Intermediate cell adhesion: role of calreticulin and LRP

描述（由申请人提供）：细胞功能由细胞骨架的化学和机械信号调节。中间细胞粘附是由血栓反应蛋白（TSP）诱导的状态，在这种状态下，局灶性粘附被分解，但细胞仍然扩散和整合素聚集。TSP活性位点是肝素结合域（HBD）中的一个19aa序列（hep I肽），通过细胞表面钙调蛋白（CRT）和低密度脂蛋白受体相关蛋白（LRP）的受体共复合体发出信号。Hep I信号表明局灶黏附破坏，刺激细胞运动和侵袭，抗凋亡信号的产生，以及内皮细胞管的形成。因此，我们认为I型肝炎病毒的中间粘附状态信号是对细胞应激的适应性反应，这在组织重塑、修复和发育中很重要。本课题的主要目的是通过对体内组织修复中TSP/hep信号的研究，确定通过CRT-LRP复合物介导的TSP信号的分子机制以及中间粘附状态的生理意义。在Specific Aim 1中，将通过识别CRT中的LRP结合位点，并通过在生物试验中使用表达缺乏hep I或LRP结合位点的CRT的转基因细胞来研究TSP-CRT-LRP信号复合物的分子基础。在Specific Aim 2中，将通过研究tsp依赖性G蛋白/适配器蛋白结合、自磷酸化、脂筏定位以及lrp介导的CRT内吞和局灶黏附调节来研究CRT- lrp信号传导。TSP信号对中间粘附的生理意义将在Specific Aim 3中讨论。我们将进一步研究TSP/HBD/hep I信号对细胞侵袭和促进粘附依赖性存活的影响。在体内海绵肉芽肿模型中，传递表达hep I或HBD的质粒，为入侵细胞提供局部、持续的转染，将用于确定该信号在组织修复过程中细胞入侵、凋亡和血管生成中的作用。这些研究将首次从生物学角度了解中间粘连在组织重塑中的作用，并对伤口修复、慢性损伤疾病（动脉粥样硬化）、转移和组织工程具有指导意义。

DESCRIPTION (provided by applicant): Cell function is regulated by chemical and mechanical signaling from the cytoskeleton. Intermediate cell adhesion is the state induced by thrombospondins (TSP), in which focal adhesions are disassembled, but cells remain spread and integrin clustered. The TSP active site is a 19 aa sequence (hep I peptide) in the heparin-binding domain (HBD) which signals through the receptor co-complex of cell surface calreticulin (CRT) and LDL receptor-related protein (LRP). Hep I signals focal adhesion disassembly, stimulation of cell motility and invasion, generation of anti-apoptotic signals with rescue from anoikis, and endothelial cell tube formation. Thus, we propose that hep I signaling of the intermediate adhesive state is an adaptive response to cellular stress which is important in tissue remodeling, repair, and development. The major goals of this proposal are to determine the molecular mechanisms of TSP signaling through CRT-LRP complexes and the physiological significance of the intermediate adhesive state through study of TSP/hep I signaling in tissue repair in vivo. In Specific Aim 1, the molecular basis of the TSP-CRT-LRP signaling complex will be studied by identifying the LRP binding site in CRT and through use of transgenic cells expressing CRT lacking the hep I or LRP binding sites in biological assays. In Specific Aim 2, CRT-LRP signaling will be studied by investigation of TSP-dependent G protein/adapter protein binding, autophosphorylation, localization to lipid rafts, and study of LRP-mediated CRT endocytosis and focal adhesion regulation. The physiological significance of TSP signaling of intermediate adhesion will be addressed in Specific Aim 3. TSP/HBD/hep I signaling of cell invasion and promotion of adhesion-dependent survival will be studied further. An in vivo sponge granuloma model which delivers plasmid expressing hep I or the HBD to provide local, sustained transfection of invading cells will be used to determine the role of this signaling in cell invasion, apoptosis, and angiogenesis during tissue repair. These studies will provide the first biologic insights into the role of intermediate adhesion in tissue remodeling, with implications for wound repair, diseases of chronic injury (atherosclerosis), metastasis, and tissue engineering.