* Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka-shi, Tokyo, Japan; and Department of Nephrology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto-shi, Kumamoto, Japan
Address correspondence to: Dr. Yoshikatsu Kanai, Department of Pharmacology and Toxicology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Phone: +81-422-47-5511; Fax: +81-422-79-1321; E-mail: ykanai{at}kyorin-u.ac.jp
Received for publication March 22, 2005.
Accepted for publication September 1, 2005.
Human organic anion transporter 4 (OAT4) is an apical organicanion/dicarboxylate exchanger in the renal proximal tubulesand mediates high-affinity transport of steroid sulfates suchas estrone-3-sulfate (E1S) and dehydroepiandrosterone sulfate.Here, two multivalent PDZ (PSD-95/Discs Large/ZO-1) proteinsPDZK1 and NHERF1 were examined as interactors of OAT4 by a yeasttwo-hybrid assay. These interactions require the extreme C-terminalregion of OAT4 and the first and fourth PDZ domains of PDZK1and the first PDZ domain of NHERF1. These interactions wereconfirmed by surface plasmon resonance assays (KD: 36 nM, 1.2µM, and 41.7 µM, respectively). In vitro bindingassays and co-immunoprecipitation studies revealed that theOAT4 wild-type but not a mutant lacking the PDZ motif interacteddirectly with both PDZK1 and NHERF1. OAT4, PDZK1, and NHERF1proteins were shown to be localized at the apical membrane ofrenal proximal tubules. The association with PDZK1 or NHERF1enhanced OAT4-mediated E1S transport activities in HEK293 cells(1.2- to 1.4-fold), and the deletion of the OAT4 C-terminalPDZ motif abolished this effect. The augmentation of the transportactivity was accompanied by alteration in Vmax of E1S transportvia OAT4 and was associated with the increased surface expressionlevel of OAT4 protein. This study indicates that the functionalactivity of OAT4 is modulated through the PDZ interaction withthe network of PDZK1 and NHERF1 and suggests that OAT4 is involvedin the regulated apical organic anion handling in the renalproximal tubules, provided by the PDZ scaffold.
The human organic anion transporter OAT4 (encoded by SLC22A11)is expressed in the kidney and the placenta and mediates thehigh-affinity transport of steroid sulfates such as estrone-3-sulfate(E1S) and dehydroepiandrosterone sulfate (DHEAS) (1). Becauseof its apical membrane localization in the renal proximal tubules(2), OAT4 had been presumed to be the apical exit pathway oforganic anions that are taken up by the basolateral entrancepathway such as OAT1 and OAT3 (3). Recently, Ugele et al. (4)found the OAT4 protein expression at the fetal side of the syncytiotrophoblastsin the placenta and proposed a role for OAT4 in the placentaluptake of fetal-derived steroid sulfates. On the basis of ourfinding that OAT4 is an apical organic anion/dicarboxylate exchanger(5), we suggested that OAT4 mainly functions as an apical entrancepathway for some organic anions in renal proximal tubules drivenby an outwardly directed dicarboxylate gradient created by Na+/dicarboxylateco-transporters (6). One possible role of OAT4 is as an apicalbackflux pathway (7) for some organic anions such as steroidsulfates, functionally coupled to the apical efflux transportersfor organic anions, such as MRP2, MRP4, and NPT1 (putative humanhomologue of OATV1) (8–13).
At the extreme C-terminal (CT) end, OAT4 has a specific protein–proteininteraction peptide sequence named the PDZ (PSD-95/Discs Large/ZO-1)motif (S-T-S-L) (14). PDZ domains have been identified in variousproteins and are known to be modular protein–protein recognitiondomains that play roles in protein targeting and protein complexassembly (15–17). These multidomain molecules not onlytarget and provide scaffolds for protein–protein interactionsbut also modulate the function of receptors and ion channels,by which they associate. Recently, we reported that the urate/anionexchanger URAT1 (18), which has a similar PDZ motif (S-T-Q-F)at its C-terminus, interacts with multivalent PDZ protein PDZK1(19,20). In the same study, we also identified that PDZK1 interactedwith the C-terminus of OAT4, which has a PDZ motif, but notwith those of human OAT1, OAT2, or OAT3, which do not have PDZmotifs (20). Both URAT1 and OAT4 are considered to functionto generate a reabsorptive pathway for organic anions localizedat the apical membrane of renal proximal tubules. Therefore,it is likely that these transporters bind with the same or otherPDZ protein(s) via their CT PDZ motifs.
