title: Modulation of renal apical organic anion transporter 4 function by two PDZ domain-containing proteins.
abstract: Human organic anion transporter 4 (OAT4) is an apical organic anion/dicarboxylate exchanger in the renal proximal tubules and mediates high-affinity transport of steroid sulfates such as estrone-3-sulfate (E1S) and dehydroepiandrosterone sulfate. Here, two multivalent PDZ (PSD-95/Discs Large/ZO-1) proteins PDZK1 and NHERF1 were examined as interactors of OAT4 by a yeast two-hybrid assay. These interactions require the extreme C-terminal region of OAT4 and the first and fourth PDZ domains of PDZK1 and the first PDZ domain of NHERF1. These interactions were confirmed by surface plasmon resonance assays (K(D): 36 nM, 1.2 microM, and 41.7 microM, respectively). In vitro binding assays and co-immunoprecipitation studies revealed that the OAT4 wild-type but not a mutant lacking the PDZ motif interacted directly with both PDZK1 and NHERF1. OAT4, PDZK1, and NHERF1 proteins were shown to be localized at the apical membrane of renal proximal tubules. The association with PDZK1 or NHERF1 enhanced OAT4-mediated E1S transport activities in HEK293 cells (1.2- to 1.4-fold), and the deletion of the OAT4 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by alteration in V(max) of E(1)S transport via OAT4 and was associated with the increased surface expression level of OAT4 protein. This study indicates that the functional activity of OAT4 is modulated through the PDZ interaction with the network of PDZK1 and NHERF1 and suggests that OAT4 is involved in the regulated apical organic anion handling in the renal proximal tubules, provided by the PDZ scaffold.
authors.affiliation: Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka-shi, Tokyo, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Miyazaki
authors.forename: Naohiko
authors.initials: N
authors.lastname: Anzai
authors.forename: Sophapun
authors.initials: S
authors.lastname: Ekaratanawong
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakata
authors.forename: Ho Jung
authors.initials: HJ
authors.lastname: Shin
authors.forename: Promsuk
authors.initials: P
authors.lastname: Jutabha
authors.forename: Taku
authors.initials: T
authors.lastname: Hirata
authors.forename: Xin
authors.initials: X
authors.lastname: He
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nonoguchi
authors.forename: Kimio
authors.initials: K
authors.lastname: Tomita
authors.forename: Yoshikatsu
authors.initials: Y
authors.lastname: Kanai
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Endou
mesh_terms: D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D002478:Cells, Cultured; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D027361:Organic Anion Transporters / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D027361:Organic Anion Transporters; C484659:PDZK1 protein, human; D012333:RNA, Messenger