Understanding Polycystin-1 Structure-GPCR Function for the Development of New Therapeutic Approaches in ADPKD

总结 常染色体显性多囊肾病（ADPKD）是最常见的潜在致命遗传病 疾病。 85% 的 ADPKD 病例是由于 PKD1 基因突变所致，其中 大约 1/4-1/3 是由于错义突变和小的框内缺失，这些缺失可能适合 功能性救援。 PKD1 编码多囊蛋白-1 (PC1)，这是一种大而复杂的蛋白质，被认为具有 非典型 G 蛋白偶联受体 (GPCR)。现在已知 PC1-G 蛋白调节的丧失 PC1 是 PKD 发病机制的基础，但我们对 PC1 的机制基本上一无所知 调节 G 蛋白信号传导活性。我们的长期目标是了解 PC1-G 的底层机制 蛋白质调节以靶向并功能性恢复或增强该功能作为 ADPKD 的治疗。 基于 PC1 和 GPCR 粘附类别之间的结构和功能相似性，以及 从我们的初步研究中得出有趣的结果，我们提出了一种涉及神秘拴系的机制 肽配体负责 PC1 调节 G 蛋白信号传导，此外还参与 肾囊肿形成的发病机制。因此，本提案的主要目标是确定 PC1 系留配体介导的 G 蛋白信号传导调节背后的结构-功能关系 并证明这种调节机制在发病机制和潜在治疗中的相关性 ADPKD。具体来说，我们打算确定 PC1 束缚肽配体的基本特性 负责 G 蛋白信号传导的调节，绘制参与束缚配体的 PC1 区域图 依赖性信号传导，并确定肽配体依赖性肾脏救援的条件 囊肿发生。拟议的研究将采用已建立的细胞系的转染以及标准 分子生物学方法和生化测定来评估修饰表达的信号传导能力 PC1 和可溶性栓系配体衍生肽的构建体，并将包括囊性囊肿的治疗 在离体和体内模型系统中具有可溶性拴系配体衍生肽的肾脏。成功 该项目的完成预计将揭示 PC1 调节 G 蛋白信号传导的分子细节 通过其神秘的、系留的配体，并证明这种调节机制作为一种新型的潜力 治疗目标。阐明 PC1 调节的 G 蛋白信号传导机制将推进我们的研究 了解 ADPKD 的分子发病机制，这对于开发新的药物至关重要 用于治疗或预防这种疾病的疗法。

Summary Autosomal dominant polycystic kidney disease (ADPKD) is the most common potentially lethal genetic disease. Eighty-five percent of the cases of ADPKD are due to mutation of the PKD1 gene of which approximately ¼-1/3 are due to missense mutations and small, in-frame deletions that may be amenable to functional rescue. PKD1 encodes polycystin-1 (PC1), a large and complex protein thought to function as an atypical G protein-coupled receptor (GPCR). The loss of PC1-G protein regulation is now known to be fundamental to the pathogenesis of PKD, yet we know essentially nothing about the mechanism whereby PC1 regulates G protein signaling activity. Our long-term goal is to understand the mechanism underlying PC1-G protein regulation in order to target and functionally restore or augment this function as a treatment for ADPKD. Based on the structural and functional similarities between PC1 and the adhesion class of GPCRs, and intriguing results from our preliminary studies, we propose that a mechanism involving a cryptic tethered peptide ligand is responsible for the regulation of G protein signaling by PC1, and furthermore, is involved in the pathogenesis of renal cyst formation. The major goals of this proposal, therefore, are to determine the structure-function relationships behind the tethered ligand-mediated regulation of G protein signaling by PC1 and to demonstrate the relevance of this regulatory mechanism in the pathogenesis and potential treatment of ADPKD. Specifically, we intend to determine the essential properties of the tethered peptide ligand of PC1 responsible for its regulation of G protein signaling, map the regions of PC1 involved in tethered ligand- dependent signaling, and determine the conditions for the peptide ligand-dependent rescue of renal cystogenesis. The proposed studies will employ transfection of established cell lines along with standard molecular biology methods and biochemical assays to assess the signaling capability of modified expression constructs of PC1 and of soluble tethered ligand-derived peptides, and will include the treatment of cystic kidneys with soluble tethered ligand-derived peptides in both ex vivo and in vivo model systems. Successful completion of this project is expected to reveal molecular details of the regulation of G protein signaling by PC1 via its cryptic, tethered ligand, and to demonstrate the potential of this regulatory mechanism as a novel therapeutic target. Elucidating the mechanism of PC1-regulated G protein signaling will advance our understanding of the molecular pathogenesis of ADPKD, which is imperative for the development new therapies for the treatment or prevention of this disease.