In this study, we examined the interaction between OAT4 andtwo PDZ proteins, PDZK1 and NHERF1, using a yeast two-hybridassay, an in vitro pulldown assay, co-immunoprecipitation, andsurface plasmon resonance assay. We observed that the OAT4-mediatedtransport function is modulated equivalently by binding withtwo PDZ proteins via the C-terminus of OAT4. These results,together with recent emerging findings concerning PDZ proteins,will provide important insight into the renal apical handlingof organic anions through the transporter complexes supportedby PDZ protein networks.
Plasmid Construction
DNA encoding residues 513 to 550 (wt, T548A, L550A) or 513 to547 (d3) of human OAT4 were amplified by PCR using specificprimers (Table 1) and cloned into the EcoRI and XhoI sites ofthe pEG202 plasmid (bait) and into pGEX-6P-1 (Amersham Biosciences,Inc., Piscataway, NJ) to generate OAT4-CT-wt, OAT4-CT-d3, OAT4-L550A,or OAT4-T548A. The full-length coding sequence of human OAT4(wt) as well as its CT-3-amino-acid deletion mutant (d3) wereinserted into the mammalian expression plasmid pcDNA3.1 (Invitrogen,Gaithersburg, MD) for functional analysis and into the pEGFP-C2plasmid (Clontech, Palo Alto, CA) for GFP-fused OAT4 proteinpreparation. Proper folding of GFP-fused proteins was confirmedby their significant transport activities of E1S (data not shown).The full-length coding sequence of human NHERF1 was amplifiedfrom human kidney cDNA (Clontech) and subcloned into pcDNA3.1(Invitrogen) to generate pcDNA3.1-NHERF1 and into pJG4–5,a B42 activation domain fusion vector, to generate pJG4–5-NHERF1.Prey vectors (pJG4–5) that contained the individual PDZdomains of human NHERF1 were generated by PCR using specificprimers (Table 1). The pcDNA3.1 vector that contained the full-lengthhuman PDZK1 and prey vectors that contained a single PDZ domainof human PDZK1 were prepared as described previously (20).
Yeast Two-Hybrid Assay
Yeast two-hybrid assays were performed in the EGY48 strain withthe LexA-based GFP two-hybrid system (Grow‘n’Glowsystem; MoBiTec, Göttingen, Germany) as described elsewhere(21).
GST Fusion Protein Binding Assays
The OAT4-CT domains for GST fusion protein production in bacteriawere prepared as reported previously (21). In vitro translationwas performed from a plasmid carrying the full-length PDZK1and NHERF1 with the TNT T7 Quick for PCR DNA system (Promega,Madison, WI) in the presence of Transcend Biotinylated tRNA(Promega), as described elsewhere (20). Five microliters ofin vitro–translated products was applied into ProFoundPull-Down GST Protein:Protein Interaction Kit (Pierce, Rockford,IL) with 50 µl of GST-glutathione-Sepharose resin, andprotein complexes were eluted according to the manufacturer’sinstructions.
Surface Plasmon Resonance
The interactions of OAT4-CT with the first and fourth PDZ domainsof PDZK1 and the first PDZ domains of NHERF1 were investigatedusing a BIAcore 3000 analytical system (BIAcore AB, Uppsala,Sweden) based on principles described previously (22). Usingan amine coupling kit, GST-fused OAT4-CT wild-type or GST proteinalone was attached to a CM5 sensor chip according to the manufacturer’sinstructions, giving a gain of 13,269 resonance units (RU) forGST-OAT4-CT or 8566 RU for GST alone.
Tissue Distribution
Aliqouts of 1.5 µl of Human Multiple Tissue cDNA PanelsI and II (Clontech) were amplified as described previously (20).The OAT4 and NHERF1 primers used for PCR amplification are shownin Table 1.
