MOLECULAR CHARACTERIZATION OF MUSCARINIC ACH RECEPTORS

毒蕈碱 ACH 受体的分子表征

• 批准号: 3402166
• 负责人: WILLIAM L KLEIN
• 金额: $ 8.27万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-23 至 1988-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3402166
• 关键词: acetylcholine; affinity chromatography; autoradiography; chemical binding; electron microscopy; gel electrophoresis; genetic library; genetic translation; histochemistry /cytochemistry; hybridomas; immunochemistry; ligands; molecular weight; monoclonal antibody; neural information processing; neurochemistry; synapses

The long-range goal of this work is to understand how signal transduction is established and maintained at CNS cholinergic synapses; its focus is on the biochemistry, development and cellular regulation of muscarinic ACh receptors. These receptors mediate a large proportion of CNS cholinergic signaling, have been implicated in many behaviorally and clinically significant phenomena and show a broad spectrum of transductional and regulatory responses. At present, it appears most appropriate to isolate and begin characterizing receptor system proteins and to generate receptor specific antibodies for studies of muscarinic cell and developmental biology. The following three aims have therefore been chosen: AIM #1--To generate a library of muscarinic receptor-specific monoclonal antibodies. Partially purified (250-fold) bovine brain receptors will be used for antibody induction using a combination of methods already tested for their effectiveness. Binding assays and western blots will be used in testing antibody specificity. AIM #2--To purify and begin characterization of receptor molecules. Immunoaffinity chromatography will be added to tested separations based on subcellular fractionation, hydrophobicity, charge, molecular weight and specific glycosylation. 2D-Gel, sedimentation equilibrium and N-terminal analyses will be used in testing purity. The ligand specificity, number of subunits, number of binding sites, and overall amino acid composition of the purified molecules will be assessed. AIM #3--To localize receptors as a function of synaptic development, post-translational modification, and cholinergic stimulation. The monoclonal antibodies obtained above will be used to localize receptors on avian retina neurons at the subcellular and ultrastructural levels. A novel approach employing isolation of differentiated neurons and HVEM whole mount analysis will be used.

这项工作的长期目标是了解信号转导如何