STRUCTURE AND FUNCTION OF THE TSP - CD36 SYSTEM

我们实验室最近的工作使我们成功地分离出了 血小板膜糖蛋白LV(GPlV、CD36、GPl11b)的鉴定 凝血酶原蛋白膜受体(TSP)此应用程序建议 TSP及其与细胞受体相互作用的研究 细胞黏附过程与血小板聚集、细胞黏附和 肿瘤转移。TSP是一种450kD的三聚体糖蛋白，是一种主要的 血小板α颗粒组成及活化后的表达 支持不可逆的血小板聚集的血小板表面。TSP 是由多种细胞类型合成的，并被整合到 生长细胞的基质，在那里它似乎调节细胞的生长， 分化和粘连。定义TSP与ITS的相互作用 细胞受体是理解TSP功能的关键。 在初步研究中，我们在TSP中定义了一个多肽结构域，即 负责其与细胞受体CD36的相互作用。这 修订后的申请包含了用CD36转基因Jurkat获得的新数据 细胞为这种相互作用提供了进一步的证据。治疗性 一种可能干扰TSP在血小板中作用的多肽的意义 肿瘤转移的聚集性是深刻的。在拟议的实验中， 参与细胞内TSP相互作用的TSP结构域的研究 将会被追查。具体地说，该提案中概述的实验 将解决以下问题： 1.TSP细胞结合域的鉴定 A.参与互动的TSP域(S) 纯化的CD36将被鉴定。 B.TSP在血小板功能中的作用将进一步 在血小板聚集和结合研究中探索以确定 更准确地说，是负责TSP的受体 活化的血小板表面的表达和功能。 合成肽结合研究将用于识别 TSP分子的功能区。 2.CD36TSP结合结构域(S)的鉴定 A.使用纯化的受体和配基进行的交联研究。 在标识符合以下条件的TSP域之后 负责观察到TSP和CD36之间的相互作用， 负责TSP绑定的CD36域将是 在交联性研究中确定。 B.筛选随机CD36表位表达文库 作为身份识别的替代方法执行 TSP结合结构域。 C.CD36缺失突变体将在Jurkat细胞中表达，以 确认交联性研究。 3.TSP和CD36在肿瘤细胞中作用的研究 生物学。 CD36在肿瘤恶性表型表达中的作用 细胞将在体外和裸鼠模型中进行研究。

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.