Identification of a Novel Human Organic Anion Transporting Polypeptide as a High Affinity Thyroxine Transporter
F. Pizzagalli,
B. Hagenbuch,
B. Stieger,
U. Klenk,
G. Folkers and
P. J. Meier
Division of Clinical Pharmacology and Toxicology (F.P., B.H., B.S., P.J.M.), Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland; Institute of Pathology (U.K.), University of Dresden, D-01307 Dresden, Germany; and Institute of Pharmaceutical Chemistry (G.F.), Department of Applied Biosciences, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland
Address all correspondence and requests for reprints to: Prof. P. J. Meier-Abt, Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland. E-mail: meierabt{at}kpt.unizh.ch.
 |
ABSTRACT
|
---|
Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 4748% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T4 and reverse T3 with apparent Km values of approximately 90 nM and 128 nM, respectively. Substrates less well transported by OATP-F include T3, bromosulfophthalein, estrone-3-sulfate, and estradiol-17ß-glucuronide. Furthermore, OATP-F-mediated T4 uptake could be cis-inhibited by L-T4 and D-T4, but not by 3,5-diiodothyronine, indicating that T4 transport is not stereospecific, but that 3',5'-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.
 |
INTRODUCTION
|
---|
ORGANIC ANION TRANSPORTING polypeptides (Oatps in rodents, OATPs in humans) are a rapidly growing family of polyspecific membrane transporters that are expressed in multiple organs of all mammalian species. They are classified within the solute carrier gene family Slc21a/SLC21A (www.gene.ucl.ac.uk/nomenclature; www.informatics.jax.org), which until very recently included eight mouse/rat Oatps and seven human OATPs (1). All Oatps/OATPs represent 12 transmembrane domain glycoproteins with apparent molecular masses between 80 and 90 kDa. They mediate sodium-independent transmembrane transport of a wide range of amphipathic organic compounds, including organic anions [e.g. bromosulfophthalein (BSP), bile salts, bilirubin and bilirubinglucuronides, estrogen-conjugates, thyroid hormones, linear and cyclic oligopeptides, angiotensin-converting enzyme inhibitors, and numerous other drugs], neutral steroids (e.g. the cardiac glycosides ouabain and digoxin), and selected lipophilic (bulky) organic cations (e.g. N-methylquinine, rocuronium; Refs. 1, 2, 3, 4). Although most Oatps/OATPs exhibit overlapping substrate specificities among each other, some family members show preferential or even selective transport for certain substrates such as, for example, rat Oatp2 (Slc21a5) and human OATP8 (SLC21A8) for digoxin, human OATP-A (SLC21A3) for N-methylquinine, and human OATP-E (SLC21A12) for taurocholate and the thyroid hormones T4, T3, and reverse T3 (rT3; Refs. 1, 5). Furthermore, although multiple tissue expression (MTE) is a common feature of most Slc21a/SLC21A family members, some Oatps/OATPs are predominantly or even exclusively expressed in one tissue only. The latter include especially the rat Oatp4 (Slc21a10) and the human OATP-C/liver specific transporter 1/OATP2 (SLC21A6) and OATP8, which are all selectively expressed at the basolateral (sinusoidal) plasma membrane of hepatocytes where they mediate uptake of a wide range of amphipathic albumin-bound compounds that are destined for biotransformation and/or biliary excretion within the liver (4, 6, 7, 8, 9). In contrast to these liver-specific transporters, the expression and physiological functions of extrahepatic Oatps/OATPs are considerably less well understood.
Obviously, a prerequisite for a clear understanding of the physiological and pathophysiological significance of Oatp/OATP-mediated substrate transport is the identification and functional characterization of all members of the Slc21a/SLC21A gene family. With the successful cloning of the entire human genome (10, 11), a vast array of expressed sequence tags (ESTs) and genomic sequences has become available that can now be screened to detect novel transporters in human tissues. This approach has recently been used to identify and isolate several human OATPs, including the novel transporters OATP-B (SLC21A9), OATP-D (SLC21A11), and OATP-E (12). In this study, we have followed a similar strategy and report the original identification of an additional human OATP, called OATP-F (SLC21A14), from a human brain library. Although the genomic organization and the structure of OATP-F are similar to other OATP family members, the comparison of the amino acid sequence identities indicate that OATP-F belongs to a new OATP subfamily. Furthermore, based on Northern blot analysis, OATP-F is predominantly expressed in brain (multiple brain regions) and testis (Leydig cells). And finally, functional expression studies in Xenopus laevis oocytes and Chinese hamster ovary (CHO) cells indicate that OATP-F represents a high affinity transporter for the thyroid hormones T4 and rT3. Thus, OATP-F represents a new member of the Oatp/OATP gene family that exhibits preferential transport of T4 and rT3 in brain and testis.
 |
RESULTS
|
---|
Cloning of the Human OATP-F (SLC21A14) cDNA
Through screening of an EST-database (Incyte, Palo Alto, CA) we identified and isolated the novel OATP-F from a human brain library. The OATP-F cDNA clone contained 3059 bp, including a 5'-leader sequence of 62 bp, an open reading frame (ORF) of 2136 bp, and a 3'-trailer sequence of 861 bp. OATP-F is a 712-amino acid protein (Fig. 1
) and contains 12 predicted transmembrane domains with the C- and N-termini located inside the cell. This feature is in accordance with the topology of other members of the Oatp/OATP gene family (13). In addition OATP-F, contains four postulated N-glycosylation sites, two of them at positions 520 and 530 being conserved between OATP-F, OATP-A, OATP-C, and OATP8. The amino acid sequence identity of OATP-F with other human OATPs was highest for OATP-A (48%; Ref. 14) and OATP8 (48%; Ref. 9), followed by OATP-C (47%; Refs. 6, 8, 15), OATP-D (37%; Ref. 12), human prostaglandin transporter (36%; Ref. 16), OATP-B (34%; Ref. 12), and OATP-E (34%; Ref. 12). OATP-F also shares about 85% amino acid identity with three additional sequences present in the GenBank under the accession numbers NM 021471 (mouse), AF306546 (rat), and BAB12153 (monkey). These animal proteins might represent orthologs of the human OATP-F.

