![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CELLULAR AND MOLECULAR
Department of Pharmaceutics, Rutgers, The State University of New Jersey (M.H., F.Z., K.L., G.Y.), and Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey (G.Y.)
Received November 26, 2006; accepted December 11, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Burckhardt and Burckhardt, 2003; Eraly et al., 2004; Sweet, 2005; Sekine et al., 2006). Six OATs (OAT1–6) have been identified by different laboratories (Lopez-Nieto et al., 1997; Sekine et al., 1997, 1998; Sweet et al., 1997; Wolff et al., 1997; Reid et al., 1998; Cihlar et al., 1999; Kusuhara et al., 1999; Lu et al., 1999; Cha et al., 2000; Ekaratanawong et al., 2004; Monte et al., 2004; Youngblood and Sweet, 2004). These OATs have distinct organ and cellular localizations and different substrate specificities. OAT1 and OAT3 are predominantly expressed at the basolateral membrane of kidney proximal tubule cells and the apical membrane of brain choroid plexus (Kojima et al., 2002; Motohashi et al., 2002; Nagata et al., 2002). OAT4, which is only found in humans, is expressed at the apical membrane of kidney proximal tubule cells (Babu et al., 2002) and the basolateral membrane of placental trophoblast (Ugele et al., 2003). OAT2 is expressed in hepatocytes and in the kidney. The cellular localization of OAT2 in the kidney is still controversial. Human OAT2 was localized to the basolateral membrane of kidney proximal tubule (Enomoto et al., 2002), whereas mouse and rat OAT2 were localized to the apical membrane of the kidney proximal tubule (Ljubojevic et al., 2006). OAT5 is expressed only in the kidney (Youngblood and Sweet, 2004; Anzai et al., 2005). OAT6 is expressed in the olfactory mucosa (Monte et al., 2004).
In the kidney, OAT1 and OAT3 use a tertiary transport mechanism to move organic anions across the basolateral membrane into the proximal tubule cells for subsequent exit across the apical membrane into the urine for elimination (You, 2002; Burckhardt and Burckhardt, 2003; Eraly et al., 2004; Sweet, 2005; Sekine et al., 2006). Through this tertiary transport mechanism, Na+K+-ATPase maintains an inwardly directed (blood-to-cell) Na+ gradient. The Na+ gradient then drives a sodium dicarboxylate cotransporter, sustaining an outwardly directed dicarboxylate gradient that is used by a dicarboxylate/organic anion exchanger (termed OAT) to move the organic anion substrate into the cell. This cascade of events indirectly links organic anion transport to metabolic energy and the Na+ gradient, allowing the entry of a negatively charged substrate against both its chemical concentration gradient and the electrical potential of the cell.
OAT family and another closely related organic cation transporter OCT family belong to a large group of related proteins, the major facilitator superfamily (MFS). The members of MFS share common structural features, including 12 putative transmembrane-spanning domains and intracellular carboxyl and amino termini. Recent elucidation of high-resolution crystal structures of two other MFS members, LacY (Abramson et al., 2003) and GlpT (Huang et al., 2003), suggests that all MFS members may share a common fold. Based on such assumption, three-dimensional structure models of rat OCT1 (Popp et al., 2005) and rabbit OCT2 (Zhang et al., 2005) have been developed using the structural template of LacY and GlpT. In such a model, transmembrane-spanning domains 1, 2, 4, 5, 7, 8, 10, and 11 form a large hydrophilic cleft for substrate binding. By analogy, the corresponding transmembrane domains in OATs may also be involved in the binding of organic anions. The putative transmembrane domain 7 has been indicated to be important for substrate selectivity of rat OAT3 (Feng et al., 2002). However, rat OAT3 is distinct from human OAT1 (hOAT1) in terms of substrate specificity and species difference. Therefore, whether the putative transmembrane domain 7 affects hOAT1 function is unknown. In the present study, we performed alanine-scanning mutagenesis of all the residues in the putative transmembrane domain 7 of hOAT1. We identified a group of residues critical for substrate binding and stability of hOAT1.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Site-Directed Mutagenesis. Mutant transporters were generated by site-directed mutagenesis using hOAT1-myc as a template. Epitope myc was tagged to the carboxyl terminus of hOAT1 to facilitate immunodetection of the transporter. We previously showed that hOAT1-myc retained the functional properties of the unmodified hOAT1 (Tanaka et al., 2004). The mutant sequences were confirmed by the dideoxy chain termination method.
