WNT signaling in Autosomal Dominant Polycystic Kidney Disease

项目摘要/摘要 了解PKD1和TRPP2突变如何导致常染色体显性遗传性多囊肾病 仍然是寻找治疗这一流行的人类疾病的有效疗法的主要障碍。这种蛋白质 这些基因的产物(PKD1和TRPP2)形成受体-通道复合体，对该复合体的激活 机制仍不清楚。出于这个原因，针对功能失调的下游影响的调查 建筑群继续构成挑战。TRPP2作为非选择性阳离子通道发挥作用 在一种整合或疾病中，人们对PKD1和PKD1/TRPP2作为一个复合体的理解还不完全。 相关视角。已证明PKD1在1：3PKD1/TRPP2复合体中形成离子通道亚单位， 并独立地被认为是一种非典型的G蛋白偶联受体。WNT配体已 最近发现激活PKD1/TRPP2复合体导致TRPP2介导的电流。这一发现 为研究PKD1/TRPP2的激活机制和PKD1的作用提供了新的机会。这个 本研究的目的是解决WNT诱导的PKD1/TRPP2的激活机制 很复杂。这一目标将通过检验WNT与PKD1结合导致 通过中介G蛋白信号途径下游激活TRPP2。其基本原理是来自 这些研究将有助于深入了解PKD1/TRPP2的功能和调控。这一知识将推动未来 研究以确定损害功能的突变如何导致囊性肾脏疾病。为了测试 在这一假设下，将追求以下三个具体目标。AIM 1将测试WNT与PKD1的结合是否 这是PKD1/TRPP2通道激活的必要条件和充分条件。最小WNT结合域中的残基 将使用下拉和原位结合的方法确定与WNT相互作用所必需的PKD1的 化验。然后将使用膜片钳技术来确定破坏WNT结合的突变 随后阻断WNT诱导的通道激活。目标2将研究WNT在WNT的功能中的作用 PKD1是一个非典型的G蛋白偶联受体。这将使用NanoBRET分析来完成 确定WNT配体是否诱导PKD_1介导的G蛋白信号转导以及WNT与PKD_1结合是否诱导 PKD1受体脱敏。目的3将确定PKD1介导的G蛋白信号在TRPP2中的作用 激活。这将通过使用NanoBRET和膜片钳来测试PKD1介导的G蛋白是否丢失 信号转导干扰通道激活，研究G蛋白与TRPP2的关联，并确定 抑制G蛋白信号可阻止WNT诱导的TRPP2活性。拟议的研究将提供一种独特的 生物化学、分子生物学、绿色聚合酶链式反应和离子通道功能的培训机会 最常见的遗传病。这项提议的成功完成将产生一个明确的模式 WNT诱导的PKD1/TRPP2复合体的激活以指导未来针对ADPKD的功能分析 发病机制。

PROJECT ABSTRACT/SUMMARY Understanding how mutations in PKD1 and TRPP2 lead to Autosomal Dominant Polycystic Kidney Disease remains a major obstacle in the search for effective therapies to treat this prevalent human disease. The protein products of these genes (PKD1 and TRPP2) form a receptor-channel complex for which the activation mechanism remains unknown. For this reason, investigations targeting the downstream effects of dysfunctional complexes continue to pose a challenge. TRPP2 functions as a nonselective cation channel yet the functions ascribed to PKD1 and to PKD1/TRPP2 as a complex are incompletely understood in an integrated or disease- relevant perspective. PKD1 has been shown to form an ion channel subunit in the 1:3 PKD1/TRPP2 complex, and independently has been proposed to act as an atypical G protein-coupled receptor. WNT ligands have recently been shown to activate the PKD1/TRPP2 complex leading to TRPP2-mediated currents. This finding presents a novel opportunity to investigate the mechanism of PKD1/TRPP2 activation and the role of PKD1. The objective of the proposed research is to resolve the WNT-induced activation mechanism of the PKD1/TRPP2 complex. This objective will be attained by testing the hypothesis that WNT binding to PKD1 leads to the downstream activation of TRPP2 via intermediary G protein signaling. The rationale is that information from these studies will yield insight into PKD1/TRPP2 function and regulation. This knowledge will drive future investigations to determine how mutations that compromise function can lead to cystic kidney disease. To test the hypothesis, the following three specific aims will be pursued. Aim 1 will test if WNT binding to PKD1 is necessary and sufficient for PKD1/TRPP2 channel activation. Residues within the minimal WNT binding domain of PKD1 that are necessary for the interaction with WNT will be identified using pull-down and in situ binding assays. Patch clamp techniques will then be used to determine if mutations that disrupt WNT binding subsequently disrupt WNT-induced channel activation. Aim 2 will investigate the role of WNT in the function of PKD1 as an atypical G protein-coupled receptor. This will be accomplished using NanoBRET assays to determine whether WNT ligands induce PKD1-mediated G protein signaling and if WNT binding to PKD1 induces PKD1 receptor desensitization. Aim 3 will determine the role of PKD1-mediated G protein signaling in TRPP2 activation. This will be achieved using NanoBRET and patch clamp to test if loss of PKD1-mediated G protein signaling disrupts channel activation, investigate the association of G proteins with TRPP2, and determine if inhibition of G protein signaling prevents WNT-induced TRPP2 activity. Proposed studies will provide a unique training opportunity in biochemistry, molecular biology, GPCR and ion channel function, in the context of one of the most common genetic diseases. The successful completion of this proposal will yield a defined model for WNT-induced activation of the PKD1/TRPP2 complex to direct future functional analyses targeting ADPKD pathogenesis.