Modulation of renal apical organic anion transporter 4 function by two PDZ domain-containing proteins.

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.

Human organic anion transporter 4 is a renal apical organic anion/dicarboxylate exchanger in the proximal tubules.

Human organic anion transporter OAT4 is expressed in the kidney and placenta and mediates high-affinity transport of estrone-3-sulfate (E1S). Because a previous study demonstrated no trans-stimulatory effects by E1S, the mode of organic anion transport via OAT4 remains still unclear. In the present study, we examined the driving force of OAT4 using mouse proximal tubular cells stably expressing OAT4 (S2 OAT4). OAT4-mediated E1S uptake was inhibited by glutarate (GA) (IC50:1.25 mM) and [14C]GA uptake via S2 OAT4 was significantly trans-stimulated by unlabeled GA (5 mM) (P<0.001). [3H]E1S uptake via S2 OAT4 was significantly trans-stimulated by preloaded GA (P<0.001) and its [14C]GA efflux was significantly trans-stimulated by unlabeled E1S in the medium (P<0.05). In addition, both the uptake and efflux of [14C]p-aminohippuric acid (PAH) and [14C]GA via S2 OAT4 were significantly trans-stimulated by unlabeled GA or PAH. The immunoreactivities of OAT4 were observed in the apical membrane of proximal tubules along with those of basolateral organic anion/dicarboxylate exchangers such as hOAT1 and hOAT3 in the same tubular population. These results indicate that OAT4 is an apical organic anion/dicarboxylate exchanger and mainly functions as an apical pathway for the reabsorption of some organic anions in renal proximal tubules driven by an outwardly directed dicarboxylate gradient.