Analysis of CD81-PGRL complex on T cells

描述(申请人提供)：CD81是一种具有多种免疫调节功能的河豚毒素。在T细胞上，CD81抗体可激活LFA-1，并与TCR/CD3提供共刺激信号，促进T细胞活化。CD81抗体也可以阻止T细胞的早期发育。CD81-/-小鼠Th2反应受损，对过敏原诱导的呼吸道高反应性具有抵抗力。CD81是丙型肝炎病毒包膜蛋白E2的受体，其与CD81的结合作用与抗CD81抗体的作用相似。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与一个新的免疫球蛋白超家族成员PGRL有物理联系。PGRL可调节T细胞对前列腺素(PG)的反应。概括地说，这项提议寻求特定的PGs、CD81和通过LFA-I调节T细胞黏附和运动之间的联系的证据。具体地说，这个项目将：1)确定CD81介导的信号，导致T细胞上LFA-1的激活。棕榈酰化的作用和参与信号转导的特定支架蛋白的招募将被研究。使用流式细胞术和ICAM包被的荧光微球的一种新的LFA-1亲和力测定将用于识别CD81和LFA-1之间的信号中间体。2)明确CD81-PGRL络合物的结构和分子特征。这一目标将利用CD81和PGRL的突变，以及包括MALDI-TOF MS在内的生化技术来验证CD81作为跨膜适配器蛋白连接PGRL和细胞内信号蛋白的假设。3)确定CD81和/或PGRL是否与EP3相互作用或调节其活性。EP3的亲和力将在有或没有CD81或PGRL抗体的情况下进行测试，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ICAM上T细胞整合素活性的协同作用

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.