View larger version (106K):
[in this window]
[in a new window]
|
Figure 1. Comparison of the Amino Acid Sequence of OATP-F with the Other Human OATPs
Putative transmembrane domains were determined with the PredictProtein Programm (37 ) and are underlined. Potential N-glycosylation sites for OATP-F at positions 146, 510, 520, and 530 are shown by . The OATP-F cDNA sequence has been deposited at GenBank with the accession no. AF260704.
|
|
Comparison of the Genomic Organization of the Human OATP-F (SLC21A14), OATP-A (SLC21A3), OATP-C (SLC21A6), and OATP8 (SLC21A8) Genes
The cloned OATP-F cDNA was used to screen a human genomic DNA sequence database (GenBank). This resulted in the identification of a genomic clone (GenBank/European Bioinformatics Institute accession no. AC064821) that covers the complete OATP-F gene and is localized on chromosome 12p12. The organization of the OATP-F gene is shown in Table 1
. It is approximately 58 kb long and includes 15 exons and 14 introns. The first exon is localized in the 5' untranslated region (UTR), whereas the other 14 exons contribute to the ORF. The longest intron (9.7 kb) is located between exons 9 and 10, and the shortest (1.0 kb) between exons 8 and 9. All intron/exon boundaries are compatible with the canonical donor and acceptor consensus motifs. With one exception, all introns start with a GTA at the 5'-splice donor site, and nine introns end with a CAG at the 3'-splice acceptor site. Besides OATP-F, the genes of OATP-A, OATP-C, and OATP8 are also localized on chromosome 12p12 (9). As illustrated in Fig. 2
, all OATP genes localized on chromosome 12 have a similar organization. In all four transporter genes, the ORF is composed of 14 exons (Fig. 2A
). Nine of the 14 shared exons have exactly the same length, indicating that they are conserved in all four transporter genes. Furthermore, in all four genes, the longest exon is exon 15 (1079 bp), which includes the 3' UTR, and the shortest exon is exon 13 (65 bp). Interestingly, due to variable intron lengths, the total gene size increases continuously from OATP-F (58 kb) to OATP-A (66 kb), OATP-C (98 kb), and OATP8 (101 kb). These four transporter genes and an additional pseudogene form a gene cluster on chromosome 12p12 that spans approximately 700 kb of genomic sequence and is oriented as illustrated in Fig. 2B
.

View larger version (29K):
[in this window]
[in a new window]
|
Figure 2. Genomic Organization of OATP-F in Comparison to OATP-A, OATP-C, and OATP8 (A) and Genomic Structure of the Human OATP-Cluster Located on Chromosome 12p12 (B)
A, Light gray blocks represent exons with identical length, and dark gray blocks represent exons with different lengths for the indicated genes. The gene length is given at the right. B, Length of arrows corresponds to gene length. The overall length of the cluster is approximately 700 kb.
|
|
Tissue Distribution of Human OATP-F
The tissue distribution of the human OATP-F was determined by Northern blot analysis. The strongest hybridization signals were obtained with human brain and testis mRNAs (Fig. 3A
). A faint hybridization reaction was also obtained with heart mRNA. In all positive tissue samples, the RNA probe hybridized to an approximately 3.3-kb mRNA species, which most likely corresponds to the full-length OATP-F mRNA. The exact nature of the additional hybridization signal at approximately 7.0 kb in human brain is not known, but it might represent a partially spliced or unspliced mRNA. As further indicated on the MTE array (Fig. 3B
), OATP-F mRNA was detected in numerous brain regions with the exceptions of pons and cerebellum. Although this wide intracerebral distribution of OATP-F might indicate expression at the blood brain barrier, the exact cellular localization of OATP-F in human brain remains to be investigated.

View larger version (35K):
[in this window]
[in a new window]
|
Figure 3. Tissue Distribution of OATP-F
Commercially available human multiple tissue Northern-blots (CLONTECH Laboratories, Inc.) containing 2 µg of Poly (A+) RNA (A) and a human MTE array (CLONTECH Laboratories, Inc.) (B) were hybridized overnight with an OATP-F antisense-RNA probe and a DNA probe, respectively (see Materials and Methods), and after high stringency washing were exposed to autoradiography film at -70 C for 3 d.
|
|
To finely localize OATP-F in testis, we performed immunohistochemistry in paraffin sections of biopsy tissue obtained from a 68-yr-old man with hyperplasia of Leydig cells. As can be seen in Fig. 4
, Oatp-F could be selectively localized in nests of Leydig cells, indicating that OATP-F represents a constitutive Leydig cell transporter.

View larger version (113K):
[in this window]
[in a new window]
|
Figure 4. Immunohistochemical Localization of OATP-F in Leydig Cells of Human Testis
Paraffin sections of human testis were used for indirect immunohistochemical detection with peroxidase to probe for OATP-F with a polyclonal antiserum as described in Materials and Methods. Positive labeling was only found in interstitial cells, but not in the seminiferous tubuli (top). Higher magnification shows OATP-F expression in Leydig cells (bottom).
|
|
Functional Expression of OATP-F in X. laevis Oocytes and in CHO Cells
To delineate the transport function of OATP-F, a wide range of substrates that have been previously shown to be transported by one or several Oatps/OATPs (1, 3, 4, 7, 17, 18, 19, 20) were tested in OATP-F cRNA-injected oocytes. As summarized in Table 2
, only the thyroid hormone T4 and BSP were transported significantly above uptake in water-injected control oocytes. These OATP-F-mediated T4 and BSP uptake activities were independent of the presence of sodium in the uptake medium (data not shown). No stimulation of uptake was found in OATP-F cRNA-injected oocytes for bile salts, steroid conjugates, anionic peptides, cardiac glycosides, leuktriene C4, prostaglandin E2, T3, N-methylquinine, and methotrexate. Furthermore, typical substrates of the organic anion transporter family (SLC22A), folate and methotrexate (21, 22), were also not significantly higher transported in OATP-F cRNA as compared with water-injected oocytes. These data indicate that OATP-F has a very limited substrate specificity and may represent a selective T4 transporter in human brain and testis.
