CHARACTERIZATION OF VIP RECEPTORS & VIP AUTOANTIBODIES

VIP 受体的表征

• 批准号: 3074388
• 负责人: Sudhir Paul
• 金额: $ 6.37万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3074388
• 关键词: G protein; adenylate cyclase; affinity chromatography; antibody formation; antibody receptor; antireceptor antibody; asthma; autoantibody; autoimmune disorder; binding proteins; blood tests; crosslink; cyclic AMP; glycosides; guanine nucleotide binding protein; guinea pigs; hybridomas; immunochemistry; laboratory mouse; lung; monoclonal antibody; phospholipase A2; phospholipids; protein structure function; radioimmunoassay; receptor; sulfides; tissue /cell culture; vasoactive intestinal peptide

The candidates long term aims are to make contributions in the understanding of: (1) receptor mediated information transfer, and (2) airway smooth muscle hyperactivity. The immediate objectives are: 1) To solublize functional receptors for VIP from lung; (2) To raise anti-receptor antibody; (3) To purify the receptor and analyze its structure; (4) To rreconstitute signal transduction by the receptor; (5) To determine whether GTP-binding proteins (G- proteins) and phospholipids regulate receptor affinity, selectivity, and coupling with adenylate cyclase; (6) To compare the VIP-neutralizing ability of VIP-autoantibodies identified in asthma patients and healthy subjects, by measuring the effect of these antibodies on VIP binding to receptors, VIP-stimulated cyclic AMP synthesis, and VIP-induced tracheal relaxation; (7) To correlate changes in the affinity and titer of VIP antibodies with the severity of asthma in individual patients. (8) To assess whether autoantibody to the VIP-receptor may be a pathogenetic factor in asthma. An RCDA will ensure that the bulk of the candidate's time (approximately 90%) is applied to achieving these objectives. The candidate's academic environment supplies support mechanisms necessary for intensive research and offers good opportunities of close interaction with investigators in pharmacology, biochemistry, molecular biology, and pulmonary medicine. The candidate has already established fruitful collaborations with established researchers working on neuropeptides, receptor regulation and information transfer, and airway disease. The experimental design is: (1) Solubilization of VIP receptors; (2) Characterization of their 125I-VIP binding properties; (3) Anti-receptor antibody production by hybridoma techniques; (4) Receptor-purification by HPLC and affinity chromatography; (5) Structural analysis by analytical biochemical methods; (6) Receptor-reconstitution with G-proteins, adenylate cyclase and phospholipids followed by assay of GTP-sensitive VIP-binding, and VIP-sensitive GTP binding, GTPase activity and cyclic AMP synthesis; (7) Comparison of receptor affinity, peptide selectivity and receptor-adenylate cyclase coupling, after: (i) reconstitution with and without G-protein, (ii) reconstitution with different phospholipids, (iii) phospholipase-treatment of reconstituted receptors; (8) RIA of VIP-receptor autoantibody; (9) RIA of VIP binding autoantibodies and measurement of their affinity and titer; (10) Spirometric evaluation of the severity of asthma; (11) Delineation of the functional properties of VIP-autoantibodies by measuring their, (i) effect on VIP receptor binding, cyclic AMP synthesis, and tracheal relaxation.

候选人的长期目标是在 理解：（1）受体介导的信息传输，以及 （2）气道平滑肌多动症。 直接目标 是：1）从肺中溶解功能受体的VIP； （2） 提高抗受体抗体； （3）净化受体和 分析其结构； （4）rreconstitute信号转导 受体； （5）确定GTP结合蛋白是否（G-）是否 蛋白质）和磷脂调节受体亲和力， 选择性，并与腺苷酸环化酶偶联； （6）比较 在 哮喘患者和健康受试者，通过测量 VIP与受体，VIP刺激的循环的这些抗体 AMP合成和VIP诱导的气管松弛； （7）至 将VIP抗体的亲和力和滴度的变化与 个别患者哮喘的严重程度。 （8）评估 对VIP受体的自身抗体是否可能是一种致病性 哮喘的因素。 RCDA将确保大部分 候选人的时间（约90％）用于实现这些 目标。 候选人的学术环境提供支持 深入研究所需的机制，并提供良好的机制 与调查人员进行密切互动的机会 药理学，生物化学，分子生物学和肺部 药品。 候选人已经确立了富有成果的 与研究人员合作 神经肽，受体调节和信息传递，以及 气道疾病。 实验设计是：（1）VIP受体的溶解化； （2）表征其125-VIP结合特性； （3） 杂交瘤技术产生抗受体抗体； （4） HPLC和亲和色谱法的受体纯化； （5） 通过分析生化方法进行结构分析； （6） 与G蛋白，腺苷酸环化酶和受体恢复原状 磷脂，然后测定GTP敏感的VIP结合，并 VIP敏感的GTP结合，GTPase活性和环状AMP 合成; （7）比较受体亲和力，肽选择性 和受体 - 腺苷酸环化酶耦合，之后：（i）重建 在有或没有G蛋白的情况下，（ii）重建不同 磷脂，（iii）重组的磷脂酶处理 受体； （8）VIP受体自身抗体的RIA； （9）VIP的RIA 结合自身抗体及其亲和力和滴度的测量； （10）哮喘严重程度的肺活量评估； （11） 通过vip-autotibodies的功能特性描述 测量其（i）对VIP受体结合，环状AMP的影响 合成和气管松弛。