Recent work in our laboratory has resulted in the successful isolation and identification of platelet glycoprotein lV (GPlV, CD36, GPlllb) as a membrane receptor for thrombospondin (TSP). This application proposes study of TSP and its interaction with cellular receptors as a paradigm for cell adhesion processes relevant to platelet aggregation, cell adhesion and tumor metastasis. TSP is a 450 kD trimeric glycoprotein that is a major platelet alpha granule constituent and is expressed on the activated platelet surface where it supports irreversible platelet aggregation. TSP is synthesized by a variety of cell types and is incorporated into the matrix of growing cells where it appears to regulate cell growth, differentiation, and adhesion. Defining the interactions of TSP with its cellular receptors is critical to an understanding of the function of TSP. In preliminary studies, we have defined a peptide domain within TSP that is responsible for its interaction with the cellular receptor CD36. This revised application contains new data obtained with CD36-transfected Jurkat cells that adds further evidence for this interaction. The therapeutic implications of a peptide that might interfere with TSP's role in platelet aggregation of tumor metastasis are profound. In the proposed experiments, an examination of TSP domains that participate in cellular TSP interactions will be pursued. Specifically, the experiments outlined in this proposal will address the following: 1. Identification of the cellular binding domains of TSP. a. TSP domain(s) that participate in the interaction with purified CD36 will be identified. b. The role of TSP in platelet function will be further explored in platelet aggregation and binding studies to define more precisely the receptors that are responsible for TSP expression and function on the activated platelet surface. Synthetic peptide binding studies will be used to identify functional regions of the TSP molecule. 2. Identification of the ligand (TSP) binding domain(s) of CD36. a. Cross-linking studies using purified receptor and ligand. Following the identification of a TSP domain that is responsible for the observed interaction between TSP and CD36, the domain of CD36 responsible for TSP binding will be identified in cross-linking studies. b. Screening a """"""""random CD36 epitope expression library"""""""" will be performed as an alternative approach to the identification of a TSP binding domain. c. Deletion mutants of CD36 will be expressed in Jurkat cells to confirm the cross-linking studies. 3. Investigation of the role of TSP and CD36 in tumor cell biology. The role of CD36 in the expression of the malignant phenotype of tumor cells will be investigated in vitro and in a nude mouse model.
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