The thrombospondins (TSPs) are a family of five proteins that are closely and transiently associated with the interface between the cell surface and extracellular matrix (ECM) during tissue genesis and remodeling. The long-term goal of this project is to elucidate the structure and function of the members of the TSP gene family. Specific focus for the next period of support will be on the following areas.
Specific Aim1. Determination of the structure of COMP. For various TSP family members, structural and functional data indicate that the type 3 repeats and the C-terminal domain form a single complex. The goal of the proposed study is to use X-ray crystallography and NMR to determine the structure of the type 3 repeat/C-terminal complex of cartilage oligomeric matrix protein (COMP). Based on molecular modeling approaches, we hypothesize that the C-terminal of COMP folds to form a four-bladed beta propeller and the type 3 repeats form looped structures that are closely associated with one surface of the beta propeller. Furthermore, we hypothesize that the juxtaposition of the type 3 repeats and the C-terminal domain is essential for optimal collagen, proteoglycan (PG) and integrin binding.
Specific Aim 2. Identification of key amino acids for the interaction of COMP with proteins and PGs. The hypothesis that COMP is a matricellular protein implies that it participates in ECM assembly or structure and binds to cell surface proteins and PGs to modulate cellular phenotype. The goal of this aim is a detailed understanding of the molecular basis for the interaction of COMP with collagen II, fibronectin, integrins and glycosaminoglycans (GAGs). Key amino acids for these interactions within the type 3 repeat/C-terminal complex will be identified by site-directed mutagenesis within the context of the intact molecule and in the individual domains.
Specific Aim 3. Identification of cell surface receptors for COMP. Preliminary data indicate that COMP (1) interacts with PGs and integrins on chondrocytes, (2) modulates chondrogenesis, (3) activates ERK 1/2 and p38 pathways to stimulate vascular smooth muscle cell (VSMC) migration, and (4) is highly expressed by fibroblasts in the stroma of mammary tumors. The goal of this aim is to determine the molecular basis for the interaction of COMP with chondrocytes, VSMCs and fibroblasts. Specific blocking reagents for various candidate receptors will be assayed, proteins that coimmunoprecipitate with COMP from detergent solubilized cells will be identified by mass spectrometry and cell extracts will be chromatographed on COMP-Sepharose columns.
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