In Vitro Reconstitution of Calcium-Mediated Membrane Reorganization by Annexin A2

描述(申请人提供)：受体和配体通过内吞作用内化需要细胞膜的戏剧性重组，由细胞外和细胞内信号协调。尽管膜重组是笼蛋白和小窝介导的内吞作用的一个特征，但实现它的机制-以及它如何与特定的信号相协调-仍然不清楚。在小窝介导的内吞作用之前，参与膜重组的一组候选蛋白是膜联蛋白，这是一个蛋白质家族，以钙依赖的方式转运到膜上，在那里它们与脂质微域结合，并可能形成二维支架[Gerke，NAT]。莫尔牧师。细胞生物，2005年]。这一行为表明，膜联蛋白可能在组织和维持小窝中发挥作用，小窝是膜上的区域，在内化之前，外层小叶中的脂质微域和GPI锚定蛋白被认为聚集在一起[Pelkmann，Traffer，2002]。虽然某些膜联蛋白以钙不依赖的方式结合胆固醇并定位于小凹的细胞质一侧[Mayran，EMBO Journal，2003；Lisanti，J.Cell Biol.，1994]，但对于如何平衡膜联蛋白与胆固醇的钙非依赖性相互作用与它们与其他膜组分的钙依赖相互作用，以及膜联蛋白本身是否在协调脂质微域和GPI锚定的蛋白质从细胞膜一侧进入小窝方面发挥积极作用，仍然是一个悬而未决的问题。这项研究建议通过在体外重建膜-膜联蛋白相互作用的元素来解决这个问题，其中刺激钙信号可以在空间和时间上被控制，并且膜联蛋白的反应可以在生物化学的明确定义的条件下确定。这将通过将膜联蛋白AnxA2包裹在膜成分可控的脂泡中(目标1)，研究胆固醇对AnxA2转位的影响(目标2)，以及调查Annexin A2转位和分布对连接到膜外层小叶的蛋白质分布的影响(目标3)来实现。实现这些目的将有助于阐明膜联蛋白参与小窝介导的内吞作用的机制。更好地从机制上理解这一基本的细胞过程可能有助于确定治疗与凹陷相关的病理的新靶点，包括许多脂质储存疾病[Marks，Trends Cell Biol.，2002]。 与公共健康相关：这项研究的完成将推进细胞如何协调其内部复杂的脂质运输，以及外部信号在这种协调中的作用的现有知识。了解细胞如何对外部提示做出反应来调节其内部成分，将有助于深入了解这一过程如何在疾病期间发生故障，并可能建议可以开发出纠正这些故障的治疗方法。

DESCRIPTION (provided by applicant): The internalization of receptors and ligands via endocytosis requires dramatic reorganization of the cell membrane, coordinated by extracellular and intracellular signals. Although membrane reorganization is a feature of both clathrin- and caveolae-mediated endocytosis, the mechanism by which it is achieved - and how it is coordinated with specific cues - remains unclear. One set of candidates involved in membrane reorganization prior to caveolae-mediated endocytosis are the annexins, a family of proteins which translocate to the membrane in a calcium-dependent manner, where they associate with lipid microdomains and may form two-dimensional scaffolds [Gerke, Nat. Rev. Mol. Cell Biol., 2005]. This behavior suggests a possible role for annexins in organizing and maintaining caveolae, regions of the membrane where lipid microdomains and GPI-anchored proteins in the outer leaflet are believed to cluster prior to internalization [Pelkmans, Traffic, 2002]. While certain annexins bind cholesterol in a calcium-independent manner and localize to the cytoplasmic side of caveolae [Mayran, EMBO Journal, 2003; Lisanti, J. Cell Biol., 1994], it remains an open question as to how the calcium-independent interactions of annexins with cholesterol are balanced with their calcium-dependent interactions with other membrane components, and if annexins themselves play an active role in coordinating the clustering of lipid microdomains and GPI-anchored proteins into caveolae from the cytoplasmic side of the membrane. This research proposes to address this question by reconstructing elements of membrane- annexin interactions in vitro, where the stimulating calcium signal can be spatially and temporally controlled, and the annexin response determined under biochemically well-defined conditions. This will be accomplished by encapsulating the annexin AnxA2 in lipid vesicles with controlled membrane composition (Aim 1), studying the effects of cholesterol on AnxA2 translocation (Aim 2), and investigating the effects of annexin A2 translocation and distribution on the distributions of proteins linked to the outer leaflet of the membrane (Aim 3). Accomplishing these aims will help to elucidate the mechanism through which annexins participate in caveolae-mediated endocytosis. A better mechanistic understanding of this fundamental cellular process could help in identifying new targets for treating pathologies associated with caveolae, including many lipid storage diseases [Marks, Trends Cell Biol., 2002]. PUBLIC HEALTH RELEVANCE: The completion of this research will advance current knowledge of how cells coordinate the complex traffic of lipids in their interior, and the role of external signals in this coordination. Understanding how cells regulate their internal composition in response to external cues will provide insight into how this process can malfunction during disease, and may suggest treatments that could be developed to correct these malfunctions.