Cell Culture and Transfections. COS-7 cells were grown at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. Confluent cells were transfected with DNA plasmids using Lipofectamine 2000 reagent (Invitrogen). For each well of 12-well plates, 2 µg of plasmid DNA was added into 125 µl of Opti-MEM medium (Invitrogen) for 5 min and then mixed with another 125 µl of Opti-MEM medium containing 5 µl of Lipofectamine 2000 reagent. The mixture was incubated at room temperature for 20 min and diluted with 1250 µl of Dulbecco's modified Eagle's medium before added into cell monolayer. As for cells plated on 48-well plates for uptake assays, 0.32 µg of plasmid DNA and 0.8 µl of Lipofectamine 2000 reagent were used for the transfection. The transfection efficiency was 
60% in all experiments as estimated by visualization by fluorescence microscopy after staining of OAT1-myc-transfected cells using anti-myc antibody in conjunction with fluorescein isothiocyanate-conjugated secondary antibody. Transfected cells were incubated for 48 h at 37°C and then used for transport assay and cell surface biotinylation.
Transport Measurements. For cells grown in 48-well plate, uptake solution was added. The uptake solution consisted of phosphate-buffered saline (PBS)/CM (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.1 mM CaCl2, and 1 mM MgCl2, pH 7.4) and PAH (1 µM[3H]PAH and 19 µM unlabeled PAH). Uptake was allowed to proceed for 10 min (within the linear range of the uptake), and then it was stopped by rapidly washing the cells with ice-cold PBS. The cells were then solubilized in 0.2 N NaOH, neutralized in 0.2 N HCl, and aliquoted for liquid scintillation counting. Uptake count was standardized by the amount of protein in each well. Values were mean ± S.E. (n = 3).
Cell Surface Biotinylation. Cell surface expression levels of hOAT1 and its mutants were examined using the membrane-impermeable biotinylation reagent NHS-SS-biotin (Pierce Chemical). hOAT1 and its mutants were transfected into cells grown in 12-well plates using Lipofectamine 2000 as described above. After 48 h, the medium was removed, and the cells were washed twice with 1 ml of ice-cold PBS/CM, pH 8.0. The plates were kept on ice, and all solutions were ice-cold for the rest of the procedure. Each well of cells was incubated with 0.5 ml of NHS-SS-biotin (0.5 mg/ml in PBS/CM) in two successive 20-min incubations on ice with very gentle shaking. The reagent was freshly prepared for each incubation. After biotinylation, each well was briefly rinsed with 1 ml of PBS/CM containing 100 mM glycine and then incubated with the same solution for 20 min on ice to ensure complete quenching of the unreacted NHS-SS-biotin. The cells were dissolved on ice for 1 h in lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100 with 1/100 protease inhibitor cocktail, pH 7.4), and the cell lysates were cleared by centrifugation at 16,000g at 4°C for 20 min. Streptavidin-agarose beads (Pierce Chemical) were then added to the supernatant to isolate cell membrane protein. hOAT1 and its mutants were detected in the pool of surface proteins by SDS-PAGE and immunoblotting.
To ensure that biotin was only labeling surface proteins, the integrity of the cell membrane during biotinylation was tested in each experiment by immunoblotting with an anti-actin antibody as we described previously (Xu et al., 2006). In all experiments, actin immunoreactivity was only detected when cell membranes were permeabilized by 0.1% Nonidet P-40, confirming the impermeability of the biotinylation reagent.
Electrophoresis and Immunoblotting. Protein samples (30 µg) were resolved on 7.5% SDS-PAGE minigels and electroblotted on to polyvinylidene difluoride membranes. The blots were blocked for 1 h with 5% nonfat dry milk in PBS/0.05% Tween 20. They were then washed and incubated for 1 h at room temperature with appropriate primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. The signals were detected by SuperSignal West Dura Extended Duration Substrate kit (Pierce Chemical).