To exclude the possibility that the limited transport activity of OATP-F in cRNA-injected oocytes was caused by insufficient expression of OATP-F at the oocyte surface, we performed additional transport studies in stably transfected CHO-K1 cells (so called CHO-F cells). Thereby, a polyclonal antiserum against the C-terminal end of OATP-F was raised in rabbits and used for detection of surface expression of OATP-F. As illustrated in Fig. 5A
, stably transfected and butyrate-induced CHO-F cells showed immunopositive surface staining, indicating marked expression of OATP-F at the plasma membrane. No immunopositive reaction was observed in wild-type CHO-K1 cells. Furthermore, the OATP-F expressing CHO-F cells showed an 11-fold stimulation of T4 and a 14-fold stimulation of rT3 uptake, whereas uptakes of T3, BSP, estron-3-sulfate, and estradiol-17ß-glucuronide were stimulated only approximately 2-fold as compared with uptakes in wild-type CHO-K1 cells (Fig. 5
, B and C, and Table 3
). Similar to cRNA-injected X. laevis oocytes, no other OATP-F substrate could be identified in transfected CHO-F cells (Table 3
). Thus, high expression in CHO-F cells identified rT3 in addition to T4 as preferential OATP-F substrate, whereas transport of other typical Oatp/OATP substrates was stimulated only to a low extent (e.g. 2-fold) or not at all. In general, these results demonstrate that high expression levels are crucial for the exact delineation of the substrate preference of a given transport protein.

View larger version (51K):
[in this window]
[in a new window]
|
Figure 5. Correlation of OATP-F Expression (A) and Transport Function (B, C) in Stably Transfected CHO Cells
A, Control CHO cells (CHO-K1, left) or OATP-F-expressing CHO cells (CHO-F, right) were grown to confluency on coverslips and treated as described in Materials and Methods. On separate dishes, [125I]T4 (10 nM) (B) or [125I]T3 (5 nM) (C) uptake was measured, after a 24-h incubation in 5 mM sodium butyrate, for 3 min in a choline chloride medium. Uptake values represent means ± SD of triplicate determinations.
|
|
Next, we determined the kinetics of OATP-F-mediated T4 and rT3 transport in stably transfected CHO-F cells. Preliminary experiments indicated initial linear uptake rates for 1 min in OATP-F-expressing CHO-F cells (data not shown). As shown in Fig. 6
, initial uptake rates (30 sec for T4, 40 sec for rT3) exhibited clear saturability with increasing substrate concentrations. The calculated apparent Km values were 90 ± 28 nM for T4 (Fig. 6A
) and 128 ± 38 nM for rT3 (Fig. 6B
). These results further substantiate that OATP-F represents a high affinity transporter for both T4 and rT3.

View larger version (15K):
[in this window]
[in a new window]
|
Figure 6. Kinetics of OATP-F-Mediated T4 (A) and rT3 (B) Uptake into Stably Transfected CHO Cells
Wild-type CHO-K1 cells or OATP-F-expressing CHO cells were grown to confluency on 3-cm dishes. After a 24-h incubation in 5 mM sodium butyrate, the cells were incubated with increasing concentrations of [125I]T4 (A) and [125I]rT3 (B) at 37 C for 30 sec and 40 sec, respectively, in a choline chloride medium. Net OATP-F mediated uptake values ( ) were calculated by subtracting the values obtained with the wild-type CHO-K1 cells from those obtained with the stably transfected CHO-F cells (values are means ± SEM of three determinations). The data were fitted and plotted to the Michaelis Menten equation using nonlinear regression analysis.
|
|
Finally, we performed cis-inhibition studies with L-T4, D-T4, and 3,5-diiodothyronine (3,5-T2). As illustrated in Fig. 7
, both L-T4 and D-T4 markedly inhibited OATP-F-mediated (L-) T4 uptake, whereas 3,5-T2 had virtually no inhibitory effect. Together with the active transport of rT3 (Table 3
and Fig. 6B
), these data indicate that 1) OATP-F-mediated T4 transport is not stereospecific, and 2) iodination at the 3',5' positions is an important requirement for efficient transport of iodothyronines by OATP-F.

View larger version (12K):
[in this window]
[in a new window]
|
Figure 7. Inhibition of OATP-F-Mediated T4 Uptake by L-T4, D-T4, and 3,5-T2 in Stably Transfected CHO Cells
Control CHO cells (CHO-K1) or OATP-F-expressing CHO cells (CHO-F) were grown to confluency on 3-cm dishes. After a 24-h incubation in 5 mM sodium butyrate, [125I]T4 (10 nM) uptake was measured during 3 min in the absence (control) or in the presence of 10 µM L-T4, D-T4, or 3,5-T2, respectively. Data represent the means ± SD of three determinations.
|
|
 |
DISCUSSION
|
---|
In this study, we have identified and isolated a novel human member of the Oatp/OATP-gene family of membrane transporters. According to the suggested provisional alphabetic designation of human OATPs (1, 12), the novel organic anion transporting polypeptide has been called OATP-F, and its gene symbol is SLC21A14. OATP-F consists of 712 amino acids (Fig.1
) and exhibits similar structural features on both the protein (Fig. 1
) and genomic (Fig. 2A
) levels as the other OATPs. OATP-F shares the highest amino acid sequence identities with OATP-A (48%), OATP8 (48%), and OATP-C (47%), and the genes of all four of these transporters are clustered on chromosome 12p12 (Fig. 2B
). OATP-F is predominantly expressed in various brain regions (Fig. 3B
) and in Leydig cells of the testis (Fig. 4
). Functionally, OATP-F mediates preferential sodium-independent transport of T4 and rT3 (Table 2
and Figs. 5
and 6
), whereas T3 and other typical Oatp/OATP substrates (e.g. BSP, estron-3-sulfate, estradiol-17ß-glucuronide) are less well transported or not transported at all (Tables 2
and 3
). On the basis of cis-inhibition studies, OATP-F-mediated thyroid hormone transport is not stereospecific, but appears to require intact 3',5'-iodination of the substrates (Figs. 6
and 7
). OATP-F was found to have a 10- to 100-fold higher affinity for T4 (Fig. 6
) than any previously characterized sodium-independent T4 transporter (23). Hence, OATP-F appears to be a rather selective high affinity T4 and rT3 transporter that might play an important role in the transport and disposition of iodothyronines in brain and testis.
