Intermediate cell adhesion: role of calreticulin and LRP

描述(申请人提供)：细胞功能由细胞骨架的化学和机械信号调节。中间细胞黏附是由血小板反应素(TSP)引起的一种状态，在这种状态下，局部黏附被解体，但细胞仍散布，整合素聚集。TSP活性部位是肝素结合域(HBD)上的19个氨基酸序列(Hep I肽)，通过细胞表面钙网蛋白(CRT)和低密度脂蛋白受体相关蛋白(LRP)的受体共复合体传递信号。Hep I信号涉及局部黏附解体、刺激细胞运动和侵袭、通过解救失巢凋亡而产生抗凋亡信号以及内皮细胞管的形成。因此，我们认为，中间黏附状态的Hep I信号是对细胞压力的适应性反应，这在组织重塑、修复和发育中是重要的。本研究的主要目的是通过研究TSP/Hep I信号在体内组织修复中的作用，探讨TSP通过CRT-LRP复合体传递TSP信号的分子机制以及中间黏附状态的生理意义。在具体目标1中，将通过鉴定CRT中的LRP结合位点和利用生物学检测中缺乏Hep I或LRP结合位点的表达CRT的转基因细胞来研究TSP-CRT-LRP信号复合体的分子基础。在具体目标2中，将通过研究依赖于TSP的G蛋白/适配子蛋白结合、自身磷酸化、定位于脂筏以及研究LRP介导的CRT内吞作用和焦点黏附调节来研究CRT-LRP信号。TSP信号转导中间黏附的生理意义将在特定目的3.TSP/HBD/HEP I信号转导细胞侵袭和促进黏附依赖生存将被进一步研究。体内海绵状肉芽肿模型将用于确定该信号在组织修复过程中在细胞侵袭、凋亡和血管生成中的作用。该模型提供表达Hep I或HBD的质粒以提供局部、持续的侵袭细胞。这些研究将首次为中间黏附在组织重塑中的作用提供生物学见解，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重要意义。

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.