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

描述（由申请人提供）：通过胞吞作用的受体和配体内化需要细胞膜的显著重组，由细胞外和细胞内信号协调。虽然膜重组是网格蛋白和小窝介导的内吞作用的一个特征，但其实现机制以及如何与特定线索协调仍然不清楚。在小窝介导的内吞作用之前参与膜重组的一组候选物是膜联蛋白，其是以钙依赖性方式易位至膜的蛋白质家族，其中它们与脂质微结构域缔合并且可以形成二维支架[Gerke，Nat. Rev. Mol.细胞生物学，2005年]。这种行为表明膜联蛋白可能在组织和维持小窝中发挥作用，小窝是膜的区域，据信在内化之前，外小叶中的脂质微结构域和GPI锚定蛋白聚集在该区域[Pelkmans，Traffic，2002]。虽然某些膜联蛋白以钙非依赖性方式结合胆固醇并定位于小窝的细胞质侧[Mayran，EMBO Journal，2003; Lisanti，J. Cell Biol.，1994]，关于膜联蛋白与胆固醇的钙非依赖性相互作用如何与它们与其他膜组分的钙依赖性相互作用平衡，以及膜联蛋白本身是否在协调脂质微结构域和GPI锚定蛋白从膜的细胞质侧聚集成小窝中起积极作用，仍然是一个悬而未决的问题。本研究提出通过在体外重建膜-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控制，并且膜联蛋白响应在生物化学定义良好的条件下确定。这将通过将膜联蛋白AnxA 2封装在具有受控膜组成的脂质囊泡中（目标1），研究胆固醇对AnxA 2易位的影响（目标2），并研究膜联蛋白A2易位和分布对与膜外小叶连接的蛋白质分布的影响（目标3）来实现。实现这些目标将有助于阐明膜联蛋白参与小窝介导的内吞作用的机制。对这种基本细胞过程的更好的机械理解可以帮助鉴定用于治疗与小窝相关的病理的新靶标，包括许多脂质储存疾病[Marks，Trends Cell Biol.，2002年]。 公共卫生相关性：这项研究的完成将推进细胞如何协调其内部脂质的复杂运输以及外部信号在这种协调中的作用的现有知识。了解细胞如何调节其内部组成以响应外部线索将提供对疾病期间这一过程如何发生故障的深入了解，并可能建议可以开发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.