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

项目总结/摘要 我们的长期目标是了解异源二聚体（PC 1cFL）和未切割（PC 1U）的特定功能。 多囊蛋白-1（PC 1）在肾脏发育、稳态和PKD发病机制中的状态。该项目构建 我们以前的关键发现，涉及切割的PC 1在GPS基序和研究分子和 生物物理机制，其中裂解调节运输和功能的PC 1。中央 假设GPS基序和相邻的接头形成一个二分力-转导模块， 在异源二聚体（PC 1cFL）和未切割（PC 1U）状态下介导PC 1的关键功能。在PC 1cFL中， GPS切割产生GPS基序内最后一条β链（β-1）的构象变化， 通过从刚性接头到N-末端的信号传递使纤毛运输和功能成为可能 跨膜结构域在PC 1U中，接头转导不依赖于切割的力。该提案将使用一个 多学科的方法来测试GPS链接器模块的功能作用。1)我们将检验这个假设 GPS内β1链的紧密结合是PC 1纤毛运输和功能所必需的， 被PKD 1相关突变破坏。我们预测，GPS基序内的β链紧密结合 是使PC 1能够募集Rabep 1/GGA 1/Ar 13、运输至纤毛并诱导体外小管形成所必需的。2)我们 将检验这样的假设，即PC 1纤毛运输需要接头中的高刚性和短长度， 功能，并被PKD 1相关突变破坏。我们预测，需要连接体的刚性， 使PC 1能够募集Rabep 1/GGA 1/Ar 13，运输到纤毛，并诱导体外小管形成。3)我们将确定 通过产生两个Pkd 1敲入小鼠模型，一个是GPS-linker模块， 其中一个具有受损的GPS关联，另一个具有灵活的链接器。我们将检查他们的肾脏表型， 发育和出生后，并分析近端和远端的突变PC 1的生物起源和运输 肾单位细胞所提出的研究将提供新的机制的见解GPS连接模块， 解剖它们在肾脏发育和不同肾单位节段中两种PC 1形式的功能作用， 产后时期。这些见解应该导致治疗策略，恢复正常的PC 1功能， 显著比例的ADPKD患者通过操纵GPS-接头模块力转导过程。

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.