The Multivalent PDZ Domain-containing Protein PDZK1 Regulates Transport Activity of Renal Urate-Anion Exchanger URAT1 via Its C Terminus
2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês
10.1074/jbc.m406724200
ISSN1083-351X
AutoresNaohiko Anzai, Hiroki Miyazaki, Rie Noshiro, Suparat Khamdang, Arthit Chairoungdua, Ho Jung Shin, Atsushi Enomoto, Shinichi Sakamoto, Taku Hirata, Kimio Tomita, Yoshikatsu Kanai, Hitoshi Endou,
Tópico(s)Porphyrin Metabolism and Disorders
ResumoThe urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents (Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., Hosoyamada, M., Takeda, M., Sekine, T., Igarashi, T., Matsuo, H., Kikuchi, Y., Oda, T., Ichida, K., Hosoya, T., Shimotaka, K., Niwa, T., Kanai, Y., and Endou, H. (2002) Nature 417, 447–452). URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (KD = 1.97–514 nm). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the Vmax of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules. The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents (Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., Hosoyamada, M., Takeda, M., Sekine, T., Igarashi, T., Matsuo, H., Kikuchi, Y., Oda, T., Ichida, K., Hosoya, T., Shimotaka, K., Niwa, T., Kanai, Y., and Endou, H. (2002) Nature 417, 447–452). URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (KD = 1.97–514 nm). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the Vmax of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules. Urate is the major inert end product of purine degradation in humans and higher primates in contrast to most other mammals because of the genetic silencing of hepatic oxidative enzyme uricase (1Roch-Ramel F. Guisan B. News Physiol. Sci. 1999; 14: 80-84PubMed Google Scholar, 2Sica D.A. Schoolwerth A.C. Brenner B.M. The Kidney. 6th Ed. Saunders, Philadelphia2000: 680-700Google Scholar). The kidney plays a dominant role in urate elimination; it excretes ∼70% of the daily urate production. Urate exists primarily as a weak acid at physiological pH (pKa 5.75), and most of it is dissociated in blood and is freely filtered through the glomerulus. Thus, urate enters the proximal tubule in its anionic form, but it hardly permeates the tubular cells in the absence of facilitated mechanisms owing to its hydrophilicity. The transport mechanisms for urate are localized in the proximal tubule. In humans, urate is almost completely reabsorbed, which results in the excretion of ∼10% of its filtered load. The absence of uricase and the presence of an effective renal urate reabsorption system contribute to higher blood urate levels in humans. Therefore, it was postulated that defects in tubular urate transport cause hypouricemia and decreased renal urate clearance leads to hyperuricemia in most hyperuricemic patients (3Maesaka J.K. Fishbane S. Am. J. Kidney Dis. 1998; 32: 917-933Abstract Full Text PDF PubMed Scopus (195) Google Scholar). Recently, we have identified the long hypothesized urate transporter in the human kidney (URAT1, 1The abbreviations used are: URAT1, urate-anion exchanger 1; OAT, organic anion transporter; GST, glutathione S-transferase; PDZ, PSD-95/Dlg/ZO-1 homology domain; NHERF, Na+/H+-exchanger regulatory factor; CT, C terminus; wt, wild type; PBS, phosphate-buffered saline; GFP, green fluorescent protein; HEK, human embryonic kidney; MBP, maltose binding protein; SPR, surface plasmon resonance; IKEPP, intestinal and kidney-enriched PDZ protein.1The abbreviations used are: URAT1, urate-anion exchanger 1; OAT, organic anion transporter; GST, glutathione S-transferase; PDZ, PSD-95/Dlg/ZO-1 homology domain; NHERF, Na+/H+-exchanger regulatory factor; CT, C terminus; wt, wild type; PBS, phosphate-buffered saline; GFP, green fluorescent protein; HEK, human embryonic kidney; MBP, maltose binding protein; SPR, surface plasmon resonance; IKEPP, intestinal and kidney-enriched PDZ protein. encoded by SLC22A12), a urate-anion exchanger localized on the apical side of the proximal tubule (4Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S.H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimotaka K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar). URAT1 is targeted by uricosuric and antiuricosuric agents that affect urate excretion (e.g. benzbromarone, probenecid, and pyrazinamide). The pharmacological properties manifested by URAT1 cRNA-injected Xenopus oocytes