The amino acid sequence identities of 4748% and the common gene cluster on chromosome 12p12 indicate that OATP-F belongs to the same gene family as OATP-A, OATP-C, and OATP8. For clarification, the evolutionary relationships between all so far known members of the Oatp/OATP-gene superfamily is illustrated in Fig. 8
. As can be seen, OATP-F forms a separate subfamily within the OATP1 family, and its rodent orthologs (i.e. Oatp14) are probably the homologous GenBank sequences with the accession numbers NM 021471 (mouse) and AF306546 (rat). The latter assumption, however, remains to be experimentally verified. The amino acid sequence identity of OATP-F with the known rodent members of the OATP1 family are also above 44%, and the mouse Oatp2 (Slc21a5) and Oatp3 (Slc21a3) genes are localized on chromosome 6, which is syntenic with human chromosome 12. Interestingly, the OATP-F is the shortest gene of all human members of the OATP1 family (Fig. 2
), indicating that it represents the most ancient family member. This assumption is supported by the occurrence of an orthologous gene product in the little skate, a low vertebrate animal species (24). In any case, the greater than 44% amino acid sequence identities among all family members, the colocalization of the OATP-A, OATP-C, OATP8, and OATP-F genes on chromosome 12p12, and their conserved gene structure indicate strongly that the OATP1 family arose through gene duplication from a common ancestor.

View larger version (20K):
[in this window]
[in a new window]
|
Figure 8. Phylogenic Tree of the Oatp/OATP Gene Superfamily
The tree was calculated using the GCG software package (38 ) and drawn with TreeView (39 ). The four different Oatp/OATP families with inter-familial amino acid sequence identities of more than 40% are indicated.
|
|
Among all Oatps/OATPs, the identified OATP-F is unique with respect to its bilocal expression in brain and testis (Fig. 3
). In brain, OATP-F exhibits a wide distribution (Fig. 3B
) similar to OATP-A, which is localized at blood brain barrier endothelial cells (24). Although the exact cellular expression of OATP-F in various brain regions remains to be investigated, it is noteworthy that OATP-F has an approximately 100-fold higher affinity for T4 as compared with OATP-A (Table 3
; Ref. 5). Thereby, the apparent Km value (
90 nmol/liter) of OATP-F for T4 corresponds to the normal average T4 concentration in plasma (
100 nmol/liter), indicating that OATP-F is not saturated under physiological conditions and, therefore, is probably not rate-limiting for the intracellular availability of T4. Interestingly, high affinity transport of T4 has been demon-strated in human glioma cells (25) as well as in various neuronal cells of rat brain (23). Thus, OATP-F could mediate cellular uptake of T4, which then is converted intracellularly into active T3 by the type II deiodinase (D2). D2 represents the only 5'-deiodinase in human brain and exhibits a similar wide cerebral distribution as OATP-F (26). The latter appears to be also true for the type III 5-deiodinase (D3) that degrades T4 and T3 into rT3 and 3',3-diiodothyronine, respectively. D3 has been reported to be expressed in neurons and in primary rat astroglial cell cultures (26). Finally, the mouse Oatp14, which represents an OATP-F ortholog, has been isolated from the inner ear (GenBank accession no. NM021471), where the cochlea is known to belong to the organs most sensitive to thyroid hormone abnormalities (26). Hence, OATP-F might play an important role in the homeostasis of thyroid hormones in various cell types of the central nervous system.
The observation of OATP-F expression in Leydig cells of the human testis (Fig. 4
) is consistent with the reported high affinity T4-binding sites in rat testis (23, 27). Thyroid hormones are well known to play an important role in the development, proliferation, and differentiation of mesenchymal, immature, and mature Leydig cells (28). Furthermore, thyroid hormones (especially T3) are essential for maintaining adult Leydig cell functions such as ongoing steroid hormone biosynthesis (29). Because, to the best of our knowledge, transport of thyroid hormones into and out of human Leydig cells has not been studied in any detail, the identification of OATP-F as a high affinity T4 and rT3 transporter opens the possibility to further explore the physiological and pathophysiological roles of plasma membrane-associated iodothyronine transport for overall Leydig cell function.
