The Multivalent PDZ Domain-containing Protein PDZK1 Regulates Transport Activity of Renal Urate-Anion Exchanger URAT1 via Its C Terminus

Abstract

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 (K D ‫؍‬ 1.97-514 nM). Coimmunoprecipitation studies revealed that the wildtype 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 V max 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 (1, 2). The kidney plays a dominant role in urate elimination; it excretes ϳ70% of the daily urate production. Urate exists primarily as a weak acid at physiological pH (pK a 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 ϳ10% 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 (3).Recently, we have id...

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“…Each cDNA sample (1.5 l) was amplified as described. 29 Primers, used for PCR amplification, are shown in Table 1.…”

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