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

在我们通过酵母双杂交(Y2H)筛选到尿酸转运蛋白URAT1的胞内结合蛋白PDZK1的初步结果的基础上，我们计划扩大这项研究的范围，以阐明尿酸转运蛋白URAT1通过其结合蛋白PDZK1的功能调节机制。(1)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Y2H实验所证实的URAT1-PDZK1相互作用。野生型URAT1蛋白可与PDZK1结合，但缺少PDZ基序(T-Q-F)后三个氨基酸残基的URAT1不能与PDZK1结合。(2)用脂质体转染法建立了表达URAT1的HEK293细胞(HEK-URAT1)，并对PDZK1共转染前后尿酸盐转运动力学进行了研究。PDZK1转染HEK-URAT1细胞后，Vmax增加，Km不变。(3)我们检测到了…的共定位用人肾连续切片进行免疫组织化学分析，发现URAT1和PDZK1在肾近端小管的顶膜上均有表达。此外，人肾总蛋白的免疫共沉淀实验证明了URAT1与PDZK1的结合。这两个发现显示了这种相互作用的生理重要性。2004年，我们进一步研究了这种URAT1-PDZK1相互作用。(1)制备了融合URAT1C-末端的GST和融合了PDZK1单独PDZ结构域的MBP，并进行了表面等离子体共振分析(Biacore)检测其结合亲和力。SPR证实其结合在PDZK1 PDZ结构域1、2和4上。它们的离解常数(K_D)为1.9-516 nm。当PDZ相互作用时，这些值是相容的。(2)通过细胞表面生物素化实验，我们认识到，至少，PDZK1基因转染后尿酸盐转运活性的增强是由于细胞质膜上URAT1蛋白表达的增加，可能是通过PDZK1相互作用稳定URAT1蛋白。(3)通过数据库检索，我们已经找到了两个人类PDZK1的SNPs。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之间的连接子的C末端。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实验清楚地表明了对Y145H突变体的亲和力降低。这两个PDZK1 SNP突变体不影响尿酸通过URAT1转运的活性。较少

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