Analysis of CD81-PGRL complex on T cells

T 细胞上 CD81-PGRL 复合物的分析

• 批准号: 6877169
• 负责人: Stephen Keith Chapes
• 金额: $ 25.46万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6877169
• 关键词: CD antigens; T lymphocyte; biological signal transduction; calcium flux; cyclic AMP; fatty acylation; human tissue; immunoprecipitation; laboratory mouse; leukocyte activation /transformation; leukocyte adhesion molecules; mass spectrometry; membrane proteins; palmitates; prostaglandin E; prostaglandin receptor; protein kinase C; transcription factor

DESCRIPTION (provided by applicant): CD81 is a tetraspanin with several immunoregulatory functions. On T cells antibody to CD81 activates LFA-1 and can provide co-stimulatory signals with TCR/CD3 to promote T cell activation. Antibody to CD81 can also block early T cell development. CD81-/- mice have impaired Th2 responses and are resistant to allergen induced airway hyperreactivity. CD81 is a receptor for the hepatitis C virus (HCV) envelope protein E2, and binding of E2 to CD81 induces effects similar to those caused by anti-CD81 antibodies. The mechanism by which CD81 regulates T cell activity is unknown. It was recently discovered that on T cells CD81 is physically associated with a novel Ig-superfamily member called PGRL. PGRL may regulate prostaglandin (PG) responses on T cells. In broad terms this proposal seeks evidence of a connection between specific PGs, CD81 and the regulation of T cell adhesion and motility via LFA-I. Specifically this project will: 1) Identify the CD81-mediated signals that cause LFA-1 activation on T cells. The role of palmitoylation and the recruitment of a specific scaffold protein involved in signal transduction will be studied. A novel LFA-1 avidity assay using flow cytometry and ICAM-coated fluorescent microspheres will be used to identify signaling intermediates between CD81 and LFA-1. 2) Define the structural and molecular features of CD81-PGRL complexes. This aim will employ CD81 and PGRL mutagenesis, and biochemical techniques including MALDI-TOF MS to test the hypothesis that CD81 serves as a transmembrane adapter protein linking PGRL with an intracellular signaling protein. 3) Determine whether CD81 and/or PGRL interact with or regulate the activity of EP3. The affinity of EP3 will be tested in the presence or absence of antibody to CD81 or PGRL and tested under conditions which control the co-expression of PGRL and CD81 or in the presence of soluble PGRL. The coordinated effect of CD81, PGRL and EP3 on T cell integrin activity measured by adhesion to or motility on ICAMs.

描述（申请人提供）：CD81是一种具有多种免疫调节功能的四跨膜蛋白。在 T 细胞上，CD81 抗体会激活 LFA-1，并可与 TCR/CD3 提供共刺激信号，以促进 T 细胞激活。 CD81 抗体还可以阻止早期 T 细胞发育。 CD81-/- 小鼠的 Th2 反应受损，并且对过敏原诱导的气道高反应性具有抵抗力。 CD81 是丙型肝炎病毒 (HCV) 包膜蛋白 E2 的受体，E2 与 CD81 的结合会产生与抗 CD81 抗体类似的效果。 CD81 调节 T 细胞活性的机制尚不清楚。最近发现，T 细胞上的 CD81 与一种称为 PGRL 的新型 Ig 超家族成员有物理关联。 PGRL 可能调节 T 细胞上的前列腺素 (PG) 反应。从广义上讲，该提案寻求特定 PG、CD81 与通过 LFA-I 调节 T 细胞粘附和运动之间联系的证据。具体而言，该项目将： 1) 识别导致 T 细胞上 LFA-1 激活的 CD81 介导的信号。将研究棕榈酰化的作用和参与信号转导的特定支架蛋白的招募。使用流式细胞术和 ICAM 涂层荧光微球的新型 LFA-1 亲合力测定将用于鉴定 CD81 和 LFA-1 之间的信号传导中间体。 2) 定义CD81-PGRL复合物的结构和分子特征。该目标将采用 CD81 和 PGRL 诱变以及包括 MALDI-TOF MS 在内的生化技术来测试 CD81 作为连接 PGRL 与细胞内信号蛋白的跨膜接头蛋白的假设。 3)确定CD81和/或PGRL是否与EP3相互作用或调节EP3的活性。 EP3的亲和力将在存在或不存在针对CD81或PGRL的抗体的情况下进行测试，并且在控制PGRL和CD81的共表达的条件下或在可溶性PGRL的存在下进行测试。 CD81、PGRL 和 EP3 对 T 细胞整合素活性的协调作用通过 ICAM 的粘附或运动来测量。