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、复杂的结构，含有bbbb250蛋白，包括几种多囊肾病（PKD）相关蛋白。在肾上皮细胞中，初级根尖纤毛似乎是分化信号的主要效应器，并在PKD发病中起关键作用。最近的体外研究表明，初级纤毛作为细胞传感器，通过多囊蛋白-1/多囊蛋白-2依赖性ca++信号通路传导根尖机械信号。然而，纤毛形成、稳定和信号转导的确切机制尚不明确，甚至对这些纤毛相关蛋白如何靶向纤毛并进行功能组装的了解更少。我们已经在cpk小鼠中鉴定出Cys1为致病基因；证实其新蛋白产物胱氨酸定位于初根尖纤毛；并确定胱氨酸通过n端结构域与脂筏分异，这可能是预测的n -肉豆蔻酰化/多碱基基序。我们假设胱氨酸通过脂质筏介导的机制运输到初级纤毛，与纤毛膜相关，并作为稳定纤毛轴突微管支架的分子框架的一部分。使用一组稳定转染的细胞系，表达野生型胱氨酸和各种截断突变体作为gfp标记的融合蛋白，我们已经确定n端结构域对于将胱氨酸靶向纤毛是必要的，但不是充分的，并且需要第二个新的信号。由于胱氨酸的表达水平较低，目前还没有功能检测方法，因此我们开发了一套创新的策略来进一步表征这种新型蛋白及其细胞内运输路线，作为确定其功能的第一步。具体而言，在本课题中，我们将：1)确定绿色荧光蛋白（cytin - gfp）标记的胱氨酸（cytin - gfp）是否能在体内拯救cpk表型，并正确靶向肾上皮的初级纤毛；2)从预测的n -肉豆蔻酰化位点、推测的纤毛靶向信号和推测的相互作用伙伴等方面对胱氨酸进行表征；3)检查囊素在细胞内运输到初级根尖纤毛的动力学。所提出的研究的中心假设是，初级纤毛功能的缺陷损害了肾小管上皮分化的终末阶段，上皮对这种发育停滞的反应是囊肿形成。因此，原发性根尖纤毛代表了解剖肾囊性疾病复杂机制的新焦点，并最终可能成为治疗干预的新靶点。

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.