BIOLOGY OF THE CD2/E-RECEPTOR COMPLEX

CD2/E-受体复合物的生物学

• 批准号: 3130641
• 负责人: Malek Kamoun
• 金额: $ 19.15万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3130641
• 关键词: T lymphocyte; chemical binding; chemical structure function; electroporation; erythrocytes; gel electrophoresis; gene deletion mutation; gene expression; genetic mapping; genetic markers; human subject; immune adherence reaction; immunochemistry; interleukin 2; leukocyte activation /transformation; membrane activity; membrane proteins; mitogens; molecular cloning; monoclonal antibody; phosphorylation; protein engineering; protein sequence; receptor; tissue /cell culture; transfection; transposon /insertion element

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.

该提案是我们之前研究的延续，旨在 分析 CD2/E 受体的特性和功能 人类T细胞激活途径。 我们的兴趣是研究 CD2的结构-功能关系并定义和更好 了解与相关的分子和生理事件 促细胞分裂剂和单克隆抗体触发 CD2 受体 抗体。 我们将检查 1) CD2 的磷酸化 在已知方案存在的情况下，人类 T 细胞上的分子 影响细胞活化，包括促有丝分裂的触发 抗CD2单克隆抗体的组合。 我们将分析 32P CD2 的磷酸氨基酸组成。 2）此外，我们 将进行 CD2 cDNA 克隆的分析和功能测试 使用DNA转染技术。 我们将尝试重建 CD2 阴性淋巴 T 中通过 DNA 转移产生活性 CD2 受体 细胞系。 我们已经证明了全长 CD2 cDNA 克隆可以在 Cos-7 细胞中瞬时表达，使用 含有 SV40 启动子的 pcEXV-CD2 载体。 表面CD2 转染细胞分子将粘附位点表达为 以及由单克隆抗体定义的激活位点。 我们将 尝试分离稳定的淋巴细胞系转染子 使用表达式表达高水平的细胞表面 CD2 含有人巨细胞病毒的载体 pBC12/CMV/CD2 立即早期启动子以及其他表达载体。 稳定转染子的阳性选择将通过共同实现 含有哺乳动物的转染子质粒（pcEXV-Neo） 选择标记neo。 CD2缺失突变体的产生 使用核酸外切酶 III 消化或位点特异性诱变获得 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 结合位点（T111）、激活位点（T112/T113）和作用 细胞质区域中的特定氨基酸，包括 那些通过磷酸化修饰的，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 分化使得CD2结构-功能的研究变得极为重要 对于理解和潜在地操纵免疫很重要 回复。