WNT signaling in Autosomal Dominant Polycystic Kidney Disease

常染色体显性多囊肾病中的 WNT 信号传导

• 批准号: 10387670
• 负责人: Emily P Hardy
• 金额: $ 3.48万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10387670
• 关键词: Address; Autosomal Dominant Polycystic Kidney; Binding; Biochemistry; Biological Assay; C-terminal; Cations; Cells; Complex; Cystic Kidney Diseases; Disease; Event; Future; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Proteins; Genetic Diseases; Goals; Heterotrimeric GTP-Binding Proteins; In Situ; Investigation; Ion Channel; Knowledge; Lead; Ligands; Mediating; Modeling; Molecular Biology; Mus; Mutation; Outcome; Patch-Clamp Techniques; Pathogenesis; Protein Binding Domain; Protein Subunits; Proteins; Regulation; Research; Role; Signaling Protein; Testing; WNT Signaling Pathway; desensitization; design; effective therapy; extracellular; human disease; insight; new therapeutic target; novel; patch clamp; prevent; receptor; receptor-mediated signaling; response; training opportunity

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.

项目摘要/总结 了解PKD 1和TRPP 2突变如何导致常染色体显性多囊肾病 仍然是寻找治疗这种流行的人类疾病的有效疗法的主要障碍。蛋白质 这些基因的产物（PKD 1和TRPP 2）形成受体通道复合物， 机制仍然未知。因此，针对功能失调的下游影响的调查 综合体继续构成挑战。TRPP 2作为非选择性阳离子通道发挥作用，但其功能 归因于PKD 1和PKD 1/TRPP 2作为复合物在整合或疾病中的不完全理解- 相关视角。PKD 1已显示在1：3 PKD 1/TRPP 2复合物中形成离子通道亚基， 并且已经独立地提出作为非典型G蛋白偶联受体。WNT配体具有 最近显示激活PKD 1/TRPP 2复合物，导致TRPP 2介导的电流。这一发现 为研究PKD 1/TRPP 2激活机制和PKD 1的作用提供了新的机会。的 本研究的目的是阐明WNT诱导PKD 1/TRPP 2活化的机制 复杂.这一目标将通过检验WNT与PKD 1结合导致PKD 1表达的假设来实现。 通过中间G蛋白信号传导的TRPP 2的下游活化。理由是， 这些研究将有助于深入了解PKD 1/TRPP 2的功能和调节。这些知识将推动未来 研究以确定损害功能的突变如何导致囊性肾病。测试 根据这一假设，将追求以下三个具体目标。目标1将测试WNT与PKD 1的结合是否 这是PKD 1/TRPP 2通道激活的必要条件和充分条件。最小WNT结合结构域内的残基 与WNT相互作用所必需的PKD 1的表达将使用下拉和原位结合来鉴定 测定。然后将使用膜片钳技术来确定破坏WNT结合的突变是否 随后破坏WNT诱导通道激活。目的2将研究WNT在功能中的作用， PKD 1是一种非典型的G蛋白偶联受体。这将使用NanoBRET测定来完成， 确定WNT配体是否诱导PKD 1介导的G蛋白信号传导，以及WNT与PKD 1的结合是否诱导 PKD 1受体脱敏。目的3将确定PKD 1介导的G蛋白信号转导在TRPP 2中的作用。 activation.这将使用NanoBRET和膜片钳来实现，以测试PKD 1介导的G蛋白的丢失是否 信号传导破坏通道激活，研究G蛋白与TRPP 2的关联，并确定 G蛋白信号传导的抑制阻止了WNT诱导的TRPP 2活性。拟议的研究将提供一个独特的 在生物化学，分子生物学，GPCR和离子通道功能的培训机会，在一个背景下， 最常见的遗传病。该提案的成功完成将产生一个明确的模式， WNT诱导PKD 1/TRPP 2复合物的活化以指导未来针对ADPKD的功能分析 发病机制