Regulatory mechanism of urate transport function by urate transporter binding protein PDZK1

基于我们利用酵母双杂交（Y2 H）技术筛选到的肾脏尿酸转运蛋白URAT 1胞内结合蛋白PDZK 1的初步结果，我们计划进一步研究结合蛋白PDZK 1对尿酸转运蛋白URAT 1转运尿酸的功能调控机制。(1)We通过免疫共沉淀法、GST-pull down法等体外生物化学结合实验，证实了Y2 H实验所鉴定的URAT 1-PDZK 1相互作用。野生型URAT 1蛋白能与PDZK 1结合，而缺失PDZ基序（T-Q-F）的URAT 1不能与PDZK 1结合。(2)We通过脂质体转染法建立表达URAT 1的HEK 293细胞（HEK-URAT 1），并在有或没有PDZK 1共转染的情况下进行尿酸盐转运动力学研究。PDZK 1转染HEK-URAT 1细胞后，Vmax增加，而Km不变。(3)我们检测到了 ...更多信息 URAT 1和PDZK 1在肾近端小管的顶膜通过免疫组织化学分析使用人肾连续切片。此外，使用人肾总蛋白的免疫共沉淀实验证明了URAT 1和PDZK 1之间的结合。2004年，我们进一步研究了URAT 1-PDZK 1的相互作用。(1)We制备GST融合的URAT 1C-末端和MBP融合的PDZK 1的各个PDZ结构域，并进行表面等离子体共振测定（Biacore）以检查它们的结合亲和力。通过SPR在PDZK 1 PDZ结构域1、2和4中证实了结合。它们的解离常数（K_D）为1.9 - 516 nM。这些值作为PDZ相互作用是相容的。(2)By通过细胞表面生物素化实验，我们认识到，至少PDZK 1转染后尿酸盐转运活性的增强是由于HEK 293细胞质膜上URAT 1蛋白表达的增加，可能是通过PDZK 1相互作用稳定URAT 1蛋白。(3)By通过数据库检索，我们已经发现了人类PDZK 1的两个SNPs。Y145 H位于PDZK 1的第二个PDZ结构域的中间，V372 E位于PDZ结构域3和4之间的接头中的C末端侧。在Y2 H测定中，我们不能发现三个克隆（野生型、Y145 H和V372 E）之间的差异，而SPR实验清楚地显示对Y145 H突变体的亲和力降低。这两个PDZK 1 SNP突变体不影响尿酸盐通过URAT 1的转运活性。少

Based on our preliminary results that the identification of intracellular binding protein PDZK1 for renal urate transporter URAT1 by yeast two-hybrid (Y2H) screening, we were planning to extend this research to clarify the functional regulatory mechanism of urate transport via urate transporter URAT1 by its binding protein PDZK1.In 2003, we clarified these three points. (1)We carried out the in vitro biochemical binding experiments such as co-immunoprecipitation assay and GST-pull down assay to confirm the URAT1-PDZK1 interaction identified by Y2H experiments. Wild type URAT1 proteins could bind with PDZK1, but URAT1 lacking the last three amino acid residues including PDZ motif (T-Q-F) failed to bind with PDZK1. (2)We established URAT1-expressing HEK293 cells (HEK-URAT1) by lipofection method and performed urate transport kinetic study with or without PDZK1 cotransfection. PDZK1 transfection into HEK-URAT1 cell increased Vmax without changing Km. (3) We detected the colocalization of … More both URAT1 and PDZK1 at the apical membrane of renal proximal tubules by immunohistochemical analysis using human kidney serial sections. In addition, coimmunoprecipitation experiments using human kidney total protein demonstrated the binding between URAT1 and PDZK1. These two findings showed the physiological importance of this interaction.In 2004, we further examined this URAT1-PDZK1 interaction. (1)We prepared the GST fused URAT1 C-terminal and MBP fused individual PDZ domains of PDZK1 and performed Surface Plasmon Resonance assay (Biacore) to check their binding affinities. The bindings were confirmed in PDZK1 PDZ domain 1, 2 and 4 by SPR. Their dissociation constant (K_D) is 1.9 - 516 nM. These values were compatible as PDZ interactions. (2)By the cell surface biotinylation experiments, we recognize that, at least, the enhancement of urate transport activity after the transfection of PDZK1 is due to the increased expression of URAT1 proteins at the plasma membrane of HEK293 cells, may be through the stabilization of URAT1 proteins by PDZK1 interaction. (3)By the database searches, we have already found two SNPs of human PDZK1. Y145H is located in the middle of the second PDZ domain of PDZK1 and V372E is located at the C-terminal side in the linker between PDZ domain 3 and 4. In the Y2H assay, we could not find the difference among three clones (wild type, Y145H, and V372E), whereas SPR experiments clearly exhibited the reduced affinity for Y145H mutants. These two PDZK1 SNP mutants did not affect the urate transport activity via URAT1. Less