Immunohistochemical Analysis
We used human single-tissue slides (Biochain) for light microscopicimmunohistochemical analysis, and they were treated with 10µg/ml primary rabbit polyclonal antibodies against OAT4and hPDZK1 and with EBP50 (NHERF1)-specific mAb purchased fromBD Bioscience (San Jose, CA) (23) at 4°C overnight, as reportedpreviously (5).
Cell culture and Transfections
Human embryonic kidney 293 (HEK293) cells were maintained inDMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 100 units/mlpenicillin, and 100 mg/ml streptomycin (Invitrogen) at 37°Cand 5% CO2. Transient transfection with Lipofectamine 2000 (Invitrogen)was performed according to the manufacturer’s recommendations.For the establishment of OAT4-expressing cells, stable transfectantswere selected for 2 wk by adding 1 mg/ml G418 to the medium.
Immunoprecipitation and Immunoblotting
Immunoprecipitation analysis was performed as described previously(20). GFP-fused OAT4 and associated proteins in HEK293 celllysate were immunoprecipitated by the anti-GFP antibody (full-lengthA.v. polyclonal antibody; Clontech) using the Seize Classic(A) Immunoprecipitation kit (Pierce). The eluates were treatedas described in the GST Fusion Protein Binding Assays section.The affinity-purified rabbit PDZK1 antibodies, EBP50 antibody(23), and horseradish peroxidase–conjugated goat anti-rabbitIgG (Amersham Biosciences, Inc.) were used for immunoblottingwith enhanced chemiluminescence reagents (ECL Plus; AmershamBiosciences, Inc.).
E1S Transport Activity Assays
HEK293 cells, plated on 24-well culture plates at a densityof 2 x 105 24 h before transfection, were incubated in Lipofectamine2000 as described above. After 36 h, the culture medium wasremoved and the cells were incubated in serum-free Dulbecco’smodified PBS (D-PBS; containing [in mM] 137 NaCl, 3 KCl, 8 Na2HPO4,1 KH2PO4, 1 CaCl2, and 0.5 MgCl2 [pH 7.4]) supplemented with5.5 mM d-glucose for 10 min. The uptake study was started byadding 500 µl of D-PBS that contained 50 nM [3H]E1S intothe plate. After 2 min, the cells were washed twice in ice-coldD-PBS and lysed in 0.1 N NaOH for 20 min for scintillation counting.
For determining the kinetic parameters, the concentrations ofE1S were varied from 50 nM to 3000 nM. OAT4-mediated E1S uptakewas calculated as the difference between the values of uptakeinto HEK293 cells that stably expressed OAT4 (HEK-OAT4) andthose of uptake into HEK293 cells that were transfected withvector only (HEK-mock). The kinetic parameters for the uptakevia OAT4 were estimated using the following equation: v = Vmax[S]/(Km + [S]), where v is the uptake rate of substrates, [S]is the substrate concentration (µM) in the medium, andKm is the Michaelis-Menten constant (µM). These valueswere determined using the Eadie-Hofstee equation.
Cell Surface Biotinylation
Surface biotinylation of OAT4 at the plasma membrane was performedas described elsewhere (20). Surface proteins in HEK-OAT4 cellsthat were transfected with pcDNA3.1(+)-hPDZK1, pcDNA3.1(+)-hNHERF1,or pcDNA3.1(+) empty vector were biotinylated with Sulfo-NHS-SS-Biotin(0.5 mg/ml; Pierce) in PBS for 30 min at 4°C. Cell lysatesthen were incubated with Ultralink-immobilized NeutrAvidin beads(Pierce) to precipitate biotinylated proteins. The bound proteinswere eluted with SDS sample buffer and were subjected to SDS-PAGEand Western blotting followed by ECL (Amersham Biosciences).OAT4 was detected with affinity-purified polyclonal OAT4 antibody(1:5000) (5).
Statistical Analyses
Uptake experiments were conducted three times, and each uptakeexperiment was performed in triplicate. Values are presentedas the means ± SEM. Statistical significance was determinedby t test.
