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

基于我们通过酵母双杂交（Y2H）筛选鉴定出肾尿酸转运蛋白URAT1的胞内结合蛋白PDZK1的初步结果，我们计划进一步扩展本研究，阐明其结合蛋白PDZK1通过尿酸转运蛋白URAT1转运尿酸的功能调控机制。2003年，我们明确了这三点。(1)我们进行了体外生化结合实验，如共免疫沉淀实验和GST-pull - down实验，以证实Y2H实验鉴定的URAT1-PDZK1相互作用。野生型URAT1蛋白可以与PDZK1结合，但URAT1缺乏PDZ基序（T-Q-F）等最后3个氨基酸残基，无法与PDZK1结合。(2)通过脂质转染法建立表达urat1的HEK293细胞（HEK-URAT1），并在PDZK1共转染或未转染的情况下进行urat1转运动力学研究。PDZK1转染HEK-URAT1细胞后，Vmax升高，Km不变。(3)利用人肾连续切片，通过免疫组化分析，检测到URAT1和PDZK1在肾近端小管顶端膜上的共定位。此外，利用人肾总蛋白进行的共免疫沉淀实验证实了URAT1与PDZK1之间的结合。这两个发现表明了这种相互作用在生理上的重要性。2004年，我们进一步研究了URAT1-PDZK1的相互作用。(1)制备GST融合URAT1 c端，MBP融合PDZK1的PDZ域，并采用表面等离子体共振（Biacore）检测其结合亲和力。SPR证实了PDZK1 PDZ结构域1、2和4的结合。它们的解离常数K_D为1.9 ~ 516 nM。这些值作为PDZ交互是兼容的。(2)通过细胞表面生物素化实验，我们认识到，至少，转染PDZK1后，尿酸转运活性的增强是由于HEK293细胞质膜上URAT1蛋白的表达增加，可能是通过PDZK1相互作用稳定URAT1蛋白。(3)通过数据库检索，我们已经找到了2个人类PDZK1的snp。Y145H位于PDZK1的第二个PDZ结构域的中间，V372E位于PDZ结构域3和4之间的连接器的c端侧。在Y2H实验中，我们没有发现三个克隆（野生型、Y145H和V372E）之间的差异，而SPR实验明确显示对Y145H突变体的亲和力降低。这两个PDZK1 SNP突变体不影响URAT1的尿酸转运活性。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