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.
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