The Molecular Mechanisms of Polycystin-1 Proteolytic Cleavage in Kidney Health and Polycystic Kidney Disease

项目摘要/摘要 我们的长期目标是了解异二聚体(PC1cFL)和未裂解的(PC1U)的特定功能 多囊蛋白-1(PC1)在肾脏发育、动态平衡和PKD发病机制中的状态。此项目构建 在我们之前关于GPS基序上PC1裂解的关键发现上，并研究了分子和 卵裂调节PC1的运输和功能的生物物理机制。中环 假设GPS基序和相邻的连接子形成一个二分力传递模块，该模块 调节PC1在异二聚体(PC1cFL)和非切割(PC1U)状态下的关键功能。在PC1cFL中， Gps裂解产生gps基序中最后一条β链(β-1)的构象变化，这 通过从刚性接头到N-端的信号传递实现纤毛运输和功能 跨膜结构域。在PC1U中，连接子传递不依赖于切割的力。此提案将使用 多学科方法测试GPS链接器模块的功能作用。1)我们将检验这一假设 β纤毛运输和功能所需的PC11-链的紧密结合是 被PKD1相关突变破坏。我们预测这条β链在全球定位系统基序中的紧密结合 2)我们 将检验PC1纤毛运输需要高刚性和短连接子长度的假设 功能，并被PKD1相关突变所破坏。我们预测连接子的刚性需要 使PC1能够招募Rabep1/GGA1/Ar13，流量到纤毛，并在体外诱导小管生成。3)我们将确定 通过建立两个PKD1敲打小鼠模型来研究GPS-Linker模块在体内的作用和机制 带有受损的GPS关联，而另一个具有灵活的链接器。我们将在以下时间检查它们的肾脏表型 分析突变型PC1在近端和远端的生物发生和转运 肾单位细胞。拟议的研究将提供对GPS链接器模块和 解剖两种PC1形式在肾脏发育和肾单位不同节段中的功能作用 产后期。这些见解应该会导致治疗策略，恢复正常的PC1功能 有相当比例的ADPKD患者通过操纵GPS-Linker模块的力传导过程。

Project Summary/Abstract Our long-term goal is to understand specific functions of the heterodimeric (PC1cFL) and uncleaved (PC1U) states of Polycystin-1 (PC1) in kidney development, homeostasis, and PKD pathogenesis. This project builds on our previous key findings involving cleavage of PC1 at the GPS motif and investigates molecular and biophysical mechanisms by which cleavage regulates the trafficking and function of PC1. The central hypothesis is that the GPS motif and the adjacent linker form a bipartite force-transduction module that mediates critical functions of PC1 in both the heterodimeric (PC1cFL) and uncleaved (PC1U) states. In PC1cFL, GPS cleavage generates a conformational change of the last β-strand (β-1) within the GPS motif, which enables ciliary trafficking and function via signal transmittance from the rigid linker to the N-terminal transmembrane domain. In PC1U, the linker transduces cleavage-independent forces. This proposal will use a multi-disciplinary approach to test the functional role of the GPS-linker module. 1) We will test the hypothesis that a tight association of the β1-strand within the GPS is required for PC1 ciliary trafficking and function, and is disrupted by PKD1-associated mutations. We predict that tight association of this β-strand within the GPS motif is required to enable PC1 to recruit Rabep1/GGA1/Arl3, traffic to cilia, and induce in vitro tubulogenesis. 2) We will test the hypothesis that high rigidity and short length in the linker is required for PC1 ciliary trafficking and function, and is disrupted by PKD1-associated mutations. We predict that the rigidity of the linker is required to enable PC1 to recruit Rabep1/GGA1/Arl3, traffic to cilia, and induce in vitro tubulogenesis. 3) We will determine the in vivo role and mechanism of the GPS-linker module by generating two Pkd1 knockin mouse models, one with impaired GPS association and the other with a flexible linker. We will examine their kidney phenotypes at development and after birth, and analyze the biogenesis and trafficking of mutant PC1 in proximal and distal nephron cells. The proposed studies will provide novel mechanistic insights into the GPS-linker module and dissect their functional role for the two PC1 forms at kidney development and in different nephron-segments in postnatal periods. These insights should result in therapeutic strategies that restore normal PC1 function in a significant proportion of ADPKD patients by manipulating the GPS-linker module force-transduction process.