Protease Treatment. hOAT1 and its mutants were transfected into COS-7 cells grown in 12-well plates using Lipofectamine 2000. Cells were then incubated in Dulbecco's modified Eagle's medium containing proteasome inhibitor MG132 (10 µM) or lysosomal inhibitors leupeptin/pepstatin A (50 and 100 µg/ml), NH4Cl (2, 5, and 15 mM), and chloroquine (100 and 200 µM) individually. The inhibitors under these concentrations efficiently inhibited degradation of wild-type hOAT1 and therefore were used to test the stability of the mutant transporters. Treated cells were collected at specific times as indicated in the figure legends and lysed. Equal amount of proteins were loaded on 7.5% SDS-PAGE minigels and analyzed by immunoblotting as described previously.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Surface and Total Cell Expression. Because cellular uptake of PAH requires that the transporter be localized to the plasma membrane, we tested whether the abolished transport activity of these mutants was due to abnormal expression of the transporter at the plasma membrane. For this purpose, cell surface biotinylation was performed using a cell-impermeable reagent NHS-SS-biotin. Our results (Fig. 2a) showed that mutants Y353A and Y354A were abundantly expressed at the cell surface, whereas the surface expression of the mutants W346A and T349A was undetectable.
|
The diminished cell surface expression of mutants W346A and T349A could result from an impaired membrane targeting or from a decreased total cell expression of the transporter protein. To differentiate between these possibilities, we determined the amount of the protein in whole cell extracts. Our results (Fig. 2b) showed that the total cell expression of these mutants correlated well with their cell surface expression. The 80-kDa band corresponds to the mature and cell surface form of hOAT1 (Tanaka et al., 2004), and the 60-kDa band corresponds to the immature form of hOAT1, which resides in the endoplasmic reticulum (Tanaka et al., 2004). The low intensity of the 60-kDa band in wild-type (Wt) hOAT1 and its mutants Y353A and Y354A suggests the efficient conversion of hOAT1 from its immature form to its mature form.
The Role of Tyr-353 and Tyr-354. The above-mentioned studies showed that substitution of Tyr-353 and Tyr-354 with alanine resulted in a complete loss of transport activity. Yet, these nonfunctional mutants had significant cell surface expression. To elucidate the molecular mechanisms underlying the effect of mutation at these positions, we mutated aromatic residues Tyr-353 and Tyr-354 to two other aromatic residues Phe or Trp. As shown in Fig. 3a, top, substitution of Tyr-353 with Trp was unable to recover the transport activity lost by substitution with alanine, whereas substitution of Tyr-353 with Phe recovered the transport activity to 30% wild type. Similar to the substitution with alanine, substitution of Tyr-354 with Phe or Trp both resulted in nonfunctional transporters (Fig. 3b, top). Immunoblot analysis (Fig. 3, a and b, bottom) showed that all the mutants had significant cell surface expression, compared with that of the wild-type hOAT1.
|
; Sepp-Lorenzino et al., 1995; Ward et al., 1995), and it removes misfolded or misaggregated proteins in the endoplasmic reticulum (Kopito, 1997). The lysosome degrades membrane proteins and extracellular proteins that enter the cell via endocytosis (Ward et al., 1995). These different pathways of proteolysis can be determined by their sensitivity to different inhibitors. Degradation of polypeptides by the proteasome can be inhibited by MG132. Lysosomal proteolysis can be inhibited by leupeptin, pepstatin A, NH4Cl, or chloroquine. As shown in Fig. 4, treatment of cells expressing wild-type hOAT1 with lysosomal inhibitors (50 µg/ml leupeptin/pepstatin A, 2 mM NH4Cl, and 100 µM chloroquine) led to the accumulation of a 80-kDa protein in the total cell extracts, which corresponds to the mature and cell surface form of hOAT1 (Tanaka et al., 2004). However, the same inhibitors did not result in the accumulation of 80-kDa mutant proteins. We also tried these inhibitors at higher concentrations (100 µg/ml leupeptin/pepstatin A, 5 and 15 mM NH4Cl, and 200 µM chloroquine). Again, no accumulation of mutant proteins was observed (data not shown). In contrast, treatment of cells with proteasomal inhibitor MG132 resulted in the accumulation of 60-kDa proteins in the total cell extracts for both the wild type and its mutants (Fig. 5). The 60-kDa protein corresponds to the immature form of hOAT1, which resides in the endoplasmic reticulum (ER) (Tanaka et al., 2004).