A further characteristic feature of OATP-F in comparison with other Oatps/OATPs is its rather narrow spectrum of transport substrates (Tables 2
and 3
). In fact, especially within the OATP1 family, OATP-F is an exception, because all other family members are paradigmatic for their wide and overlapping substrate spectrum (1, 4). Common substrates of all OATP1 family members include the thyroid hormones T4, T3, and rT3, which are transported with different affinities by rat Oatp1 (Ref. 19), Oatp2 (Km values, T4,
6.5 µM; T3,
5.9 µM; Ref. 30), Oatp3 (Km values, T4,
4.9 µM; T3,
7.3 µM; Ref. 30) and Oatp4 (Ref. 7) and by the human OATP-A (Km values, T4,
8.0 µM; T3,
6.5 µM; Ref. 1), OATP-C (Km values, T4,
3.0 µM; T3,
2.7 µM; Refs. 1, 6), and OATP8 (Ref. 1), and even by OATP-E (Km value, T3,
0.9 µM; Ref. 5), which belongs to a different OATP family (Fig. 8
). On the basis of these studies, it was suggested that tissue-specific Oatps/OATPs might be involved in thyroid hormone uptake in various organs such as, for example, OATP-A in brain, OATP-C/OATP8 in liver, and OATP-E in peripheral tissues (5). Our study now increases further the diversity of thyroid hormone transporters and supports the concept that several transporters are involved in the local disposition of thyroid hormones in a single organ (23). Thus, the preferred substrates of OATP-F are T4 and rT3 (Table 3
and Fig. 6
). Furthermore, L-T4 and D-T4 cis-inhibited OATP-F-mediated T4 uptake to similar extents, whereas 3,5-T2 had no inhibitory effects (Fig. 7
). These data indicate that OATP-F-mediated T4 transport is not stereospecific, and that iodination at the 3' and 5' positions is required for efficient iodothyronine transport by OATP-F. The latter conclusion is consistent with the general preference of Oatps/OATPs for organic anions, because dual iodination of the outer ring must be assumed to decrease the pKa value of iodothyronines. Furthermore, based on the assumption that all Oatps/OATPs may function as organic anion exchangers (2), OATP-F could well mediate cellular uptake of T4 as well as efflux of its D3-dependent degradation product rT3, and thus provide an additional mechanism for protecting tissues from an excess of thyroid hormones. Although the latter suggestion requires more extensive investigations of a possible colocalization of D2, D3, and OATP-F in identical cell types and of the exact molecular transport properties of OATP-F, coupled T4 uptake and rT3 efflux in conjunction with coordinated D2 and D3 activities would represent an efficient mechanism for adjustment of intracellular concentrations of active T3 to the actual metabolic needs of the cells. Furthermore, because human brain is devoid of type I deiodinase, which represents the principal pathway for rT3 clearance in liver and kidney, increased OATP-F mediated rT3 efflux from D3-expressing brain cells could well contribute to the known reciprocal changes of T3 (decrease) and rT3 (increase) in serum during caloric stress and moderate to severe illness, which are associated with decreased intracellular T4-to-T3 conversion most probably as a consequence of D2 deficiency (26).
In conclusion, we have isolated a novel organic anion transporting polypeptide from human brain. This so-called OATP-F represents a high affinity T4 and rT3 transporter and is expressed predominantly in brain and Leydig cells of the testis. The OATP-F gene colocalizes together with the OATP-A, OATP-C, and OATP8 genes on chromosome 12p12, indicating that this OATP-gene cluster has evolved by gene duplication from a common ancestor. Its limited spectrum of transport substrates and its high affinities for T4 and rT3 indicate that OATP-F may play an important role in the local disposition of iodothyronines at specific cellular sites in brain and testis.
 |
MATERIALS AND METHODS
|
---|
Materials
[35S]Bromosulfophthalein (BSP) was synthesized with a specific activity of 9 Ci/mmol as described previously (31). [3H(G)]taurocholate (2.0 Ci/mmol), [3H]dehydroepiandrosterone sulfate (DHEAS; 60 Ci/mmol), [6,7-3H(N)]estrone-3-sulfate (53 Ci/mmol), [6,7-3H(N)]estradiol-17ß-glucuronide (44 Ci/mmol), [Tyrosyl-3,5-3H]deltorphin II (41 Ci/mmol), [Tyrosyl-2,6-3H(N)] D-penicillamine2,5 enkephalin (DPDPE; 45 Ci/mmol), [3H]digoxin (19 Ci/mmol), [3H(G)]ouabain (16.5 Ci/mmol), [3H(N)]leukotriene C4 (158 Ci/mmol), [3H(N)]prostaglandin E2 (200 Ci/mmol), L-[125I]T3 (779 Ci/mmol), L-[125I]T4 (969 Ci/mmol), and L-[125I]rT3 (757 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). [Prolyl-3,4(n)-3H]Cyclo[D-Trp-D-Asp-L-Pro-D-Val-L-Leu] (BQ-123; 36 Ci/mmol), [3',5', 7-3H]methotrexate (8 Ci/mmol), and [3',5',7,9-3H]folic acid (51 Ci/mmol) were obtained from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). [3H]N-methylquinine (85 Ci/mmol) was synthesized as described (17). [3H]glycocholic acid (14 Ci/mmol) was a kind gift from Dr. A. F. Hofmann of the University of California at San Diego. Secondary antibody fluorescein-conjugated goat IgG fraction to rabbit IgG was obtained from ICN Biomedicals, Inc. (Eschwege, Germany). All cell culture media and reagents were obtained from Life Technologies, Inc. (Paisley, UK). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.
Cloning of the Human Full-Length OATP-F cDNA
The novel human OATP-F was identified by searching the Incyte EST database for sequences homologous to human OATP-A. One EST sequence, Incyte accession number 024020, was found to encode a protein with a high amino acid sequence identity with the human OATP-A. Based on this sequence information, two primers, the forward primer 024020f (5'-CAGAAAGACAATGGATGTCC-3') and the reverse primer 024020r (5'-CACATCTTTTAAATCCCCATTTGAGGC-3'), were designed and used to screen a human adult brain Rapid-Screen cDNA library panel (OriGene Technologies, Rockville, MD). This resulted in the isolation of a single new cDNA clone that was sequenced on both strands using cycle sequencing and an ALF Express (Amersham Pharmacia Biotech). This cDNA lacked the 5' UTR and 271 bp of the ORF. To isolate the full-length OATP-F, additional 5'-upstream primers were used to rescreen the library (forward primer 024020f3, 5'-CATTGAAAGGAACTGGCTATCTTTG-3'; and reverse primer 024020r3, 5'-TTGACTGCTTGACTCTAGGAGACAC-3'). During the PCR optimization of these new primers by RT-PCR with total RNA from human brain (CLONTECH Laboratories, Inc., Palo Alto, CA), we cloned and sequenced a fragment that was 250 bp longer than expected from the cDNA sequence. This fragment contained the missing base pairs of the ORF and additional 5' untranslated DNA. Using an overlap extension PCR approach, the full-length OAPT-F cDNA was assembled by two rounds of PCR and cloned into pCRII-TOPO vector (Invitrogen, Carlsbad, CA). The 3.1-kb fragment was sequenced on both strands.