Interaction between OAT4 and PDZK1/NHERF1 in a Yeast Two-Hybrid Assay
In our previous study, we reported that one of the OAT members,URAT1, binds to the multivalent PDZ domain protein PDZK1 (20).In parallel, we also identified that a bait vector that containsthe OAT4 carboxyl-terminal tail (OAT4-CT) binds to PDZK1 usinga yeast two-hybrid assay. Because the CT of OAT4 that has aPDZ motif (T-S-L) is different from the URAT1 CT (T-Q-F), thisdifference motivated us to extend the search for the interactionof OAT4 with other PDZ proteins expressed in the kidney, suchas NHERF1/EBP50, NHERF2/E3KARP, and IKEPP (24,25). We observedthe induction of two reporter genes (LEU and GFP) in the interactionof OAT4-CT with NHERF1 and NHERF2 and the induction of onlyone reporter gene (LEU) in the OAT4-CT and IKEPP interaction(data not shown). The specificities of these interactions wereconfirmed by the constructs with unrelated proteins. BecauseNHERF1 and PDZK1 but not NHERF2 are predominantly in the brushborder (26), we focused our research on the interactions ofOAT4-CT with both PDZK1 and NHERF1.
To identify the binding site of OAT4 that interacts with thetwo PDZ proteins, we constructed three mutant baits, as in ourprevious study (20). The first was a mutant that lacked thelast three residues of OAT4, which play a crucial role in PDZdomain recognition (OAT4-CT-d3). In the second and third, theextreme CT leucine (0 position) or threonine (–2 position)of OAT4 were replaced by alanine (L550A and T548A), which wasexpected to abolish or strongly reduce the binding of PDZ proteins(14). All three mutant baits were unable to interact with PDZK1full-length clone or the NHERF1 full-length clone (Table 2).Thus, these results suggested that OAT4 CT PDZ motif is thesite responsible for interactions with PDZK1 and NHERF1.
Table 2. Specificity of PDZK1 and NHERF1 for interaction with the C-terminus of OAT4 in the yeast two-hybrid assay
PDZK1 PDZ-1 and -4 Domains and NHERF1 PDZ-1 Domain Are Required for Interaction with OAT4-CT
PDZK1 possesses four PDZ domains and NHERF1 possesses two PDZdomains that assemble target proteins by binding to a CT thathas a consensus PDZ motif (14). To check the possible interactionsof the OAT4-CT with these PDZ domains of PDZK1 and NHERF1, weprepared prey vectors that contained the single PDZ domain oftwo PDZ proteins (PDZ-1, -2, -3, and -4 of PDZK1 and PDZ-1 and-2 of NHERF1). Interaction with OAT4-CT was observed when PDZ-1and -4 were present but not with PDZ-2 and -3 of PDZK1 and whenPDZ-1 was present but not with PDZ-2 of NHERF1 (Table 2).
In Vitro Binding of OAT4 and PDZK1/NHERF1
To confirm the ability of OAT4-CT to bind PDZK1 or NHERF1 invitro, we next used the GST pulldown assay to validate the interaction(Figure 1). GST fusion proteins that bear the wild-type CT regionof OAT4 (OAT4-CT-wt) or CT region mutants (OAT4-CT-d3) wereused to pull down the full-length PDZK1 or the full-length NHERF1proteins that were generated from in vitro translation experiments.The data demonstrated the same specificity of interaction forPDZK1/NHERF1 and OAT4 in the yeast two-hybrid assays. We didnot observe the binding of PDZK1 and NHERF1 to OAT4-CT thatlacked the PDZ motif (OAT4-CT-d3; Figure 1, A and B).