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Burckhardt and Burckhardt, 2003; Eraly et al., 2004; Sweet, 2005; Sekine et al., 2006). In the present study, we probed the contribution of the putative TM7 to the function of hOAT1 by combined approaches of site-directed mutagenesis, transport analysis, cell surface biotinylation, and protease inhibition. These studies provided new insight into the functional importance of this transmembrane segment. Alanine-scanning mutagenesis was first used to examine the functional importance of each residue within the putative TM7 of hOAT1. This approach identified four critical amino acid residues: Trp-346, Thr-349, Tyr-353, and Tyr-354. Substitution of these residues with alanine led to a complete loss of transport activity (Fig. 1). Other mutants with reduced transport activity (A348V, F351A, A352V, G355A, and L356A) may also be functionally important and need to be further investigated.
The lack of transport activity in mutants W346A, T349A, Y353A, and Y354A could result from the lack of expression of the mutant transporter protein or could result from impaired binding abilities of the mutants for their substrates. By directly measuring transporter expression at the cell surface and in the total cell extracts, we observed two distinct phenomena (Fig. 2): substitution of Trp-346 and Thr-349 with alanine resulted in a complete loss of transporter expression at the cell surface and in the total cell extracts, whereas substitution of Tyr-353 and Tyr-354 with alanine resulted in transporter proteins that had significant expression at the cell surface and in the total cell extracts compared with that of the wild-type hOAT1.
The effects of the side chain of Tyr-353 and Tyr-354 were further investigated by replacing these residues with Phe or Trp (Fig. 3). The side chain of Tyr contains an aromatic ring and a -OH group. The side chain of Phe contains only an aromatic ring, whereas the side chain of Trp contains both an aromatic ring and an indole ring. We showed that substitution of Tyr-353 with Trp was unable to recover the transport activity lost by substitution with alanine, whereas substitution of Tyr-353 with Phe recovered the transport activity to 30% of wild type. In contrast, substitution of Tyr-354 with either Phe or Trp could not recover the transport activity lost by substitution with alanine, suggesting that both the -OH group and the size of the side chain at position 353 and 354 are essential for the transport function. The -OH group often participates in substrate binding or maintaining the protein structure through forming hydrogen bonds (Diez-Sampedro et al., 2001; Witt et al., 2002; Leu et al., 2003).
To investigate the mechanisms underlying the total loss of protein expression when Trp-346 and Thr-349 were replaced by alanine, we examined the degradation of these mutants using both the proteasomal and the lysosomal inhibitors. Although lysosomal inhibitors did not result in the accumulation of the mutant proteins (Fig. 4), proteasomal inhibitor led to the accumulation of the immature form of the mutants (Fig. 5). Proteasome serves in a "proof-reading" process in the ER and degrades misfolded or incompletely oligomerized polypeptides. Therefore, mutation at Trp-346 and Thr-349 may impose a folding defect on hOAT1, which is recognized by the ER quality control machinery as non-native and therefore marked for degradation by proteasome. As a result, escape from the ER for maturation is severely compromised.
In conclusion, we demonstrated that Tyr-353 and Tyr-354 play critical roles in the substrate binding of hOAT1, whereas Trp-346 and Tyr-349 are essential for maintaining the stability of the transporter. This is the first detailed molecular identification and characterization of critical amino acid residues in the putative TM7 of hOAT1 and may provide important insight into the structure-function relationships of the organic ion transporter family.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: OAT, organic anion transporter; MFS, major facilitator superfamily; h, human; PAH, p-aminohippuric acid; NHS-SS-biotin, sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate; PBS, phosphate-buffered saline; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane; Wt, wild-type; ER, endoplasmic reticulum.
Address correspondence to: Dr. Guofeng You, Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Rd., Piscataway, NJ 08854. E-mail: gyou{at}rci.rutgers.edu.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abramson J, Smirnova I, Kasho V, Verner G, Kaback HR, and Iwata S (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science (Wash DC) 301: 610-615.
Anzai N, Jutabha P, Enomoto A, Yokoyama H, Nonoguchi H, Hirata T, Shiraya K, He X, Cha SH, Takeda M, et al. (2005) Functional characterization of rat organic anion transporter 5 (Slc22a19) at the apical membrane of renal proximal tubules. J Pharmacol Exp Ther 315: 534-544.
