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&gt；250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者，并在PKD的发病机制中发挥关键作用。最近的体外研究表明，初级纤毛作为细胞传感器，通过依赖于多囊蛋白-1/多囊蛋白-2的钙信号通路传递心尖机械信号。然而，纤毛形成、稳定和信号转导的确切机制还不是很清楚，更不知道这些纤毛相关蛋白是如何靶向纤毛和功能组装的。我们已经确定Cys1是CPK小鼠的致病基因；证明了其新的蛋白产物Cystin定位于初级顶端纤毛；并确定了Cys1通过N-末端结构域与脂筏分离，可能是预测的N-肉豆蔻化/多碱基序。我们推测，Cystin通过脂筏介导的机制运输到初级纤毛，与纤毛膜结合，并作为稳定纤毛轴丝微管支架的分子框架的一部分。利用一组稳定表达野生型Cystin和各种截断突变体作为GFP标记的融合蛋白的细胞系，我们已经确定N-末端结构域是将Cystin靶向纤毛的必要条件，但不是充分的，并且需要第二个新的信号。由于Cystin的表达水平很低，目前还没有功能分析方法，我们开发了一套创新的策略来进一步表征这种新的蛋白质及其细胞内运输路线，作为确定其功能的第一步。具体地说，在这项建议中，我们将：1)确定绿色荧光蛋白标记的囊藻蛋白(cystin-GFP)是否拯救CPK表型，并在体内正确地靶向肾上皮细胞的初级纤毛；2)根据预测的N-肉豆蔻酰化位点、假定的纤毛靶向信号和假定的相互作用伙伴来表征囊藻蛋白；以及3)检测囊藻毒素在细胞内向初级顶端纤毛的运输的动力学。支持这项研究的中心假设是，初级纤毛功能缺陷损害了肾小管上皮细胞分化的终末阶段，而对这种发育停滞的上皮性反应是囊性形成。因此，初级根尖纤毛是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.