Figure 1. Interaction of PDZK1 and NHERF1 with organic anion transporter 4 (OAT4). (A and B) The in vitro translation products of full-length PDZK1 (A) or NHERF1 (B) using Transcend Biotinylated Lysine tRNA (Promega) were incubated with the GST alone (lane 1), GST-OAT4-CT-wt (lane 2), or GST-OAT4-CT-d3 (lane 3). The pulldown products were analyzed by SDS-PAGE. The input corresponds to the crude in vitro translation reaction. The positions of molecular mass standards are indicated on the right. (C) Western blot analysis of crude membranes from HEK293 cells that were transfected with vector (pcDNA3.1) alone (lane 1), with pcDNA3.1-hNHERF1 full length (lane 2), and with pcDNA3.1-hNHERF2 full length (lane 3). A single strong band of approximately 50 kD, which is consistent with NHERF1, was observed in lane 2 but not in lane 3. (D and E) Co-immunoprecipitation of the OAT4 and PDZK1 or NHERF1 proteins in HEK293 cells, respectively. HEK293 cells were transfected with pEGFP-C2 vectors encoding the wild-type full-length OAT4 (GFP-OAT4-FLwt, lane 1) or the full-length OAT4 that lacked the last three amino acid residues (GFP-OAT4-FLd3, lane 2) with pcDNA3.1-PDZK1 (D) or pcDNA3.1-NHERF1 (E) and then immunoprecipitated with anti-GFP antibody. Then, the immunoprecipitates were resolved by SDS-PAGE and probed with anti-PDZK1 antibodies (D) or with anti-EBP50 antibodies (E).
Co-Immunoprecipitation of OAT4 and PDZK1/NHERF1 from Heterologous Cells
To confirm further the interaction between OAT4 and PDZK1/NHERF1,we performed a co-immunoprecipitation study using the antibodiesagainst human PDZK1 (19) and NHERF1(EBP50) in HEK293 cells.To test the specificity of EBP50 antibody, first we performedWestern blot analysis of crude membranes from HEK293 cells thatwere transfected with vector (pcDNA3.1) alone (lane 1), withpcDNA3.1-hNHERF1 full length (lane 2), and with pcDNA3.1-hNHERF2full length (Figure 1C, lane 3). A single strong band of approximately50 kD, which is consistent with hNHERF1, was observed in lane2 but not in lane 3. We coexpressed full-length human OAT4 fusedwith GFP (GFP-OAT4) and full-length PDZK1/NHERF1 in HEK293 cells.PDZK1 and NHERF1 were co-immunoprecipitated with a GFP-specificantibody in the lysates from the cells transfected with GFP-OAT4-wt,but neither PDZK1 nor NHERF1 was co-immunoprecipitated in thosefrom the cells that were transfected with GFP-OAT4 that lackedthe last three amino acids (Figure 1, D and E).
Comparison of Binding Affinities between OAT4-CT and Individual PDZ Domains of PDZK1 or NHERF1
To quantify and compare the interaction of OAT4 with PDZK1 andNHERF1, we performed surface plasmon resonance experiments usingimmobilized GST-OAT4-CT and maltose-binding protein (MBP)-fusedPDZK1 PDZ domain 1, domain 4, and NHERF1 domain 1 proteins.As summarized in Table 3, the binding affinity for the PDZ domain1 of PDZK1 showed high affinity (36 nM), that for the PDZ domain4 of PDZK1 showed low affinity (1.2 µM), and that forthe PDZ domain 1 of NHERF1 exhibited the lowest affinity (41.7µM).
Table 3. Characteristics of interaction between OAT4 C-terminus and PDZK1 PDZ domain 1, domain 4, or NHERF1 PDZ domain 1a
Tissue Distribution of OAT4 and PDZK1/NHERF1 mRNA in Human Tissues
In humans, OAT4 mRNA has been detected in both the kidney andthe placenta (1); PDZK1 has been detected mainly in the liver,kidney, pancreas, gastrointestinal tract, and adrenal cortex(18); and NHERF1 has been detected ubiquitously (27). Usinghuman multiple cDNA panels, we examined the mRNA distributionof OAT4, PDZK1, and NHERF1 to compare the tissue distributionand their overlaps. OAT4 transcripts were detected in the kidney,placenta, testis, small intestine, and colon, whereas PDZK1and NHERF1 transcripts were detected in most of the tissuesanalyzed, confirming and expanding the previously describeddistribution in humans (Figure 2A). It is interesting that overlappingexpressions among OAT4, PDZK1, and NHERF1 were limited to thekidney, placenta, and small intestine. This is the first timethat OAT4 expression has been identified in the testis, smallintestine, and colon.