Babu E, Takeda M, Narikawa S, Kobayashi Y, Enomoto A, Tojo A, Cha SH, Sekine T, Sakthisekaran D, and Endou H (2002) Role of human organic anion transporter 4 in the transport of ochratoxin A. Biochim Biophys Acta 1590: 64-75.[Medline]
Burckhardt BC and Burckhardt G (2003) Transport of organic anions across the basolateral membrane of proximal tubule cells. Rev Physiol Biochem Pharmacol 146: 95-158.[Medline]
Cha SH, Sekine T, Kusuhara H, Yu E, Kim JY, Kim DK, Sugiyama Y, Kanai Y, and Endou H (2000) Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta. J Biol Chem 275: 4507-4512.
Cihlar T, Lin DC, Pritchard JB, Fuller MD, Mendel DB, and Sweet DH (1999) The antiviral nucleotide analogs cidofovir and adefovir are novel substrates for human and rat renal organic anion transporter 1. Mol Pharmacol 56: 570-580.
Diez-Sampedro A, Wright EM, and Hirayama BA (2001) Residue 457 controls sugar binding and transport in the Na+/glucose cotransporter. J Biol Chem 276: 49188-49194.
Ekaratanawong S, Anzai N, Jutabha P, Miyazaki H, Noshiro R, Takeda M, Kanai Y, Sophasan S, and Endou H (2004) Human organic anion transporter 4 is a renal apical organic anion/dicarboxylate exchanger in the proximal tubules. J Pharmacol Sci 94: 297-304.[CrossRef][Medline]
Enomoto A, Takeda M, Shimoda M, Narikawa S, Kobayashi Y, Kobayashi Y, Yamamoto T, Sekine T, Cha SH, Niwa T, et al. (2002) Interaction of human organic anion transporters 2 and 4 with organic anion transport inhibitors. J Pharmacol Exp Ther 301: 797-802.
Eraly SA, Bush KT, Sampogna RV, Bhatnagar V, and Nigam SK (2004) The molecular pharmacology of organic anion transporters: from DNA to FDA? Mol Pharmacol 65: 479-487.
Feng B, Shu Y, and Giacomini KM (2002) Role of aromatic transmembrane residues of the organic anion transporter, rOAT3, in substrate recognition. Biochemistry 41: 8941-8947.[CrossRef][Medline]
Huang Y, Lemieux MJ, Song J, Auer M, and Wang DN (2003) Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science (Wash DC) 301: 616-620.
Jensen TJ, Loo MA, Pind D, Williams DB, Goldberg AL, and Riordan JR (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129-135.[CrossRef][Medline]
Kojima R, Sekine T, Kawachi M, Cha SH, Suzuki Y, and Endou H (2002) Immunolocalization of multispecific organic anion transporters, OAT1, OAT2, and OAT3, in rat kidney. J Am Soc Nephrol 13: 848-857.
Kopito RR (1997) ER quality control: the cytoplasmic connection. Cell 88: 427-430.[CrossRef][Medline]
Kusuhara H, Sekine T, Utsunomiya-Tata N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kanai Y, and Endou H (1999) Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J Biol Chem 274: 13675-13680.
Leu YJ, Chern SS, Wang SC, Hsiao YY, Amiraslanov I, Liaw YC, and Liao YD (2003) Residues involved in the catalysis, base specificity, and cytotoxicity of ribonuclease from Rana catesbeiana based upon mutagenesis and X-ray crystallography. J Biol Chem 278: 7300-7309.
Ljubojevic M, Balen D, Breljak D, Kusan M, Anzai N, Bahn A, Burckhardt G, and Sabolic I (2006) Renal expression of organic anion transporter OAT2 in rats and mice is regulated by sex hormones. Am J Physiol 292: F361-F362.
Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, and Nigam SK (1997) Molecular cloning and characterization of NKT, a gene product related to the organic cation transporter family that is almost exclusively expressed in the kidney. J Biol Chem 272: 6471-6478.
Lu R, Chan BS, and Schuster VL (1999) Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am J Physiol 276: F295-F303.
Monte JC, Nagle MA, Eraly SA, and Nigam SK (2004) Identification of a novel murine organic anion transporter family member, OAT6, expressed in olfactory mucosa. Biochem Biophys Res Commun 323: 429-436.[CrossRef][Medline]
Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, and Inui K (2002) Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J Am Soc Nephrol 13: 866-874.
