MOLECULAR MECHANISMS OF T-LYMPHOCYTE ACTIVATION

本研究的目的是探讨耦合机制， 淋巴细胞的T细胞受体（TCR）/CD 3复合物与 磷脂酶C（PLC）是肌醇磷脂合成的关键酶 （InsPL）水解途径。 这条途径产生第二信使 这可能是诱导T细胞活化的关键。 了解 淋巴细胞活化的机制可为设计 针对信号关键要素的免疫调节策略 转导途径 细胞透化的策略已经被 我们实验室开发的，允许接触小分子（例如， 核苷酸、肽等）到细胞内环境， 淋巴细胞，同时保持TCR/CD 3复合物与PLC的偶联。 通过使用该策略，我们获得了关于调节的信息 的InsPL水解和对化学计量的assoication的src 激酶，fyn，与CD 3复合物的多肽。 因使用本 战略，我们将寻求有关PLC控制机制的信息 活动 使用抗PLC Ab和src家族产品的共沉淀 (fyn、lck.ves等）将用于 瞬态或“弱”相互作用 将通过使用同双功能化学交联剂“稳定”， 透化的细胞，然后免疫沉淀。 这一战略 先前成功用于获得CD 3/fyn的化学计量数据 互动 通过使用蛋白酪氨酸磷酸抑制剂 磷酸酶（包括可以作为潜在的磷酸酶起作用的肽） 竞争性抑制剂，如磷酸化的Fyn或LCK肽）， 完整和透化的细胞，注意将支付的作用， 这些酶，包括CD 45磷酸酶，在调节insPL 水解 由于先前的证据表明信号的异质性 不同T细胞亚群之间的转导，将注意 潜在的差异及其功能影响。 初始 将尝试构建PLC缺陷的T细胞克隆（PLC- y1）通过基因“敲除”酶。

The objective of this study is to investigate the coupling mechansism between the T cell receptor (TCR)/CD3 complex of lymphocytes and phospholipase C (PLC), the key enzyme in the inositol phospholipid (InsPL) hydrolysis pathway. This pathway generates second messengers that may be critical in inducing T cell activation. An understanding of the mechanism of lymphocyte activation may offer a basis for designing immunomodulatory strategies targeted to critical elements of the signal transducion pathway. A strategy of cell permeabilization has been developed in our laboratory which allows access to micromolecules (e.g., nucleotide, peptides, etc.) to the intracellular enviromnment of lymphocytes, while maintaining coupling of the TCR/CD3 complex with PLC. By using this strategy, we have obtained information on the regulation of InsPL hydrolysis and on the stoichiometry of the assoication of a src kinase, fyn, with polypetides of the CD3 complex. By the use of this strategy, we will seek information on the mechanism of control of PLC activity. Coprecipitation using anti-PLC Ab and src-family products (fyn, lck.ves, etc.) will be used. Transient or "weak" interactions will be "stabilized" by using homobifunctional chemical cross-linker in permeabilized cells followed by immunoprecipitation. This strategy was previously used successfully to obtain stoichionetry data of CD3/fyn interation. By using inhibitors of protein tyrosine phosphate phosphatase (including peptides which may function as potential competitive inhibitors, such as phosphorylated fyn or lck peptides) in intact and permeabilized cells, attention will be paid to the role of these enzymes, including the CD45 phosphatase, in modulating insPL hydrolysis. Since previous evidence suggests heterogeneity in signal transduction among different T cell subsets, attention will be paid to potential differences and their functional implications. AN initial attempt will be made to construct T cell clones defective in PLC (PLC- y1) by genetically "knocking out" the enzyme.