Tissue Distribution of OATP-F by Northern Blot Analysis
Northern blots containing 2 µg each of human poly(A)+ RNA (CLONTECH Laboratories, Inc.) were prehybridized for 30 min at 68 C in ULTRAhyb (Ambion, Inc., Austin, TX) hybridization solution and then hybridized overnight at 68 C in the same buffer with a 32P- labeled antisense-RNA probe (nucleotides 1545 of the published sequence, GenBank accession number AF260704) with a specific activity of 1.4 million cpm/ml. The blots were washed twice for 5 min with 2x SSC/0.1% SDS at 68 C, followed by two washes of 15 min with 0.1x SSC/0.1% SDS at 68 C, and then exposed to autoradiography film at -70 C with an intensifying screen for 3 d.
A MTE array (CLONTECH Laboratories, Inc.) containing normalized poly (A)+ RNA dots from different human tissues was prehybridized for 30 min at 65 C in ExpressHyb (CLONTECH Laboratories, Inc.) hybridization solution, and then, according to the manufacturers instructions, hybridized overnight at 65 C in the same buffer with a 32P-labeled cDNA probe (nucleotides 16881937 of the published sequence, GenBank accession number AF260704) with a specific activity of 2 Mio cpm/ml. The blot was washed five times for 20 min with 2x SSC/1% SDS at 55 C, followed by two washes of 20 min with 0.1x SSC/0.5% SDS at 55 C, and then exposed to autoradiography film at -70 C with an intensifying screen for 3 d.
Antibody Production
To produce a specific antibody against the human OATP-F, a comparison between the C-terminal 15 amino acids (HLLQPNYWPGKETQL) of OATP-F with other human proteins present in the current databases was performed. The result indicated that the C-terminal sequence of OATP-F is unique. Thus, a rabbit polyclonal antiserum was raised against the C-terminal 15 amino acids of OATP-F coupled via an additional N-terminal cystein to keyhole limpet hemocyanin (Neosystem, Strasbourg, France) as described (32).
Immunohistochemistry
Testicular tissue was obtained from routine diagnostic biopsies taken from a 68-yr-old man with Leydig cell hyperplasia. The tissue was fixed in formaldehyde, embedded in paraffin, and cut into 3-µm sections. The sections were freed from paraffin, rehydrated, and heated in a microwave oven at 600 W for 20 min in a 10 mM citrate buffer (pH 6.0). Endogenous peroxidase was blocked by immersion (30 min) into 0.3% hydrogen peroxide. To block unspecific binding, the tissue sections were incubated with rabbit nonimmune serum for 20 min. After incubation with the OATP-F antiserum (dilution 1:1000) overnight at 8 C, the slides were treated with a biotinylated anti-rabbit Ig for 30 min at room temperature and incubated with avidin-biotin-peroxidase complex (Vectastain Elite kit, Vector Laboratories, Inc., Burlingame, CA). 3,3'-Diaminobenzidine was used as a chromogen (33). Counterstaining was performed on the sections with hematoxylin. Negative controls were generated by omission of the primary OATP-F antibodies.
Expression of OATP-F in X. laevis Oocytes
Female X. laevis oocytes were purchased from the African Xenopus facility, Noordhoek, Republic of South Africa. For the expression in the X. laevis oocytes system, the OATP-F ORF was subcloned into the X. laevis expression vector (7) using a PCR approach. The ORF was amplified using the full-length OATP-F clone as template under the following conditions: the forward primer containing a NcoI restriction site (5'-GCCGCCATGGAC-ACTTCATCCAAAGAAAATATCC-3'), the reverse primer containing a HindIII restriction site (5'-GTACGTAAGCTTCTAAAGTTGAGTTTCCTTGCCTGG-3'), one cycle of 94 C for 2 min, 55 C for 1 min, 72 C for 4 min, followed by 34 cycles of 94 C for 45 sec, 55 C for 45 sec, 72 C for 3 min and 30 sec, and a final elongation of 72 C for 10 min. The amplified fragment was gel purified and digested for 2 h at 37 C with NcoI and HindIII. After an additional gel purification, the amplicon was directionally cloned into the X. laevis expression vector and sequenced on both strands.
OATP-F-capped cRNA was synthesized from NotI-linearized cDNA using the mMESSAGEmMACHINE T7 kit (Ambion, Inc.). X. laevis oocytes were prepared as previously described (34). After an overnight incubation at 18 C, healthy oocytes were microinjected with 50 nl water or with 5 ng cRNA and kept in culture at 18 C for 3 d. Thereafter, uptakes of radiolabeled substrates were measured for 30 min at 25 C in 100 µl uptake solution (100 mM NaCl or 100 mM choline chloride, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES adjusted to pH 7.5 with Tris) as described (3).
Cell Culture and Immunofluorescence
CHO cells were grown in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 µg/ml L-proline, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C with 5% CO2 and 95% humidity. Selective medium contained additionally 500 µg/ml G418 sulfate (geneticin). For immunofluorescence, wild-type CHO-K1 cells and stably transfected CHO-F cells were grown to confluency on coverslips. Sodium butyrate (5 mM) was added to the culture medium 24 h before the start of the experiments. Incubation for immunofluorescence was performed as described (32).
Stable Transfection of CHO Cells with OATP-F
The OATP-F ORF was amplified by PCR using the full-length OATP-F clone as template under the following conditions: the forward primer containing an EcoRI restriction site (5'-GCCGGAATTCGCCACCATGGACACTTCATCCAAAG-3'), the reverse primer containing a NotI restriction site (5'-TCCTTTGCGGCCGCCTAAAGTTGAGTTTCCTTGC-3'), one cycle of 94 C for 2 min, 55 C for 1 min, 72 C for 4 min, followed by 34 cycles of 94 C for 45 sec, 55 C for 45 sec, 72 C for 3 min and 30 sec, and a final elongation of 72 C for 10 min. For subcloning of the OATP-F ORF into the pIRESneo2 (CLONTECH Laboratories, Inc.) expression vector, the amplified fragment was gel purified and digested for 2 h at 37 C with EcoRI and NotI. After an additional gel purification, the digested amplicon was directionally subcloned into the EcoRI/NotI digested pIRESneo2 vector and verified by sequencing. The construct was introduced into CHO-K1 cells by electroporation, and after 24 h stably transfected cells were selected by adding G418 to the culture medium. From the resulting transfected cell pool, single clones were isolated with the use of cloning cylinders and tested for sodium-independent T4 uptake. Clone CHO-F exhibited the highest T4 transport activity and was selected for use in all further experiments.
