BIOLOGY OF THE CD2/E-RECEPTOR COMPLEX

这项建议是我们先前研究的延续，目的是 CD2/E受体的特性和功能分析 人类T细胞活化的途径。我们的兴趣是研究 CD2的结构-功能关系及其界定和完善 了解与以下各项相关的分子和生理事件 有丝分裂原和单抗对CD2受体的触发作用 抗体。我们将研究1)CD2的磷酸化 人类T细胞上的分子在已知方案存在的情况下 影响细胞激活，包括由有丝分裂原触发 抗CD2单抗的组合。我们将分析 ~(32)P-CD2的磷酸氨基酸组成。2)此外，我们 将继续对CD2基因克隆进行分析和功能测试 使用DNA转基因技术。我们将尝试重建 在CD2阴性淋巴T细胞中通过DNA转移获得活性CD2受体 细胞系。我们已经证明了一个全长的CD2 克隆的基因可在Cos-7细胞中瞬时表达 含SV40启动子的pcEXV-CD2载体。表面CD2 转染体细胞的分子表达粘附点为 以及由单抗定义的激活位点。我们会 分离稳定的淋巴样细胞系转染体的尝试 利用表达载体高效表达细胞表面CD2 人巨细胞病毒载体pBC12/CMV/CD2 即早启动子以及其他表达载体。 对稳定的转染体进行正向选择将通过联合 含哺乳动物的转基因载体(pcEXV-Neo) 可选标记NEO。CD2基因缺失突变体的产生 使用核酸外切酶III酶切或定点突变的cdna 将使我们能够检查必要的特定氨基酸序列 为了CD2的正常功能，特别是配体 结合部位(T111)、活化部位(T112/T113)和作用 细胞质区域的特定氨基酸，包括 通过磷酸化修饰的那些，以及不寻常的簇 碱性残基和脯氨酸。 许多人描述了CD2在T细胞激活和表达中的作用 分化使CD2结构与功能的研究异常活跃 对于理解和潜在地操纵免疫很重要 回应。

This proposal is a continuation of our previous studies aimed at analyzing the properties and the function of the CD2/E-receptor pathway of human T cell activation. Our interest is to study the structure-function relationship of CD2 and to define and better understand the molecular and physiological events associated with the triggering of CD2 receptors by mitogens and monoclonal antibodies. We will examine 1) the phosphorylation of CD2 molecules on human T cells in the presence of regimens known to affect cell activation, including the triggering by mitogenic combinations of anti-CD2 monoclonal antibodies. We will analyze the phosphoamino acid composition of 32P CD2. 2) Furthermore, we will pursue the analysis and functional testing of CD2 cDNA clones using DNA transfection techniques. We will attempt to reconstitute an active CD2 receptor by DNA transfer in CD2-negative lymphoid T cell lines. We already have demonstrated that a full-length CD2 cDNA clone can be transiently expressed in Cos-7 cells, using the pcEXV-CD2 vector containing the SV40 promoter. Surface CD2 molecules of transfectant cells expressed the adhesion sites as well as activation sites defined by monoclonal antibodies. We will attempt to isolate stable lymphoid cell line transfectants expressing high levels of cell surface CD2 using the expression vector pBC12/CMV/CD2 containing the human cytomegalovirus immediate-early promoter as well as other expression vectors. Positive selection for stable transfectant will be achieved by co- transfectant plasmids (pcEXV-Neo) containing the mammalian selectable marker neo. The generation of deletion mutants of CD2 cDNA using Exonuclease III digestion or site specific mutagenesis will allow us to examine specific amino acid sequences necessary for the proper functioning of CD2, in particular, the ligand binding site (T111), the activation site (T112/T113), and the role of particular amino acids in the cytoplasmic region, including those modified by phosphorylation, as well as the unusual clusters of basic residues and prolines. Many described roles of CD2 in T cell activation and differentiation make the study of CD2 structure-function extremely important for understanding and potentially manipulating the immune response.