Analysis of CD81-PGRL complex on T cells

描述（由申请人提供）：CD81是一种具有多种免疫调节功能的四白蛋白。在T细胞上，CD81抗体激活LFA-1，并与TCR/CD3提供共刺激信号，促进T细胞活化。CD81抗体也可以阻断早期T细胞的发育。CD81-/-小鼠Th2反应受损，对过敏原诱导的气道高反应性有抵抗力。CD81是丙型肝炎病毒（HCV）包膜蛋白E2的受体，E2与CD81结合可产生与抗CD81抗体相似的作用。CD81调节T细胞活性的机制尚不清楚。最近发现，在T细胞上，CD81与一种名为PGRL的新型igg超家族成员存在物理关联。PGRL可能调节前列腺素（PG）对T细胞的反应。从广义上讲，该建议寻求特异性pg， CD81和通过lfa - 1调节T细胞粘附和运动之间联系的证据。具体而言，本项目将：1)识别cd81介导的T细胞LFA-1激活信号。棕榈酰化的作用和特定支架蛋白的募集参与信号转导将被研究。使用流式细胞术和icam包被荧光微球的新型LFA-1亲和力测定将用于鉴定CD81和LFA-1之间的信号中间体。2)明确CD81-PGRL复合物的结构和分子特征。本研究将利用CD81和PGRL诱变，以及包括MALDI-TOF质谱在内的生化技术来验证CD81作为连接PGRL和细胞内信号蛋白的跨膜适配蛋白的假设。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CD81或PGRL抗体存在或不存在的情况下进行测试，并在控制PGRL和CD81共表达的条件下或在可溶性PGRL存在的情况下进行测试。CD81、PGRL和EP3对T细胞整合素活性的协同作用（通过icam的粘附或运动来测量）。

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.