are consistent with those of the previously described urate transport activities in human brush-border membrane vesicles. We also found that defects in SLC22A12 lead to idiopathic renal hypouricemia (Mendelian Inheritance in Man number 220150), and patients with such defects show a high fractional urate excretion such as 95 ± 10% (normally <10%). These results indicate that URAT1 regulates blood urate level and vice versa, that is, to control blood urate levels, the URAT1 transport function should be regulated. A newly found genetic alteration in SLC22A12 from a patient of an idiopathic renal hypouricemia has prompted us to consider the importance of the URAT1 extreme intracellular C-terminal region for its function (5Ichida K. Hosoyamada M. Hisatome I. Enomoto A. Hikita M. Endou H. Hosoya T. J. Am. Soc. Nephrol. 2004; 15: 164-173Crossref PubMed Scopus (313) Google Scholar). A 5-bp deletion near the URAT1 C-terminal end (1639–1643del) causes frameshift, and the seven amino acids in the terminal sequences have changed into eight different amino acids. The URAT1 transport activity of this mutation is low in the Xenopus oocyte expression system. Interestingly, the PDZ binding motif at the C-terminal end of URAT1, which is known to participate in protein-protein interaction, disappears by this amino acid sequence modification. PDZ (PSD-95, DglA, and ZO-1)-binding domains have been identified in various proteins and are known to be modular protein-protein recognition domains that play a role in protein targeting and protein complex assembly (6Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 7Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 8Fanning A.S. Anderson J.M. Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (273) Google Scholar). These domains range from 80 to 90 amino acids in length and bind typically to proteins containing the tripeptide motif (S/T)XΦ (X = any amino acid and Φ = a hydrophobic residue) at their C termini (9Harris B.Z. Lim W.A. J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar, 10Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1212) Google Scholar). These multidomain molecules not only target and provide scaffolds for protein-protein interactions but also modulate the function of receptors and ion channels, by which they associate (11Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 12Jackson M. Song W. Liu M.-Y. Jin L. Dykes-Hoberg M. Lin C.-l.G. Bowers W.J. Federoff H.J. Sternweis P.C. Rothstein J.D. Nature. 2001; 410: 89-93Crossref PubMed Scopus (203) Google Scholar, 13Raghuram V. Mak D.D. Foskett J.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1300-1305Crossref PubMed Scopus (197) Google Scholar, 14Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 15Horio Y. Hibino H. Inanobe A. Yamada M. Ishii M. Tada Y. Satoh E. Hata Y. Takai Y. Kurachi Y. J. Biol. Chem. 1997; 272: 12885-12888Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 16Kurschner C. Mermelstein P.G. Holden W.T. Surmeier D.J. Mol. Cell Neurosci. 1998; 11: 161-172Crossref PubMed Scopus (101) Google Scholar). The disruption of the association between PDZ proteins and their targets contributes to the pathogenesis of a number of human diseases, most probably because of the failure of PDZ proteins to appropriately target and modulate the actions of associated proteins (6Hung A.Y. Sheng M. J. Biol. Chem. 2002; 277: 5699-5702Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar). In this study, we use the yeast two-hybrid approach to investigate the putative URAT1-associated proteins that modulate its transport function. We identify the multivalent PDZ domain-containing protein PDZK1 as an apparent partner of URAT1 in the human kidney. Moreover, we show a functional consequence of PDZK1-URAT1 interaction in transfected HEK293 cells, where URAT1 transport activities were increased by 1.4-fold by coexpression of PDZK1. We speculate that PDZK1 is a scaffolding protein that may be a physiological regulator of the function of URAT1. Plasmid Construction—The C-terminal fragments of human URAT1 cDNA, comprising three mutants (designated as d3, F555A, and T553A), were generated by PCR (antisense primers, 5′-CTC TCG AGC TAA AAC TGT GTG GAT TTT A-3′, 5′-CCC TCG AGC TAG GAT TTT AGG ACA GAG TTC-3′, 5′-CCC TCG AGC TAA GCC TGT GTG GAT TTT AGG A-3′, and 5′-CCC TCG AGC TAA AAC TGT GCG GAT TTT A-3′, respectively; sense primer, 5′-CGA