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

描述（由申请人提供）：受体和配体通过内吞作用的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。尽管膜重组是网格蛋白和小泡介导的内吞作用的一个特征，但其实现的机制-以及它如何与特定线索协调-尚不清楚。在小泡介导的内吞作用之前，一组参与膜重组的候选蛋白是膜联蛋白，这是一种以钙依赖的方式转移到膜上的蛋白质家族，在那里它们与脂质微结构域结合，并可能形成二维支架[Gerke， Nat. Rev. Mol. Cell Biol]。, 2005]。这种行为表明，膜联蛋白可能在组织和维持小泡中发挥作用，小泡是膜的一个区域，脂质微域和外小叶中的gpi锚定蛋白被认为在内化之前聚集在一起[Pelkmans， Traffic, 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泡的细胞质侧[Mayran， EMBO Journal, 2003；J.细胞生物学。[1994]，膜联蛋白与胆固醇的钙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相平衡，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到小泡中发挥积极作用，这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件来解决这个问题，在体外，刺激钙信号可以在空间和时间上被控制，并且在生化定义良好的条件下确定膜联蛋白的反应。这将通过控制膜组成将膜联蛋白AnxA2包封在脂质囊泡中（目的1），研究胆固醇对AnxA2易位的影响（目的2），以及研究膜联蛋白A2易位和分布对膜外小叶连接蛋白分布的影响（目的3）来完成。完成这些目标将有助于阐明通过膜联蛋白参与小泡介导的内吞作用的机制。对这一基本细胞过程的更好的机制理解可能有助于确定治疗与小泡相关的病理的新靶点，包括许多脂质储存疾病[Marks， Trends Cell biology]。, 2002]。

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.