Intermediate cell adhesion: role of calreticulin and LRP

描述（由申请人提供）：细胞功能由来自细胞骨架的化学和机械信号调节。中间细胞粘附是由血小板反应蛋白（TSP）诱导的状态，其中粘着斑被分解，但细胞保持扩散和整合素聚集。TSP活性位点是肝素结合结构域（HBD）中的19个氨基酸序列（hep I肽），其通过细胞表面钙网蛋白（CRT）和LDL受体相关蛋白（LRP）的受体共复合物发出信号。Hep I信号局灶性粘附解体，刺激细胞运动和侵袭，产生抗凋亡信号以从失巢凋亡中拯救，以及内皮细胞管形成。因此，我们认为，中间粘附状态的hep I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。本提案的主要目标是通过研究TSP/hep I信号在体内组织修复中的作用，确定TSP信号通过CRT-LRP复合物的分子机制和中间粘附状态的生理意义。在特定目标1中，将通过鉴定CRT中的LRP结合位点以及通过在生物测定中使用表达缺乏hep I或LRP结合位点的CRT的转基因细胞来研究TSP-CRT-LRP信号传导复合物的分子基础。在特定目标2中，将通过研究TSP依赖性G蛋白/衔接蛋白结合、自磷酸化、定位于脂筏以及研究LRP介导的CRT内吞作用和粘着斑调节来研究CRT-LRP信号传导。中间粘附的TSP信号传导的生理学意义将在具体目标3中讨论。TSP/HBD/hep I信号传导细胞侵袭和促进粘附依赖性存活将进一步研究。将使用体内海绵肉芽肿模型来确定这种信号在组织修复期间细胞侵袭、细胞凋亡和血管生成中的作用，该模型递送表达hep I或HBD的质粒以提供入侵细胞的局部、持续转染。这些研究将提供第一个生物学见解的作用，在组织重塑中的中间粘附，与伤口修复，慢性损伤（动脉粥样硬化），转移和组织工程疾病的影响。

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.