as
organic solute transporters. Our results demonstrate not only that human
OST
-OST
and mouse Ost
-Ost
are orthologues of skate
Ost
-Ost
but also that these proteins are able to functionally
complement one another across species. That is, the 7-TM domain OST
proteins from humans, mice, or skates are able to partner with any of the
OST
proteins from these three species to generate a functional
transporter. Cross-species complementation indicates a high degree of
functional conservation throughout evolution.
The mechanism by which OST and OST
interact to generate
transport activity is unknown, although the present results indicate that
co-expression is not required for trafficking to the cell surface
(Fig. 9). OST
and
OST
were able to reach the plasma membrane when expressed individually
in oocytes; however, they were not functional
(Fig. 8). These findings argue
against a chaperone function and suggest that either one protein may play a
regulatory role or the two proteins may be forming a heterodimer or
hetero-oligomers. The present results also indicate that comparable levels of
transport activity are generated by proteins that exhibit only 25% amino acid
identity, suggesting that only a few conserved amino acids may be required for
this interaction. For example, human OST
was activated equally well by
human OST
or skate Ost
(Fig.
3) despite only a 25% amino acid identity for the latter two
proteins (Fig. 2).
Alternatively, the interaction between OST
and OST
may be
determined more by their three-dimensional structures or post-translational
modifications than by primary amino acid sequences or may require the
participation of a third, as yet unidentified protein or cofactor.
Interestingly, the predicted transmembrane domain architectures of human,
mouse, and skate OST are similar, as are those of the three OST
proteins (Figs. 1 and
2), providing additional
evidence that these proteins carry out the same biological functions. Each of
the predicted TM domains and hydrophilic loops in OST
and OST
from human, mouse, and skate are similar in length and relative position
within the polypeptides (Figs.
1 and
2). The only significant
exception is skate Ost
, which has a longer amino terminus region;
however, the first 27 amino acids of skate Ost
are predicted to be a
signal peptide (8) such that
the mature protein may be comparable in length to the human and mouse
proteins. The conserved membrane architecture between these evolutionarily
divergent proteins indicates that this membrane structure is essential for
function.
As indicated above, the amino acid identity between the human, mouse, and
skate proteins is not restricted to the TM helices but is also seen in
putative intracellular and extracellular loops (Figs.
1 and
2). For OST, several
amino acid regions appear highly conserved in the hydrophilic loops, including
an unusual stretch of 67 cysteine residues that reside in a predicted
cytosolic loop between TM domains 3 and 4 (TGPCCCCCPC(C/L)P;
Fig. 1). The significance of
this cysteine motif in OST
is not known, although it may function
either as a ligand or substrate binding site, a site of interaction with
OST
, or as a site of membrane association. In general, cysteine residues
play important roles in protein secondary structure, metal coordination,
oligomerization, and post-translational modifications. Proteins that contain
comparable cysteine-rich motifs include the human t-SNARE protein syntaxin 11
(GenBankTM/EBI accession number NP_003755
[GenBank]
), a chicken protocadherin
isoform (GenBankTM/EBI accession number AAK57196
[GenBank]
), a candidate gene for
human Cat-Eye syndrome (GenBankTM/EBI accession number AAK30049
[GenBank]
), the
cysteine-string proteins, two proteins of unknown function (Chic1 and CHIC2),
a putative human zinc transporter (GenBankTM/EBI accession number
NM_017767
[GenBank]
), and a zebra fish Na/Pi co-transporter
(GenBankTM/EBI accession number AF121796
[GenBank]
). Although each of these
proteins contains a short polycysteine motif, they share no additional
sequence identity with OST
. It is interesting to note, however, that
many of these proteins are associated with the cell membrane and are either
directly or indirectly involved in membrane transport
(1421).