Figure 2. Localization of OAT4, PDZK1, and NHERF1. (A) Distribution of OAT4, PDZK1, and NHERF1 mRNA in human multiple cDNA panels. OAT4, PDZK1, and NHERF1 expression overlaps in the kidney, placenta, and small intestine. Control amplification with -actin was performed in parallel (bottom). (B) Immunohistochemical analyses of OAT4, PDZK1, and NHERF1 in serial sections of human kidney. OAT4 was detected in the apical membrane of the proximal convoluted tubules (left), and no staining was observed in the basolateral membrane and glomeruli (G). PDZK1 (middle) and NHERF1 (right) were also detected in the apical membrane of the proximal convoluted tubules.
Coexpression of OAT4, PDZK1, and NHERF1 in Human Kidney
OAT4 is present at the apical membrane of proximal tubules (2),and PDZK1 and NHERF1 have also been reported to be expressedin the same cells (19,27). To determine whether OAT4, PDZK1,and NHERF1 localize at the apical membrane of the renal proximaltubules, we performed immunostaining of human kidney sectionswith anti-OAT4, anti-PDZK1, and anti-NHERF1 antibodies. Consistentwith previous reports, in the renal cortex, the overlappingexpression of OAT4, PDZK1, and NHERF1 was detected at the apicalmembrane in most of the proximal tubular cells (Figure 2B).
OAT4 Transport Activities Are Increased in the Presence of PDZK1/NHERF1
To determine whether OAT4 and PDZK1/NHERF1 interactions arerequired to mediate the increase in OAT4 activity, we transfectedHEK293 cells with the pcDNA3.1(+) construct that contained full-lengthOAT4 (OAT4-wt), OAT4 that lacked the last three amino acids(OAT4-d3), or OAT4 without insert (mock). At an incubation timeof 2 min, we demonstrated that the uptake of [3H]E1S via boththe OAT4-wt and OAT4-d3 was from five- to six-fold higher thanthat by the mock (Figure 3, A and B). E1S transport activitiesin OAT4-wt were significantly increased after the coexpressionof PDZK1 (1.4-fold; Figure 3A) as well as NHERF1 (1.2-fold;Figure 3B). These effects were abolished when OAT4-d3 was coexpressedwith PDZK1 (Figure 3A) and NHERF1 (Figure 3B). These resultsindicate that the interaction between the OAT4-CT and PDZK1/NHERF1is essential for the functional increase in OAT4-mediated E1Stransport.
Figure 3. Effect of PDZK1 and NHERF1 on OAT4-mediated [3H]estrone-3-sulfate (E1S) transport activity. (A) Coexpression of OAT4 and PDZK1 increased E1S uptake (50 nM) significantly compared with that by cells that were transfected with OAT4 alone. This effect was abolished when the C-terminal (CT) deletion mutant of OAT4 was co-transfected with PDZK1, confirming that the interaction of PDZK1 with the OAT4-CT domain is responsible for this effect. (B) Coexpression of OAT4 and NHERF1 increased E1S uptake (50 nM) significantly compared with that by cells that were transfected with OAT4 alone. This effect was also abolished when the CT deletion mutant of OAT4 was co-transfected with NHERF1, confirming that the interaction of NHERF1 with the OAT4-CT domain is responsible for this effect. (C) Kinetic data using HEK293 cells that stably expressed OAT4 (HEK-OAT4) showed that PDZK1 () increased Vmax from 86.5 to 123.6 pmol/mg protein per min and did not change Km (from 944 to 971 µM), as compared with vector (pcDNA3.1) transfected (). It also showed that NHERF1 () increased the Vmax from 86.5 to 110.1 pmol/mg protein per min and did not change Km (from 944 to 1058 µM), compared with vector (pcDNA3.1) transfected (). *P < 0.05.
