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KMID : 0359920080270020175
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Korean Journal of Nephrology
2008 Volume.27 No. 2 p.175 ~ p.185
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Association of rOAT1 with Caveolin-1 in Rat Kidney
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Cha Seok-Ho
Won Young-Joon
Hwang Ji-Sun
Suh Chang-Kook
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Abstract
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Purpose : A family of organic anion transporters (OAT) has been identified, and several isoforms have been reported. The regulatory mechanisms of OATs functions, however, still remain to be elucidated. The rat OAT1 contributes to move a number of negatively-charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. To address this issue, we investigated the protein-protein interaction between rOAT1 and Cav-1.
Methods : The expressions of rOAT1 and Cav-1 (mRNA and protein) were evaluated using RT-PCR and Western blot analysis. The localization of rOAT1 and Cav-1 was determined in the caveolae-rich membrane fraction isolated by sucrose density gradient ultra-centrifugation. For the direct binding between the rOAT1 and Cav-1 proteins, the immuno-precipitation method and confocal microscopy were employed. To perform functional analysis, a Xenopus oocytes expression system with the antisense oligonucleotides (ODN) technique was used.
Results : The expressions of rOAT1 and Cav-1 were detected in the kidney. The caveolae-rich membranous fractions from the kidney contained both rOAT1 and Cav-1 in the same fractions. The immuno- precipitation experiments showed the formation of a complex between the rOAT1 and Cav-1. The confocal microscopy using primary cultured renal proximal epithelial cells also supported the co-localization of rOAT1 and Cav-1 at the plasma membrane. The uptake function of rOAT1, as assessed by using a Xenopus oocytes expression system, was inhibited by the Xenopus Cav-1 antisense ODN.
Conclusion : rOAT1 co-localizes with caveolin-1 in the caveolae, and caveolin-1 plays an important role in regulating the function of rOAT1.
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KEYWORD
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Organic anion transporters, Caveolin-1, Alpha-ketoglutaric acid, Proximal renal tubule, Antisense oligonucleotides
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