It is also interesting to note that human, mouse, and skate OST and
OST
proteins all appear to have membrane-targeting sequences in their
carboxyl-terminal, putative cytosolic domains. Skate Ost
and Ost
and mouse OST
have an Arg-X-Arg (RXR) motif, whereas
human OST
and mouse OST
have an RRK sequence at the
corresponding location in the sequence (Figs.
1 and
2). RXR sequences in
hetero-oligomeric proteins function as retention or retrieval signals that
must be masked before the corresponding protein complexes can be transported
from the endoplasmic reticulum
(2224).
Although there are currently no known primary structural homologues for
OST and OST
in the mouse or human genomes, the OST
sequences from mouse and skate exhibit a low level of predicted amino acid
identity (1822%) with the carboxyl-terminal 200 amino acids
(i.e. the single-TM helix and the carboxyl-terminal domain) of
protocadherin-
, a cell surface glycoprotein that belongs to the
cadherin superfamily (8).
Cadherins are involved in cell recognition, signaling, morphogenesis, and
angiogenesis, and one study has identified a role for these proteins in
organic solute transport (25).
Dantzig and co-workers (25)
discovered a cadherin homologue that was associated with the acquisition of
peptide transport activity by transport-deficient cells (human
peptide transporter-1, HPT-1). Because HPT-1 has only
one putative TM domain, these authors speculated that this cadherin homologue
may be one component of a heteromultimeric complex or may self-associate to
form a homomultimeric complex. Although these hypotheses have not yet been
tested, these observations by Dantzig and colleagues suggest that some members
of the cadherin superfamily of proteins or perhaps structurally related
molecules such as OST
may contribute to membrane transport activity.
However, because current methods of identifying homologues rely on comparing
primary sequence data, these methods cannot identify structural or functional
homologues that exhibit only a low level of primary sequence identity. As
better modeling programs and algorithms arise, OST structural homologues may
be identified that are not currently apparent from primary sequence data.
The absence of primary structural homologues for OST and OST
and the fact that this transporter has survived evolutionary selection provide
support for the hypothesis that these genes play a necessary and perhaps
unique physiological role in humans. The present results indicate that
OST
-OST
can function as a transporter for steroids such as
estrone 3-sulfate, taurocholate, and digoxin, as well as the eicosanoid
prostaglandin E2 (Fig.
4). Because steroids and eicosanoids are involved in many cellular
functions, this transporter may play a central role in regulating these
activities. Thus, one possible role of OST
-OST
is to regulate
cellular entry and/or exit of signaling molecules.
Additional evidence for an essential physiological role of these genes is
provided by the broad tissue distribution of OST and
OST
mRNA and by the high levels of mRNA found in several human
tissues (Fig. 7). mRNA
expression was detected in 17 of 19 tissues examined with relatively high
levels found in testis, colon, liver, small intestine, kidney, ovary, and
adrenal gland. In these tissues, OST mRNA was present at 540%
of the levels of
-actin mRNA. Because
-actin is a relatively
abundant transcript, this indicates that OST
and
OST
are expressed at high levels. However, because transcript
abundance does not always correlate with high protein expression, additional
studies are needed to evaluate relative protein abundance as well as the
cellular and subcellular localization of these proteins.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants
DK48823, ES06484, DK25636, and ES07026 and by National Institute of
Environmental Health Sciences Grants ES03828 and ES01247. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
|| To whom correspondence should be addressed: Dept. of Environmental Medicine, Box EHSC, University of Rochester School of Medicine, 575 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-0262; Fax: 585-256-2591; E-mail: Ned_Ballatori{at}urmc.rochester.edu.
1 The abbreviations used are: Ost/OST, organic solute transporter;
hOST, human OST
; mOST
, mouse OST
; sOst
,
skate Ost
; ORF, open reading frame; TM, transmembrane; PBS,
phosphate-buffered saline; RT-PCR, reverse transcription PCR; SNAP, soluble
N-ethylmaleimide-sensitive fusion attachment protein; SNARE, SNAP-25
receptor.
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REFERENCES |
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