Cystin, a lipid raft and cilia-associated protein in PKD

描述（由申请人提供）：初级纤毛是动态的复杂结构，含有>250种蛋白质，包括几种多囊肾病（PKD）相关蛋白质。在肾上皮细胞中，初级顶端纤毛似乎是分化信号的主要效应器，并在PKD发病机制中发挥关键作用。最近的体外研究表明，初级纤毛作为一个细胞传感器，通过多囊蛋白-1/多囊蛋白-2依赖的Ca++信号通路转导顶端的机械信号。然而，纤毛的形成，稳定和信号转导所涉及的确切机制还没有很好的定义，甚至更少的是知道这些纤毛相关蛋白是如何靶向纤毛和功能组装。我们已经确定Cys 1作为疾病基因在cpk小鼠，证明其新的蛋白质产品，cystin，定位于初级顶端纤毛，并确定cystin分馏脂筏通过N-末端结构域，可能是预测的N-肉豆蔻酰化/ polybasic基序。我们推测，cystin交通的主要纤毛通过脂筏介导的机制，与纤毛膜，并作为分子框架的一部分，稳定的微管支架的纤毛轴丝。使用一套稳定转染的细胞系，表达野生型胱氨酸蛋白酶抑制剂和各种截短突变体的GFP标记的融合蛋白，我们已经确定，N-末端结构域是必要的，但不足以靶向胱氨酸蛋白酶抑制剂纤毛和第二，新的信号是必需的。由于cystin是在低水平表达，目前没有功能检测存在，我们已经开发了一套创新的策略，以进一步表征这种新的蛋白质和其细胞内运输行程的第一步，以确定其功能。具体而言，在本提案中，我们将：1）确定是否用绿色荧光蛋白标记cystin（cystin-GFP）拯救cpk表型并在体内正确靶向肾上皮的初级纤毛; 2）关于预测的N-肉豆蔻酰化位点、推定的纤毛靶向信号和推定的相互作用配偶体表征cystin;（3）检测胞囊蛋白向初级顶纤毛的胞内运输动力学。提出的研究的核心假设是，在初级纤毛功能的缺陷损害肾小管上皮细胞分化的终末阶段和上皮细胞对这种发展停滞的反应是囊肿形成。因此，初级顶纤毛代表了一个新的焦点解剖复杂的机制参与肾囊肿疾病，并最终，可能是一个新的目标，治疗干预。

DESCRIPTION (provided by applicant): Primary cilia are dynamic, complex structures that contain >250 proteins, including several polycystic kidney disease (PKD)-related proteins. In renal epithelial cells, the primary apical cilium appears to be a major effector of differentiation signals and to play a critical role in PKD pathogenesis. Recent in vitro studies demonstrate that the primary cilium acts as a cellular sensor, transducing apical mechanical signals through a polycystin-1/polycystin-2-dependent Ca++ signaling pathway. However, the precise mechanisms involved in cilia formation, stabilization, and signal transduction are not well-defined and even less is known about how these cilia-associated proteins are targeted to cilia and functionally assembled. We have identified Cys1 as the disease-gene in cpk mice; demonstrated that its novel protein product, cystin, localizes to the primary apical cilium; and determined that cystin fractionates with lipid rafts through an N-terminal domain, probably the predicted N-myristoylation/ polybasic motif. We hypothesize that cystin traffics to the primary cilium via lipid raft-mediated mechanisms, associates with the cilial membrane, and serves as part of the molecular framework that stabilizes the microtubular scaffold of the ciliary axoneme. Using a suite of stably transfected cell lines that express wild-type cystin and various truncation mutants as GFP-tagged fusion proteins, we have determined that the N-terminal domain is necessary but not sufficient for targeting cystin to cilia and a second, novel signal is required. Since cystin is expressed at low levels and no functional assays currently exist, we have developed an innovative set of strategies to further characterize this novel protein and its intracellular trafficking itinerary as first steps toward defining its function. Specifically, in this proposal, we will: 1) Determine whether cystin tagged with green fluorescent protein (cystin-GFP) rescues the cpk phenotype and targets correctly to the primary cilium of renal epithelia in vivo; 2) Characterize cystin with respect to the predicted N-myristoylation site, putative cilia-targeting signals, and putative interacting partners; and 3) Examine the dynamics of cystin intracellular trafficking to the primary apical cilium. The central hypotheses underlying the proposed studies are that defects in primary cilia function impair the terminal phases of renal tubulo-epithelial differentiation and the epithelial response to this developmental arrest is cyst formation. Therefore, primary apical cilium represents a new focal point for dissecting the complex mechanisms involved in renal cystic disease and ultimately, perhaps a new target for therapeutic interventions.