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

摘要 常染色体显性遗传性多囊肾病(ADPKD)是最常见的潜在致死基因 疾病。85%的ADPKD病例是由于其PKD1基因突变所致 大约1/3是由于错义突变和可能发生的微小的框内缺失所致 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蛋白调控的丧失是 是PKD发病的基础，但我们基本上对PC1 调节G蛋白的信号活性。我们的长期目标是了解PC1-G背后的机制 蛋白调节，以靶向和功能恢复或增强这一功能，作为ADPKD的治疗。 基于PC1和GPCRs粘附类在结构和功能上的相似性，以及 耐人寻味的结果来自我们的初步研究，我们提出了一种涉及隐蔽拴系的机制 多肽配体负责PC1对G蛋白信号的调节，并进一步参与 肾囊肿形成的发病机制。因此，这项提案的主要目标是确定 拴系配体介导PC1调节G蛋白信号转导的结构功能关系 并证明这一调节机制在发病机制和潜在的治疗中的相关性。 ADPKD.具体地说，我们打算确定PC1的栓系多肽配体的基本性质 负责其G蛋白信号的调节，绘制涉及系留配体的PC1区域- 依赖信号转导，并确定依赖多肽配体拯救肾脏的条件 囊变。拟议的研究将使用已建立的细胞系以及标准的 评估修饰表达信号传递能力的分子生物学方法和生化方法 构建了PC1和可溶性拴系配体衍生多肽，并将包括治疗囊性疾病 在体外和体内模型系统中，肾脏具有可溶的栓系配体衍生的多肽。成功 该项目的完成有望揭示PC1调节G蛋白信号的分子细节 通过其神秘的、系留的配体，并展示这种调控机制作为一种新的 治疗靶点。阐明PC1调节的G蛋白信号转导机制将促进我们的 认识ADPKD的分子发病机制是发展新的当务之急 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.