Uptake Studies in CHO Cells
Determination of sodium-independent substrate uptake into OATP-F-expressing CHO cells was performed as described (35). For all uptake experiments, expression of OATP-F was induced by incubation of the cells for 24 h with culture medium supplemented with 5 mM sodium butyrate (36). Because preliminary experiments did not demonstrate any sodium dependency, all uptake experiments were performed in choline chloride containing medium consisting of 116 mM choline chloride, 5.3 mM KCl, 1.1 mM KH2PO4, 0.8 mM MgSO4, 1.8 mM Ca2Cl2, 1.1 mM D-glucose, and 20 mM HEPES.
 |
ACKNOWLEDGMENTS
|
---|
We thank Kevin M. Bottomley from Roche (Welwyn, UK) for his help with database searches.
 |
FOOTNOTES
|
---|
This work was supported by the Swiss National Science Foundation (Grant 31-64140.00 to B.S. and P.J.M. and Grant 31-59204.99 to B.H.) and by an independent Ph.D. thesis fellowship provided by Roche (Welwyn, UK).
Abbreviations: BQ-123, Cyclo[D-Trp-D-Asp-L-Pro-D-Val-L-Leu]; BSP, bromosulfophthalein; CHO, Chinese hamster ovary; D2, type II deiodinase; D3, type III deiodinase; DHEAS, dehydroepiandrosterone sulfate; DPDPE, [D-penicillamine2,5]enkephalin; EST, expressed sequence tag; MTE, multiple tissue expression; OATP, human organic anion transporting polypeptide; Oatp, rodent organic anion transporting polypeptide; ORF, open reading frame; rT3, reverse T3; SLC/Slc, solute carrier gene family; 3,5-T2, 3,5-diiodothyronine; UTR, untranslated region.
Received for publication November 15, 2001.
Accepted for publication July 12, 2002.
 |
REFERENCES
|
---|
- Kullak-Ublick GA, Ismair MG, Stieger B, Landmann L, Huber R, Pizzagalli F, Fattinger K, Meier PJ, Hagenbuch B 2001 Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver. Gastroenterology 120:525533[Medline]
- Meier PJ, Eckhardt U, Schroeder A, Hagenbuch B, Stieger B 1997 Substrate specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver. Hepatology 26:16671677[Medline]
- Reichel C, Gao B, van Montfoort J, Cattori V, Rahner C, Hagenbuch B, Stieger B, Kamisako T, Meier PJ 1999 Localization and function of the organic anion-transporting polypeptide Oatp2 in rat liver. Gastroenterology 117:688695[Medline]
- Cattori V, van Montfoort JE, Stieger B, Landmann L, Meijer DKF, Winterhalter KE, Meier PJ, Hagenbuch B 2001 Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate specificity with Oatp1, Oatp2 and Oatp3. Pflügers Arch 443:188195[CrossRef][Medline]
- Fujiwara K, Adachi H, Nishio T, Unno M, Tokui T, Okabe M, Onogawa T, Suzuki T, Asano N, Tanemoto M, Seki M, Shiiba K, Suzuki M, Kondo Y, Nunoki K, Shimosegawa T, Iinuma K, Ito S, Matsuno S, Abe T 2001 Identification of thyroid hormone transporters in humans: different molecules are involved in a tissue-specific manner. Endocrinology 142:20052012[Abstract/Free Full Text]
- Abe T, Kakyo M, Tokui T, Nakagomi R, Nishio T, Nakai D, Nomura H, Unno M, Suzuki M, Naitoh T, Matsuno S, Yawo H 1999 Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1. J Biol Chem 274:1715917163[Abstract/Free Full Text]
- Cattori V, Hagenbuch B, Hagenbuch N, Stieger B, Ha R, Winterhalter KE, Meier PJ 2000 Identification of organic anion transporting polypeptide 4 (Oatp4) as a major full-length isoform of the liver-specific transporter-1 (rlst-1) in rat liver. FEBS Lett 474:242245[CrossRef][Medline]
- König J, Cui Y, Nies AT, Keppler D 2000 A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol 278:G156G164
- König J, Cui Y, Nies AT, Keppler D 2000 Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem 275:2316123168[Abstract/Free Full Text]
- Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, Gocayne JD, Amanatides P, Ballew RM, Huson DH, Wortman JR, Zhang Q, Kodira CD, Zheng XH, Chen L, Skupski M, Subramanian G, Thomas PD, Zhang J, Gabor Miklos GL, Nelson C, Broder S, Clark AG, Nadeau J, McKusick VA, Zinder N, Levine AJ, Roberts RJ, Simon M, Slayman C, Hunkapiller M, Bolanos R, Delcher A, Dew I, Fasulo D, Flanigan M, Florea L, Halpern A, Hannenhalli S, Kravitz S, Levy S, Mobarry C, Reinert K, Remington K, Abu-Threideh J, Beasley E, Biddick K, Bonazzi V, Brandon R, Cargill M, Chandramouliswaran I, Charlab R, Chaturvedi K, Deng Z, Di Francesco V, Dunn P, Eilbeck K, Evangelista C, Gabrielian AE, Gan W, Ge W, Gong F, Gu Z, Guan P, Heiman TJ, Higgins ME, Ji RR, Ke Z, Ketchum KA, Lai Z, Lei Y, Li Z, Li J, Liang Y, Lin X, Lu F, Merkulov GV, Milshina N, Moore HM, Naik AK, Narayan VA, Neelam B, Nusskern D, Rusch DB, Salzberg S, Shao W, Shue B, Sun J, Wang Z, Wang A, Wang X, Wang J, Wei M, Wides R, Xiao C, Yan C, et al. 2001 The sequence of the human genome. Science 291:13041351[Abstract/Free Full Text]
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, et al. 2001 Initial sequencing and analysis of the human genome. Nature 409:860921[CrossRef][Medline]
- Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, Tsuji A 2000 Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Biochem Biophys Res Commun 273:251260[CrossRef][Medline]
