BACKGROUND: Cystinuria has been proposed as an inherited disease causing disorders in renal cystine and basic amino acid transport in the proximal tubules. Although cystinuria-related amino acid transporter gene related to b0,+-type amino acid transporter (rBAT1) and its substrate transport properties have been reported, the functional regulatory mechanisms remain to be elucidated. In this study, protein-protein interaction between rBAT1 and caveolin (Cav)-1 was investigated. METHODS: The renal distribution of rBAT1, rBAT and Cav-1 were demonstrated by employing reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Co-localization of rBAT1 and Cav-1 was observed by immunocytochemistry in primary cultured renal proximal tubule-derived cells using a confocal microscope. This result was confirmed by Western blot analysis of isolated caveolae-rich membrane fraction and immunoprecipitation experiments using respective antibodies. RESULTS: In the separated rat kidney tissues following the corticomedullary axis, Cav-1 mRNA and protein expressions were increased from the cortex to the inner medulla. rBAT1 mRNA and protein expression were detected mainly in the outer medulla. Confocal microscopic results showed rBAT1 and Cav-1 co-localization in the plasma membrane. This result was confirmed by Western blot analysis of caveolae-rich membrane fraction and immunoprecipitates by respective antibodies. The effect of Cav-1 on rBAT1 function was evaluated using Cav-1 antisense oligodeoxynucleotide (ODN). The [14C] arginine uptake by rBAT1 was unchanged by the treatment with antisense ODN. CONCLUSIONS: From these results, rBAT1 and Cav-1 share a cellular expression in the segregated caveolae structure. As caveolae are rich in signaling molecules, BAT1 could play a role in diverse pathophysiological processes.
BACKGROUND:Cystinuria has been proposed as an inherited disease causing disorders in renal cystine and basic amino acid transport in the proximal tubules. Although cystinuria-related amino acid transporter gene related to b0,+-type amino acid transporter (rBAT1) and its substrate transport properties have been reported, the functional regulatory mechanisms remain to be elucidated. In this study, protein-protein interaction between rBAT1 and caveolin (Cav)-1 was investigated. METHODS: The renal distribution of rBAT1, rBAT and Cav-1 were demonstrated by employing reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Co-localization of rBAT1 and Cav-1 was observed by immunocytochemistry in primary cultured renal proximal tubule-derived cells using a confocal microscope. This result was confirmed by Western blot analysis of isolated caveolae-rich membrane fraction and immunoprecipitation experiments using respective antibodies. RESULTS: In the separated rat kidney tissues following the corticomedullary axis, Cav-1 mRNA and protein expressions were increased from the cortex to the inner medulla. rBAT1 mRNA and protein expression were detected mainly in the outer medulla. Confocal microscopic results showed rBAT1 and Cav-1 co-localization in the plasma membrane. This result was confirmed by Western blot analysis of caveolae-rich membrane fraction and immunoprecipitates by respective antibodies. The effect of Cav-1 on rBAT1 function was evaluated using Cav-1 antisense oligodeoxynucleotide (ODN). The [14C] arginine uptake by rBAT1 was unchanged by the treatment with antisense ODN. CONCLUSIONS: From these results, rBAT1 and Cav-1 share a cellular expression in the segregated caveolae structure. As caveolae are rich in signaling molecules, BAT1 could play a role in diverse pathophysiological processes.
Authors: John J Gildea; Brandon A Kemp; Nancy L Howell; Robert E Van Sciver; Robert M Carey; Robin A Felder Journal: Am J Physiol Renal Physiol Date: 2011-02-02