ATT CCT GCC CGA GAC CCA GAG-3′) and inserted into the EcoRI and XhoI sites of the pEG202 plasmid, a LexA DNA-binding domain fusion vector. The same human URAT1 C-terminal fragments were also inserted into the pGEX-6P-1 plasmid (Amersham Biosciences) for GST fusion protein production. The full-length coding sequences of human URAT1 (wt) as well as its C-terminal 3-amino acid deletion mutants (d3) were inserted into the mammalian expression plasmid pcDNA3.1 (Invitrogen) for functional analysis and into the pEGFP-C2 plasmid for EGFP-URAT1 (Clontech) fusion protein preparation. The full-length coding sequence of human PDZK1 (GenBank™ accession number NM_002614) was amplified from the Cap-site human kidney cDNA (Nippongene, Japan) and subcloned into pcDNA3.1, into the pJG4-5 plasmid and a B42 activation domain fusion vector. Prey vectors (pJG4-5 and pMAL-c2X (New England Biolabs) for MBP fusion protein preparation) containing a single one PDZ domain of human PDZK1 were generated by PCR (PDZ1: sense primer, 5′-CTG CAA TTG ATG ACC TCC ACC TTC AAC-3′, and antisense primer, 5′-CCC TCG AGC TAC TTC TGA CTT TGA CCC A-3′; PDZ2: sense primer, 5′-CGA ATT CCA GAA GGA GCA AGG TTT G-3′, and antisense primer, 5′-CCC TCG AGC TAT TTC AAA CTG GCT TC-3′; PDZ3: sense primer, 5′-CGA ATT CAA GCG TCA TGT TGA GCA G-3′, and antisense primer, 5′-CCC TCG AGC TAA GAA GTG GGA GTA GGA GC-3′; PDZ4: sense primer, 5′-CGA ATT CAA GGA GGC TCC AGC TCC-3′, and antisense primer, 5′-CGC TCG AGT CAC ATC TCT GTA TCT TC-3′). The sequences of all constructs were confirmed by automatic DNA sequencing (Applied Biosystems 3730xl Sequencer). Library Construction and Yeast Two-hybrid Screen—A human kidney cDNA library was constructed as described previously (11Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Briefly, human kidney poly(A)+ RNA purchased from Clontech was used to produce cDNA using a cDNA synthesis kit (Stratagene) with modified oligo(dT) primers carrying an XhoI restriction site and EcoRI adaptors to allow unidirectional cloning in an appropriate vector. The cDNAs were then purified using a CHROMA SPIN-400 column (Clontech) to pool cDNAs of 0.5 kb or longer, and ligated into the pJG4-5 prey vector previously digested with EcoRI and XhoI. After transformation in bacteria, 2.1 × 106 independent clones were obtained. Sixteen clones were randomly picked and digested with EcoRI and XhoI; all of them contained inserts with an average size of 1.3 kb (ranging from 0.5 to more than 3 kb). A URAT1 C-terminal bait corresponding to the last 39 amino acids of URAT1 was used to screen 1.8 × 107 clones of the human kidney cDNA library with the LexA-based GFP two-hybrid system (Grow'n'Glow system; MoBiTec). Several positive clones were obtained and confirmed in a second round of screening using the yeast system. The expression of all the bait constructs in yeast was confirmed by Western blot analysis of yeast protein extracts using an anti-LexA antibody (Santa Cruz Biotechnology) (data not shown). GST Fusion Protein Binding Assay—The URAT1 intracellular C-terminal region used as bait in the two-hybrid screen was inserted into the pGEX-6P-1 plasmid (Amersham Biosciences) for GST fusion protein production in bacteria as reported previously (11Anzai N. Deval E. Schaefer L. Friend V. Lazdunski M. Lingueglia E. J. Biol. Chem. 2002; 277: 16655-16661Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Briefly, the bacterial pellet was resuspended in a sonication buffer (50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 1 mm EDTA, and 1 mm dithiothreitol) and sonicated for 1 min. Triton X-100 (1% final) was added to the mixture, which was then centrifuged for 30 min at 15,000 rpm at 4 °C. The supernatant was used for protein binding assay. Maltose binding protein (MBP), fused proteins consisting of PDZK1 individual PDZ domains were purified as described above. Then they were purified using a MagExtracter MBP fusion Protein Purification kit (TOYOBO, Osaka, Japan) according to the manufacturer's instructions. In vitro translation was performed from a plasmid carrying the full-length PDZK1 preceded by a T7 promoter sequence with the TnT T7 Quick for PCR DNA system (Promega) in a final volume of 50 μl for 60 min at 30 °C in the presence of Transcend Biotinylated tRNA (Promega). 