- Hagenbuch B, Meier PJ 1996 Sinusoidal (basolateral) bile salt uptake systems of hepatocytes. Semin Liver Dis 16:129136[Medline]
- Kullak-Ublick GA, Hagenbuch B, Stieger B, Schteingart CD, Hofmann AF, Wolkoff AW, Meier PJ 1995 Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver. Gastroenterology 109:12741282[Medline]
- Hsiang B, Zhu Y, Wang Z, Wu Y, Sasseville V, Yang W-P, Kirchgessner TG 1999 A novel human hepatic organic anion transporting polypeptide (OATP2). J Biol Chem 274:3716137168[Abstract/Free Full Text]
- Lu R, Kanai N, Bao Y, Schuster VL 1996 Cloning, in vitro expression, and tissue distribution of a human prostaglandin transporter cDNA (HPGT). J Clin Invest 98:11421149[Abstract/Free Full Text]
- van Montfoort JE, Hagenbuch B, Fattinger K, Müller M, Groothuis GMM, Meijer DKF, Meier PJ 1999 Polyspecific organic anion transporting polypeptides mediate hepatic uptake of amphipathic type II organic cations. J Pharmacol Exp Ther 291:147152[Abstract/Free Full Text]
- Gao B, Hagenbuch B, Kullak-Ublick GA, Benke D, Aguzzi A, Meier PJ 2000 Organic anion-transporting polypeptides mediate transport of opioid peptides across blood-brain barrier. J Pharmacol Exp Ther 294:7379[Abstract/Free Full Text]
- Friesema EC, Docter R, Moerings EP, Stieger B, Hagenbuch B, Meier PJ, Krenning EP, Hennemann G, Visser TJ 1999 Identification of thyroid hormone transporters. Biochem Biophys Res Commun 254:497501[CrossRef][Medline]
- Bossuyt X, Muller M, Hagenbuch B, Meier PJ 1996 Polyspecific drug and steroid clearance by an organic anion transporter of mammalian liver. J Pharmacol Exp Ther 276:891896[Abstract]
- Saito H, Masuda S, Inui K 1996 Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney. J Biol Chem 271:2071920725[Abstract/Free Full Text]
- Sekine T, Watanabe N, Hosoyamada M, Kanai Y, Endou H 1997 Expression cloning and characterization of a novel multispecific organic anion transporter. J Biol Chem 272:1852618529[Abstract/Free Full Text]
- Hennemann G, Docter R, Fritschy JM, De Jong M, Krenning EP, Visser TJ 2001 Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocr Rev 22:451476[Abstract/Free Full Text]
- Cai S-Y, Wang W, Soroka CJ, Ballatori N, Boyer JL 2001 Molecular and functional characterization of a novel hepatic organic anion transporting polypeptide (OATP) from a primitive vertebrate. Hepatology 34:A1208
- Goncalves E, Lakshmanan M, Pontecorvi A, Robbins J 1990 Thyroid hormone transport in a human glioma cell line. Mol Cell Endocrinol 69:157165[CrossRef][Medline]
- Bianco AC, Salvatore D, Gereben B, Berry MJ, Larsen PR 2002 Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 23:3889[Abstract/Free Full Text]
- Gharbi-Chini J, Torresani J 1981 Thyroid hormone binding to plasma membrane preparations: studies in different thyroid states and tissues. J Endocrinol Invest 4:177183[Medline]
- Mendis-Handagama SM, Ariyaratne HB 2001 Differentiation of the adult Leydig cell population in the postnatal testis. Biol Reprod 65:660671[Abstract/Free Full Text]
- Manna PR, Poy P, Clark BJ, Stocco DM, Huhtaniemi IT 2001 Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis. J Steroid Biochem Mol Biol 76:167177[CrossRef][Medline]
- Abe T, Kakyo M, Sakagami H, Tokui T, Nishio T, Tanemoto M, Nomura H, Hebert SC, Masuno S, Kondo H, Yawo H 1998 Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2. J Biol Chem 273:2239522401[Abstract/Free Full Text]
- Kurisu H, Nilprabhassorn P, Wolkoff AW 1989 Preparation of [35S]sulfobromophthalein of high specific activity. Anal Biochem 179:7274[Medline]
- Stieger B, Hagenbuch B, Landmann L, Höchli M, Schroeder A, Meier PJ 1994 In situ localization of the hepatocytic Na+/taurocholate cotransporting polypeptide (Ntcp) in rat liver. Gastroenterology 107:17811787[Medline]
- Shi S, Key M, Kalra KL 1991 Antigen retrieval from formalin-fixed, paraffin-embedded tissue: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741748[Abstract]
- Hagenbuch B, Scharschmidt BF, Meier PJ 1996 Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes. Biochem J 316:901904[Medline]
- Eckhardt U, Schroeder A, Stieger B, Höchli M, Landmann L, Tynes R, Meier PJ, Hagenbuch B 1999 Polyspecific substrate uptake by the hepatic organic anion transporter oatp1 in stably transfected CHO cells. Am J Physiol 276:G1037G1042
- Palermo DP, DeGraaf ME, Marotti KR, Rehberg E, Post LE 1991 Production of analytical quantities of recombinant proteins in Chinese hamster ovary cells using sodium butyrate to elevate gene expression. J Biotechnol 19:3548[CrossRef][Medline]
- Rost B 1996 PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 266:525539[CrossRef][Medline]
- Devereux J, Haeberli P, Smithies O 1984 A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387395[Abstract]
- Page RDM 1996 TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357358[Medline]