Next, we examined the effect of the coexpression of two PDZproteins on the kinetics of [3H]E1S transport via HEK293 cellsthat stably expressed OAT4 (HEK-OAT4) and that had been transfectedwith pcDNA3.1 vector alone, pcDNA3.1-PDZK1, or pcDNA3.1-NHERF1.Kinetic data showed that PDZK1 and NHERF1 significantly increasedVmax from 86.5 to 123.6 and from 86.5 to 110.1 pmol/mg proteinper min, respectively (P < 0.05), and did not change Km (from944 to 971 and from 944 to 1058, respectively), as comparedwith OAT4 transfected with vector alone (Figure 3C).
Surface Expression Level of OAT4 Protein
To determine changes in the cell surface expression level ofOAT4, we used a cell membrane–impermeant biotinylationreagent to label cell-surface proteins selectively. After thetreatment, cell lysates from HEK293 cells that stably expressedOAT4 that was transfected with hPDZK1, hNHERF1, or mock wascollected. The amount of surface-biotinylated OAT4 expressionon plasma membranes increased 1.3- to 1.4-fold (mock-transfected30.0 ± 1.2, PDZK1-transfected 40.2 ± 2.6, NHERF1-transfected42.5 ± 4.0 arbitrary units; n = 3) when PDZ proteinswere coexpressed (Figure 4). These changes seem close to theone in Vmax of OAT4-mediated transport observed in Figure 3C.
Figure 4. Surface expression level of OAT4. (A) cell surface biotinylation analysis of OAT4 that transiently expressed HEK293 cells that were transfected with pcDNA3.1 (vector alone; lane 1), those that were transfected with PDZK1 (lane 2), and those that were transfected with NHERF1 (lane 3). Single bands of approximately 60 kD, which are consistent with OAT4, were observed in both lanes. (B) Quantification of immunosignal for OAT4 (n = 3; bars are SD). *P < 0.05.
Recent studies have shown that the PDZ domains function as modularprotein–protein interaction motifs that serve to localizeproteins to specific subcellular sites, to organize signalingcascades, and to regulate cell signaling (15–17). Thisdomain binds to proteins that contain the tripeptide motif (S/T)-X-Ø(X = any amino acid and Ø = a hydrophobic residue) attheir CT (14). PDZ domains are also involved in receptor andion channel clustering, to recruit kinases and phosphatasesto their membrane-associated substrates. It has been postulatedthat the organic anion transporters, such as MRP2/4, NPT1, PEPT2,Oatp1, Oat-k1/k2, and CFEX (PAT1) that localize to the apicalmembrane and have the consensus PDZ-motif sequence at theirCT, target the apical membrane mediated by PDZ proteins (28,29).Recently, we demonstrated that the urate/anion exchanger URAT1,one of the OAT family members, interacted with the PDZ proteinPDZK1 via its CT (20). URAT1 was identified by in silico cloningusing the sequence information of OAT4 (18). It is interestingthat both clones have many similarities; for example, they showhigh sequence similarity (53%) and locate at the same gene locus,and their products localize at the apical membrane of the proximaltubules and are presumed to contribute the reabsorption of endogenousorganic anions (urate and steroid sulfates) (5,18,20). Therefore,these similarities prompted us to examine the protein–proteininteraction with well-known PDZ proteins such as PDZK1 and NHERF1that are expressed at the apical side of the proximal tubules.
The E1S transport study revealed that the coexpression of PDZK1and/or NHERF1 with OAT4 in HEK293 cells leads to a significantenhancement of OAT4-mediated [3H]E1S transport. Kinetic studiesof E1S transport showed a significant increase (1.2- to 1.4-fold)in the Vmax between PDZK1-, NHERF1-, and mock-transfected HEK293cells (Figure 3C). In addition, the cell surface biotinylationexperiment revealed that the augmentation of the E1S transportactivity was associated with the increased surface expressionlevel of OAT4 protein from HEK-OAT4 cells and transfected withPDZK1 or NHERF1. These results are similar to those of the URAT1-PDZK1interaction (20). Therefore, we speculated that both PDZK1 andNHERF1 have an equivalent potential for the stabilization and/oranchoring of OAT4 protein at the cell membrane, making it lesslikely to be internalized and subsequently degraded.