5 μl each of in vitro-translated products or of MBP fusion proteins was applied together with 50 μl of GST-glutathione-Sepharose resin to Handee Spin Cup columns using the ProFound Pull-Down GST Protein:Protein Interaction kit (Pierce), and protein complexes were eluted according to the manufacturer's instructions. The eluted samples were resuspended in Laemmli buffer, heated for 5 min at 95 °C, and electrophoresed in 10% SDS-PAGE gels; the fractionated proteins were blotted onto polyvinylidene difluoride membranes. The precipitated proteins were detected with the Transcend Chemiluminescent Translation Detection System (Promega) or immunoblotting using anti-MBP antiserum (New England Biolabs) developed by enhanced chemiluminescence. Surface Plasmon Resonance—The interaction of URAT1 C terminus with individual PDZ domains of PDZK1 was investigated by the use of a BIAcore 3000 analytical system (BIAcore AB) based on principles described previously (17Malmqvist M. Nature. 1993; 361: 186-187Crossref PubMed Scopus (525) Google Scholar). URAT1 C-terminal fragment, referred to as the ligand, was immobilized on a sensor chip, and the interaction with PDZK1 individual PDZ domains fused with MBP, referred as the analytes, was detected through the mass changes of the reflective index on the sensor surface. All of the reagents such as an amine coupling kit, running buffer HBS-EP (0.01 m HEPES, pH 7.4, 0.15 m NaCl, 3 mm EDTA, 0.005% Surfactant P20), and the CM5 sensor chip were obtained from BIAcore AB. Using an amine coupling kit, GST-fused URAT1 CT wild-type or GST protein alone was attached to a CM5 sensor chip according to the manufacturer's instructions, giving a gain of 10,673 resonance units for GST-URAT1-CT or 8,566 resonance units for GST alone. Binding experiments were performed with the PDZK1 single one PDZ domains (PDZ1 to PDZ4) fused with MBP as described above. The analyte was injected at a flow rate of 30 μl/min in HBS-EP buffer at 25 °C, and the association and dissociation phases (upon switching back to buffer) were monitored for 120 and 180 s, respectively. For data acquisition, five different concentrations of each protein were used. At least two replicate experiments were performed for each fusion protein. Data were analyzed with the BIAevaluation program 3.2 (BIAcore). Preparation of Antibodies—Corresponding to the 14 amino acids of the N terminus of human PDZK1, rabbit anti-PDZK1 polyclonal antibody raised against the keyhole limpet hemocyanin-conjugated synthesized peptides, MTSTFNPREC (amino acids 1–10 of the human PDZK1 amino acid sequence), was generated. Tissue Distribution—Human Multiple Tissue cDNA Panels I and II were purchased from Clontech. Each sample (1.5 μl) was amplified in 50 μl of the PCR mix consisting of 2.5 units of Taq Polymerase (Promega), 1× reaction buffer, 1.5 mm MgCl2, and 200 μm dNTP under the following conditions: 35 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C for URAT1 and PDZK1; only 25 cycles were performed for β-actin. The PCR products (5 μl) were resolved on a 2% agarose gel. The primers used for PCR amplification are as follows: 5′-CTG GCA ACG GAC TGG AGA TTA-3′ (forward primer for hURAT1), 5′-TGT AGT AGC GCA CGG GTT GG-3′ (reverse primer for hURAT1), 5′-GAG GAG AAC TTG GAG GTG TT-3′ (forward primer for hPDZK1), 5′-TTT GTA GGC TGG TGA TGA CT-3′ (reverse primer for hPDZK1), 5′-GAG GAG AAC TTG GAG GTG TT-3′ (forward primer for human β-actin), and 5′-TTT GTA GGC TGG TGA TGA CT-3′ (reverse primer for human β-actin). Immunohistochemical Analysis—The antibodies against human URAT1 used in this study have been shown to be specific for its synthetic peptide, as previously described (4Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S.H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimotaka K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar). We used human single-tissue slides (Biochain) for light microscopic immunohistochemical analysis using the streptavidin-biotin-horseradish peroxidase complex technique (LSAB kit; DAKO, Carpentaria, CA). Sections were deparaffinized, rehydrated, and incubated with 3% H2O2 for 10 min to abrogate endogenous peroxidase activity. After rinsing in 0.05 m Tris-buffered saline containing 0.1% Tween 20 (TBST), the sections were treated with 10 μg/ml primary rabbit polyclonal antibodies against hURAT1 and hPDZK1 (4 °C overnight). Then, the sections were incubated with the secondary antibody, biotinylated goat polyclonal antibody against rabbit immunoglobulin (DAKO), diluted 1:400 for 30 min with horseradish peroxidase-labeled streptavidin. This step was followed by incubation with diaminobenzidine and hydrogen peroxide. The sections were counterstained with hematoxylin and examined by light microscopy. Cell Culture and Transfection—Human embryonic kidney 293 (HEK293) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) at 37 °C and 5% CO2. Transient transfection with LipofectAMINE 2000 (Invitrogen) was performed according to the manufacturer's instructions. After transfection, the cells were grown 36–48 h before the experiments. For the establishment of URAT1-expressing cells, stable transfectants were selected for 2 weeks by adding 0.5 mg/ml G418 to the medium. Immunoprecipitation and Immunoblotting—Twenty-four hours after cotransfection, HEK293 cells in 100-mm plates were lysed with a buffer containing 20 mm Tris (pH 7.4), 250 mm NaCl, 1% Triton X-100, 5 mm EDTA, 0.25% deoxycholate, and protease inhibitor mixture (PIC, Sigma). Lysates were centrifuged at 15,000 rpm for 5 min, and the supernatant was collected. 1 μl of the anti-GFP antibody (Full-Length A.v. Polyclonal Antibody, Clontech) was added to the supernatant, and the mixture was incubated overnight at 4 °C with continuous gentle shaking. For the coimmunoprecipitation of endogenous URAT1 and PDZK1, we used human kidney membrane fractions (Biochain) and added anti-PDZK1 antibody or control IgG to this solution. Then URAT1 and associated proteins were immunoprecipitated using the Seize Classic (A) Immunoprecipitation kit (Pierce), and protein complexes were eluted according to the manufacturer's instructions. The elution samples were resuspended in SDS sample buffer and heated for 5 min at 100 °C, and proteins were resolved on 10% SDS-PAGE gels. The resolved proteins were then electrophoretically transferred to polyvinylidene difluoride membranes. The affinity-purified rabbit PDZK1 antibody and horseradish peroxidase-coupled goat anti-rabbit IgG (Amersham Biosciences) were used for immunoblotting. Immunoblotting was developed with enhanced chemiluminescence reagents (ECL Plus, Amersham Biosciences). Cell Surface Biotinylation—Surface biotinylation of URAT1 at the plasma membrane was performed as described Huh et al. (18Huh K.-H. Wenthold R.J. J. Biol. Chem. 1999; 274: 151-157Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) with some modifications. URAT1 stably expressing HEK293 cells transfected with pcDNA3.1(+)-hPDZK1 or pcDNA3.1(+) vector alone were washed three times with phosphate-buffered saline (PBS), and surface proteins were biotinylated with Sulfo-NHS-SS-Biotin (Pierce, 0.5 mg/ml) in PBS for 30 min at 4 °C. Cells were washed with three times with ice-cold PBS containing 50 mm glycine to remove non-reacted chemicals. Cells were harvested, spun at 14,000 × g at 4 °C for 4 min to pellet insoluble material, assessed for protein contents, and adjusted to the same concentration. Cell lysates were then incubated with Ultralink-immobilized NeutrAvidin beads (Pierce) to precipitate biotinylated proteins. The bound proteins were eluted with SDS sample buffer and were subjected to SDS-PAGE and Western blotting followed by ECL (Amersham Biosciences). URAT1 was detected with polyclonal URAT1 antibody (1:5,000) (4Enomoto A. Kimura H. Chairoungdua A. Shigeta Y. Jutabha P. Cha S.H. Hosoyamada M. Takeda M. Sekine T. Igarashi T. Matsuo H. Kikuchi Y. Oda T. Ichida K. Hosoya T. Shimotaka K. Niwa T. Kanai Y. Endou H. Nature. 2002; 417: 447-452Crossref PubMed Scopus (1137) Google Scholar). Densitometric analysis was performed using Model DIANA II Imaging System (M&S Instruments Trading Inc., Tokyo, Japan). Urate Transport Activity Assay—HEK293 cells, plated on 24-well culture plates at a density of 2 × 105 cells/well 24 h prior to transfection, were incubated in LipofectAMINE 2000 as described above. After 36 h, the culture medium was removed, and the cells were incubated in serum- and chloride-free Hanks' balanced salt solution containing the following in mm: 125 sodium gluconate, 4.8 K gluconate, 1.2 KH2PO4, 1.2 MgSO4, 1.3 calcium gluconate, 5.6 glucose, and 25 HEPES, pH 7.4, for 10 min. The uptake study was started by adding HBSS containing [14C]urate to the plate. Because the uptake of urate was linear up to 5 min in transiently transfected cells and up to 2 min in stably transfected cells, the initial uptake was assessed as the uptake for 