Rat Organic Anion Transporter 3 Co-localized with Caveolin-2 in Rat Kidney / 대한신장학회잡지

ABSTRACT

BACKGROUND: The recently identified organic anion transporter 3 (rOAT3) was mainly expressed in kidney, liver and brain tissue, and it contributes the movement of endogenous or exogenous substances across the cell membrane. Although the properties of rOAT3 are gradually accumulated, the regulatory mechanism of rOAT3 remains to be elucidated. Caveolin (Cav) also plays a role as a membrane transporter and as a modulating protein for some functional proteins. Therefore, we investigated the protein-protein interaction between rOAT3 and Cav-2 in rat kidney. METHODS: The expressions of rOAT3 and Cav-2 (mRNA and protein) were observed using RT-PCR and Western blot analysis. The localization of rOAT3 and Cav-2 was determined in the caveolae-rich membrane fraction isolated by sucrose gradient ultra-centrifugation. For the direct binding between the rOAT3 and Cav-2 proteins, the immuno-precipitation method and confocal microscopy were employed. In order to perform functional analysis, a Xenopus oocytes expression system with the antisense oligodeoxynucleotides (ODN) technique was used. RESULTS: The expressions of rOAT3 and Cav-2 (mRNA and protein) were detected in the kidney. The caveolae-rich membranous fractions from the kidney contained both rOAT3 and Cav-2 in the same fractions. The immuno-precipitation experiments showed the formation of a complex between the rOAT3 and Cav-2 in the kidney. The confocal microscopic results using primary cultured renal proximal epithelial cells also supported the co-localization of rOAT3 and Cav-2 at the plasma membrane. The uptake function of rOAT3, as tested for by using a Xenopus oocytes expression system was slightly inhibited (with statistical significance) by the Xenopus Cav-2 antisense ODN. CONCLUSION: rOAT3 co-localizes with caveolin-2 in the caveolae, and caveolin-2 plays an important role in regulating the function of rOAT3.