SP RECEPTOR BINDING SITES FOR AGONISTS AND ANTAGONISTS

激动剂和拮抗剂的 SP 受体结合位点

• 批准号: 2609645
• 负责人: NORMAN D BOYD
• 金额: $ 27.19万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2609645
• 关键词: CHO cells; SDS polyacrylamide gel electrophoresis; affinity labeling; biological signal transduction; crosslink; disulfide bond; high performance liquid chromatography; immunoaffinity chromatography; immunoprecipitation; mass spectrometry; matrix assisted laser desorption ionization; molecular site; neuropeptide receptor; neurotransmitter agonist; neurotransmitter antagonist; posttranslational modifications; protein sequence; protein structure function; receptor binding; receptor coupling; site directed mutagenesis; substance P; transcription factor; western blottings

This renewal is a continuation of studies on the structure and function of the substance P (SP) receptor. The long term objective of this proposal is to understand, at the molecular level, the basis of agonist and antagonist specificity, receptor activation and downstream signaling events. Our approach will be to use photoreactive SP analogs containing p-benzoylphenylalanine substituted at different positions in the eleven amino acid sequence of SP. The site(s) of covalent attachment of these photoligands on the SP receptor will be determined by MALDI-mass spectrometric analysis of isolated photolabeled receptor fragments, which are generated by enzymatic and/or chemical cleavage of the receptor. These studies will aid in defining the SP binding pocket and orient the peptide within that pocket. We will develop benzophenone-containing derivatives of non-peptide SP antagonists as photoprobes of the antagonist binding domain(s). An understanding of the relationship between the binding sites for peptide agonist and the non-peptide antagonists forms the foundation for understanding underlying mechanisms of antagonism, which is essential for drug development. The availability of photoreactive peptide agonists, together with chemical crosslinking and immunodetection, provides us with tools to identify specific G proteins and other regulatory proteins which interact with the SP receptor. This approach will provide important information about the signal transduction machinery associated with the SP receptor. As it has been estimated that up to 60 percent of all medicines used today exert their effects through G protein signaling pathways, these studies may provide new and interesting information of basic clinical relevance. The peptide substance P has attracted considerable attention because of its multiple biological activities: as a neurotransmitter in the central, sensory and autonomic nervous systems; as an agent that causes contraction of smooth muscle in the gastrointestinal tract and as a mediator of inflammation and immune responses. SP has been implicated in a number of sensory and neurogenerative disorders. The information provided by our continuing effort should form the basis for understanding and development of novel SP receptor agonists and antagonists.

此次更新是结构和功能研究的延续 P 物质 (SP) 受体。 本次活动的长远目标 建议是在分子水平上了解激动剂的基础 和拮抗剂特异性、受体激活和下游信号传导 事件。 我们的方法是使用光反应性 SP 类似物 含有在不同位置取代的对苯甲酰基苯丙氨酸 SP的11个氨基酸序列。 共价键位点 这些光配体在 SP 受体上的附着将被确定 通过 MALDI 质谱分析分离的光标记 受体片段，由酶促和/或化学产生 受体的裂解。 这些研究将有助于定义 SP 结合口袋并将肽定位在该口袋内。 我们将 开发含二苯甲酮的非肽SP衍生物 拮抗剂作为拮抗剂结合结构域的光探针。 一个 了解肽结合位点之间的关系 激动剂和非肽拮抗剂构成了基础 了解拮抗作用的潜在机制，这一点至关重要 用于药物开发。 光反应肽的可用性 激动剂，加上化学交联和免疫检测， 为我们提供了识别特定 G 蛋白和其他蛋白的工具 与 SP 受体相互作用的调节蛋白。 这种做法 将提供有关信号转导的重要信息 与 SP 受体相关的机制。 正如已估计的那样 当今使用的所有药物中多达 60% 都发挥了作用 通过G蛋白信号通路，这些研究可能提供新的 以及基本临床相关的有趣信息。 肽物质P引起了人们的广泛关注，因为 其多种生物活性：作为中枢神经递质， 感觉和自主神经系统；作为导致 胃肠道平滑肌的收缩，并作为 炎症和免疫反应的介质。 SP已 与许多感觉和神经生成障碍有关。 这 我们不断努力提供的信息应构成以下基础： 新型 SP 受体激动剂的理解和开发 对手。