STRUCTURE/FUNCTION OF THE TSP--CD36 SYSTEM

我们实验室最近的工作成功地分离出了 鉴定血小板糖蛋白IV（GPIV，CD 36，GPIIIb）作为 血小板反应蛋白膜受体（TSP）。 本申请提出 TSP及其与细胞受体相互作用的研究作为一种范例， 与血小板聚集、细胞粘附和 肿瘤转移 TSP是一种450 kD的三聚体糖蛋白， 血小板α颗粒成分，并表达在活化的 血小板表面，支持不可逆的血小板聚集。 TSP 由多种细胞类型合成，并被整合到 生长细胞的基质，在那里它似乎调节细胞生长， 分化和粘附。 定义TSP与其 细胞受体对于理解TSP的功能至关重要。 在初步研究中，我们已经在TSP中定义了一个肽结构域， 负责其与细胞受体CD36的相互作用。 这 修订后的应用程序包含使用CD36转染的Jurkat获得的新数据 这些细胞进一步证明了这种相互作用。 治疗 一种可能干扰TSP在血小板中作用的肽的意义 肿瘤转移的聚集是深刻的。 在拟议的实验中， 对参与细胞TSP相互作用的TSP结构域的检查 将被追究。 具体而言，本提案中概述的实验 将处理以下问题： 1. TSP的细胞结合结构域的鉴定。 a.参与交互的TSP域 将鉴定纯化的CD36。 B. TSP在血小板功能中的作用将进一步研究 在血小板聚集和结合研究中探索， 定义 更准确地说是负责TSP的受体 在活化的血小板表面上的表达和功能。 合成肽结合研究将用于鉴定 TSP分子的功能区。 2. CD36的配体（TSP）结合结构域的鉴定。 a.使用纯化的受体和配体进行交联研究。 在识别TSP域之后， 负责观察到的TSP和CD36之间的相互作用， 负责TSP结合的CD36结构域将是 在交叉研究中发现。 B.筛选“随机CD36表位表达文库”将是 作为识别的替代方法， TSP结合结构域。 C. CD36的缺失突变体将在Jurkat细胞中表达， 确认交联研究。 3. TSP和CD 36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 将在体外和裸鼠模型中研究细胞。

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.