To date, some transporters that are localized at the apicalmembrane of epithelial cells, such as NaPi IIa, MRP2 (cMOAT),and CFTR, have been shown to bind with both PDZK1 and NHERF1(24,29–34). As Gisler et al. (29) demonstrated, most ofthe mouse transporters that could bind with PDZK1 in the kidneyalso interacted with NHERF1. They also indicated that PDZK1and NHERF1 could bind to each other. These results suggestedthat the scaffold under the plasma membrane is provided by thenetwork of these PDZ proteins and forms an orchestrated assemblyfor membrane transport proteins (35,36). Among various couplingsthat are mediated by the PDZ proteins, the clustering of someorganic anion transporters seems important for regulating theapical handling of organic anions (36). Although the bindingaffinities are >10-fold different (Table 3), both PDZK1 andNHERF1 demonstrated similar enhancement of OAT4-mediated E1Stransport activities. These results suggest the redundancy ofthe apical scaffolding network of the renal proximal tubulesand might explain the absence of a significant phenotype inPDZK1 knockout mice (37). Double knockout of both NHERF1 andPDZK1 may reveal the significance of their overlapping specificities.
In the kidney, a large part of organic solutes is reabsorbedand secreted by specific transporters that are localized atthe apical membrane of the proximal tubules. To achieve itsconcerted function, it seems plausible that transporters thatexhibit similar functions couple functionally and physically.Following our recent report on URAT1 (20), here we found thatOAT4 also sits on the scaffold of PDZK1 and NHERF1. In addition,the interaction between NPT1 CT and the third domain of PDZK1was found in mouse (29) and in human (P.J., unpublished observations).NPT1 is an organic anion/Cl– exchanger and may functionas an exit pathway for some organic anions (11). Because neitherOAT4 nor URAT1 bound with the third domain of PDZK1, the interactionbetween NPT1 CT and the third PDZ domain of PDZK1 enables usto speculate that the organic anion transport protein complexconsisted of reabsorptive transporters (e.g., OAT4, URAT1) andan efflux transporter (e.g., NPT1) tethered by PDZ scaffolds(38). The close spatial positioning of functionally relatedtransporter proteins may lead to the fast and efficient controlof the transtubular transport for organic anions (39). Furtherstudy is necessary to address this issue.
Acknowledgments
This work was supported in part by grants from the Ministryof Education, Culture, Sports, Science and Technology of Japan;the Japan Society for the Promotion of Science; Research onHealth Sciences focusing on Drug Innovation from the Japan HealthSciences Foundation; Mutual Aid Corporation for Private Schoolsof Japan; the Tokyo Biochemical Research Foundation; the NakatomiFoundation; the Salt Science Research Foundation (no. 0524);the Japan Foundation of Applied Enzymology; Heiwa Nakajima Foundation;and Health and Labor Sciences Research Grants for Research onAdvanced Medical Technology: Toxicogenomics Project.
Part of this work was presented at the 36th Annual Meeting andScientific Exposition of the American Society of Nephrology,San Diego, CA, October 2003.
We thank Akie Toki and Keiko Sakama for technical assistance.The anti-PDZK1 and anti-OAT4 polyclonal antibodies were suppliedby Transgenic Inc. (Kumamoto, Japan).
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Top
Abstract
Introduction
-actin was performed in parallel (bottom). (B) Immunohistochemical analyses of OAT4, PDZK1, and NHERF1 in serial sections of human kidney. OAT4 was detected in the apical membrane of the proximal convoluted tubules (left), and no staining was observed in the basolateral membrane and glomeruli (G). PDZK1 (middle) and NHERF1 (right) were also detected in the apical membrane of the proximal convoluted tubules.
) increased Vmax from 86.5 to 123.6 pmol/mg protein per min and did not change Km (from 944 to 971 µM), as compared with vector (pcDNA3.1) transfected (
). It also showed that NHERF1 (
) increased the Vmax from 86.5 to 110.1 pmol/mg protein per min and did not change Km (from 944 to 1058 µM), compared with vector (pcDNA3.1) transfected (