IMMUNOLOGICAL STUDIES OF MUSCARINIC RECEPTOR SUBTYPES

本申请寻求资金，以继续进行生产项目 并表征针对毒蕈碱的定点抗肽抗体 受体（MR）亚型。 该提案的目标是研究不同的 MR亚型的结构、结合特性、功能和定位， 哺乳动物的大脑 基于药理学假设的MR亚型 结合数据已被最近的分子生物学证据所证实 五个不同的MR基因编码独特的一级序列。 然而，药理学、生物化学和 每个亚型的功能尚不清楚，主要是因为没有 区分五种受体蛋白的可用方法。 目前可用的毒蕈碱配体仅区分三种MR亚型， 核苷酸探针不识别基因产物或受体，而仅识别 DNA或RNA受体信息。 因此，免疫学方法具有 1）区分高度同源的受体 蛋白质和2）直接识别受体蛋白。 具体地说， 针对m1-m5 MR亚型独特序列的单克隆和多克隆抗体 将被生产。 到目前为止，八个单克隆抗体从杂交瘤 产生腹水的小鼠已经产生了独特的肽 对应于M1。 还产生了多克隆抗体， 肽M2和M3。 免疫斑点，ELISA，免疫印迹， 免疫沉淀和免疫组织化学将用于筛选和 表征抗体对免疫原MR的特异性， MR亚型和分子定位。 MR的超微结构定位 啮齿动物和人脑中的亚型将在光照下进行评估， 电镜水平。 将鉴定相关的G蛋白 可溶性MR-G蛋白的特异性免疫沉淀 配合物 MR亚型之间一级结构的区别将 在选择性电泳后使用二维电泳进行 免疫沉淀或免疫亲和层析。 结果进行 研究将更清楚地了解中央MR， 记忆障碍治疗干预的重要目标 与老年痴呆症有关

This application seeks funds to continue the on-going project of producing and characterizing site-directed anti-peptide antibodies to muscarinic receptor (MR) subtypes. The goal of the proposal is to study the distinct structure, binding properties, function and location of MR subtypes in mammalian brain. MR subtypes postulated on the basis of pharmacological binding data have been verified by the recent molecular biological evidence for five distinct MR genes coding for unique primary sequences. Nevertheless, correlation between the pharmacology, biochemistry and function of each subtype remain unclear, primarily because there are no available methodologies which distinguish the five receptor proteins. Currently available muscarinic ligands distinguish only three MR subtypes, and nucleotide probes do not recognize gene product, or receptor, but only DNA or RNA receptor message. An immunological approach, therefore, has the advantage of 1) distinguishing between the highly homologous receptor proteins and 2) directly recognizing receptor proteins. Specifically, mono- and polyclonal antibodies to unique sequences of m1-m5 MR subtypes will be produced. Thus far, eight monoclonal antibodies from hybridomas and ascites-producing mice have been generated to peptide uniquely corresponding to m1. Polyclonal antibodies have also been generated to peptides m2 and m3. A combination of immunodot, ELISA, immunoblotting, immunoprecipitation and immunohistochemistry will be used to screen and characterize antibodies with respect to specificity for the immunogen, MR, MR subtype, and molecular location. Ultrastructural location of MR subtypes in the rodent and human brain will be assessed at the light and electron microscopic levels. Associated G proteins will be identified electrophoretically by specific immunoprecipitation of soluble MR-G protein complexes. Distinction in primary structure between the MR subtypes will be made using two-dimensional electrophoresis after selective immunoprecipitation or immunoaffinity chromatography. Results of these studies will provide a clearer understanding of central MR which remain important targets for therapeutic intervention in treating memory disorders associated with advanced age and Alzheimer's Disease.