1 min (data not shown). After 1 min, the cells were washed twice in ice-cold Hanks' balanced salt solution then lysed in 0.1 n NaOH for 20 min for scintillation counting. For determination of the kinetic parameters, the concentrations of urate were varied from 100 to 1000 μm. URAT1-mediated urate uptake was calculated as the difference between the values of uptake into HEK293 cells stably expressing URAT1 and those of HEK293 cells transfected vector alone. Kinetic parameters were obtained using Equation 1,υ=Vmax×SKm+S(Eq. 1) where v is the uptake rate of the substrate (picomoles/min/mg of protein), S is the substrate concentration in the medium (μm), Km is the Michaelis-Menten constant (μm), and Vmax is the maximum uptake rate (picomoles/min/mg of protein). These values were determined with the Eadie-Hofstee equation. Statistical Analysis—Uptake experiments were conducted three times, and each uptake experiment was performed in triplicate. Values were presented as means ± S.D. Statistical significance was determined by non-paired t tests. Isolation of PDZK1 in Yeast Two-hybrid Genetic Screen—In our search for potential URAT1 binding partners, we used the URAT1 C-terminal tail (URAT1-CT) as bait in a yeast two-hybrid screen of a cDNA library constructed from the human adult kidney. From 1.8 × 107 independent colonies screened, 98 positive clones were obtained. Of these positive clones, 35 yielded an identical sequence encoding some portions of the gene for human PDZK1. PDZK1 is a 519-amino acid protein that contains four PDZ domains (19Kocher O. Comella N. Tognazzi K. Brown L.F. Lab. Invest. 1998; 78: 117-125PubMed Google Scholar) (GenBank™ accession number NM_002614). The longest clone, 27A, includes PDZ2, PDZ3, and PDZ4 but lacks N-terminal sequences, including half of PDZ1. We could not detect interaction between the URAT1-CT with any other PDZ proteins known to be expressed near the apical membrane of proximal tubules, including NHERF1 and E3KARP (20Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (311) Google Scholar, 21Reczek D. Berryman M. Bretscher J. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (517) Google Scholar, 22Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman E. Welsh M.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar). For further experiments, we cloned full-length human PDZK1 by long PCR and subcloned it into a prey vector. The specificity of the interaction between PDZK1 and URAT1-CT was further confirmed by yeast two-hybrid assays. First, we tested the specificity of the interaction between PDZK1 and the C termini of other human OAT proteins. PDZK1 interacted with the C terminus of OAT4 with a different PDZ motif (TSL), but not with those of human OAT1, OAT2, or OAT3 (Fig. 1A) (23Hosoyamada M. Sekine T. Kanai Y. Endou H. Am. J. Physiol. 1999; 276: F122-F128PubMed Google Scholar, 24Cha S.H. Sekine T. Kusuhara H. Yu E. Kim J.Y. Kim D.K. Sugiyama Y. Kanai Y. Endou H. J. Biol. Chem. 2000; 275: 4507-4512Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 25Cha S.H. Sekine T. Fukushima J.-I. Kanai Y. Kobayashi Y. Goya T. Endou H. Mol. Pharmacol. 2001; 59: 1277-1286Crossref PubMed Scopus (445) Google Scholar). The C Terminus of URAT1 Is Necessary for the Interaction with PDZK1—To identify the sites in URAT1 that interact with PDZK1, we made three mutant baits. The first one (URAT1-CT-d3) is a bait that lacks the last three residues of URAT1, which are known to play a crucial role in PDZ domain recognition. The second and third ones (F555A and T553A), the extreme C-terminal phenylalanine (0 position) or threonine (–2 position) of URAT1, have been replaced by alanine, which was expected to abolish or strongly suppress the binding of PDZ proteins (10Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1212) Google Scholar). These three baits did not interact with PDZK1 (Fig. 1B). Thus, binding through the C terminus of URAT1 suggests that the PDZ motif of URAT1 is the site of interaction with PDZK1. Domain Analysis of PDZK1 Protein-Protein Interaction with URAT1 C Terminus—PDZK1 possesses four PDZ domains that assemble target proteins by binding to a C-terminal motif with a consensus sequence such as (S/T)XΦ. To determine the possible interactions of the URAT1 C-terminal region with the PDZ domains of PDZK1, we produced prey vectors each containing
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