Analysis of CD81-PGRL complex on T cells

T 细胞上 CD81-PGRL 复合物的分析

• 批准号: 7209823
• 负责人: Stephen Keith Chapes
• 金额: $ 24.14万
• 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 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7209823
• 关键词: Adhesions; Affinity; Allergens; Antibodies; Avidity; Binding; Biochemical; Biological Assay; CD81 gene; Cell Adhesion; Cell Line; Co-Immunoprecipitations; Complex; Condition; Cyclic AMP; Dinoprostone; Doctor of Philosophy; Dominant-Negative Mutation; Event; Flow Cytometry; Glutathione; Hepatitis C virus; Immunoprecipitation; Integrins; Intracellular Signaling Proteins; Label; Ligation; Link; MALDI-TOF Mass Spectrometry; Maps; Measures; Mediating; Microspheres; Molecular; Mus; Mutagenesis; Mutate; N-terminal; Peptides; Prostaglandin Receptor; Prostaglandins; Protein Isoforms; Proteins; Radio; Regulation; Resistance; Role; Scaffolding Protein; Signal Transduction; Signaling Protein; Site; T-Cell Activation; T-Cell Development; T-Lymphocyte; Techniques; Testing; Transfection; Western Blotting; adapter protein; airway hyperresponsiveness; cell motility; hepatitis C virus envelope 2 protein; human PHEMX protein; member; mimetics; novel; palmitoylation; progesterone 11-hemisuccinate-(2-iodohistamine); receptor; response; scaffold; small molecule

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（由申请人提供）：CD 81是一种四跨膜蛋白，具有多种免疫调节功能。在T细胞上，针对CD 81的抗体激活LFA-1，并且可以提供与TCR/CD 3的共刺激信号以促进T细胞活化。抗CD 81抗体也可阻断早期T细胞发育。CD 81-/-小鼠具有受损的Th 2应答，并且对变应原诱导的气道高反应性具有抗性。CD 81是丙型肝炎病毒（HCV）包膜蛋白E2的受体，E2与CD 81的结合诱导类似于抗CD 81抗体引起的效应。CD 81调节T细胞活性的机制尚不清楚。最近发现，在T细胞上，CD 81与称为PGRL的新型Ig超家族成员物理相关。PGRL可以调节T细胞上的前列腺素（PG）反应。从广义上讲，该提议寻求特定PG、CD 81与通过LFA-I调节T细胞粘附和运动性之间的联系的证据。具体而言，该项目将：1）鉴定导致T细胞上LFA-1活化的CD 81介导的信号。棕榈酰化的作用和招聘的一个特定的支架蛋白参与信号转导将被研究。一种新的LFA-1亲合力测定，使用流式细胞术和ICAM-涂层的荧光微球将被用来确定CD 81和LFA-1之间的信号中间体。2)定义CD 81-PGRL复合物的结构和分子特征。这一目标将采用CD 81和PGRL诱变，和生化技术，包括MALDI-TOF MS来测试的假设，CD 81作为跨膜衔接蛋白连接PGRL与细胞内信号蛋白。3)确定CD 81和/或PGRL是否与EP 3相互作用或调节EP 3的活性。将在存在或不存在抗CD 81或PGRL抗体的情况下测试EP 3的亲和力，并在控制PGRL和CD 81共表达的条件下或在存在可溶性PGRL的情况下测试。CD 81、PGRL和EP 3对T细胞整联蛋白活性的协同作用，通过对ICAM的粘附或运动性来测量。