Nagata Y, Kusuhara H, Endou H, and Sugiyama Y (2002) Expression and functional characterization of rat organic anion transporter 3 (rOat3) in the choroid plexus. Mol Pharmacol 61: 982-988.
Popp C, Gorboulev V, Muller TD, Gorbunov D, Shatskaya N, and Koepsell H (2005) Amino acids critical for substrate affinity of rat organic cation transporter 1 line the substrate binding region in a model derived from the tertiary structure of lactose permease. Mol Pharmacol 67: 1600-1611.
Reid G, Wolff NA, Dautzenberg FM, and Burckhardt G (1998) Cloning of a human renal p-aminohippurate transporter, hROAT1. Kidney Blood Press Res 21: 233-237.[CrossRef][Medline]
Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y, and Endou H (1998) Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429: 179-182.[CrossRef][Medline]
Sekine T, Miyazaki H, and Endou H (2006) Molecular physiology of renal organic anion transporters. Am J Physiol 290: F251-F261.
Sekine T, Watanabe N, Hosoyamada M, Kanai Y, and Endou H (1997) Expression cloning and characterization of a novel multispecific organic anion transporter. J Biol Chem 272: 18526-18529.
Sepp-Lorenzino L, Ma Z, Lebwohl DE, Vinitsky A, and Rosen N (1995) Herbimycin A induces the 20 S proteasome- and ubiquitin-dependent degradation of receptor tyrosine kinases. J Biol Chem 270: 16580-16587.
Sweet DH (2005) Organic aion transporter (Slc 22a) family members as mediators of toxicity. Toxicol Appl Pharmacol 204: 198-215.[CrossRef][Medline]
Sweet DH, Wolff NA, and Pritchard JB (1997) Expression cloning and characterization of rOAT1. The basolateral organic anion transporter in rat kidney. J Biol Chem 272: 30088-30095.
Tanaka K, Xu W, Zhou F, and You G (2004) Role of glycosylation in the organic anion transporter OAT1. J Biol Chem 279: 14961-14966.
Ugele B, St-Pierre MV, Pihusch M, Bahn A, and Hantschmann P (2003) Characterization and identification of steroid sulfate transporters of human placenta. Am J Physiol 284: E390-E398.
Ward CL, Omura S, and Kopito RR (1995) Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83: 121-127.[CrossRef][Medline]
Witt H, Schlodder E, Teutloff C, Niklas J, Bordignon E, Carbonera D, Kohler S, Labahn A, and Lubitz W (2002) Hydrogen bonding to P700: site-directed mutagenesis of threonine A739 of photosystem I in Chlamydomonas reinhardtii. Biochemistry 41: 8557-8569.[CrossRef][Medline]
Wolff NA, Werner A, Burkhardt S, and Burckhardt G (1997) Expression cloning and characterization of a renal organic anion transporter from winter flounder. FEBS Lett 417: 287-291.[CrossRef][Medline]
Xu W, Tanaka K, Sun Anqiang, and You G (2006) The functional role of the C terminus of human organic anion transporter hOAT1. J Biol Chem 281: 31178-31183.
You G (2002) Structure, function, and regulation of renal organic anion transporters. Med Res Rev 22: 602-616.[CrossRef][Medline]
Youngblood GL and Sweet DH (2004) Identification and functional assessment of the novel murine organic anion transporter Oat5 (Slc22a19) expressed in kidney. Am J Physiol 287: F236-F244.
Zhang X, Shirahatti NV, Mahadevan D, and Wright SH (2005) A conserved glutamate residue in transmembrane helix 10 influences substrate specificity of rabbit OCT2 (SLC22A2). J Biol Chem 280: 34813-34822.
This article has been cited by other articles:
|
|
 |
|
  S. K. Banu, J. Lee, M. C. Satterfield, T. E. Spencer, F. W. Bazer, and J. A. Arosh Molecular Cloning and Characterization of Prostaglandin (PG) Transporter in Ovine Endometrium: Role for Multiple Cell Signaling Pathways in Transport of PGF2{alpha} Endocrinology, January 1, 2008; 149(1): 219 - 231. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
