REGULATION OF M2 MUSCARINIC RECEPTORS

M2 毒蕈碱受体的调节

• 批准号: 6183696
• 负责人: M. M HOSEY
• 金额: $ 26.06万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6183696
• 关键词: G protein; antisense nucleic acid; arrestins; beta adrenergic receptor kinase; biological signal transduction; caveolas; chimeric proteins; confocal scanning microscopy; enzyme activity; enzyme substrate; fatty acylation; heart cell; immunocytochemistry; immunoelectron microscopy; laboratory rat; muscarinic receptor; palmitates; phosphorylation; protein structure function; receptor coupling; receptor mediated endocytosis; receptor sensitivity; site directed mutagenesis; transfection

Desensitization is a mechanism that plays an important role in turning off receptor-mediated signal transduction pathways. The goal of the proposed studies is to define the molecular basis for two processes associated with the rapid phase of desensitization of G-protein coupled receptors (GPRs), namely receptor/G-protein (R/G) uncoupling and receptor internalization. The studies will use muscarinic cholinergic receptors (mAChRs), particularly the m2 mAChRs, as a model, as enhanced or diminished receptor signalling through mAChR subtypes has been suggested to play critical roles in several diseases, including Alzheimer's and Parkinson's diseases, and the cardiovascular pathologies associated with Chagas' disease. The hypothesis that underlies this proposal is that agonists induce phosphorylation of the mAChR on defined serine/threonine (S/T) and this initiates several distinct downstream events that cause R/G uncoupling and internalization. In other systems, arrestins bind to phosphorylated GPRs to cause R/G uncoupling and guide GPRs to clathrin coated pits for endocytosis. Significant progress has been made in understanding phosphorylation-dependent events associated with desensitization of the mR mAChR, however, it is unclear how phosphorylation leads to R/G uncoupling and internalization. Compelling data suggest that arrestins do not play a major role in internalization of the m2 mAChR, leading us to question the putative role of arrestins in R/G uncoupling. Aim one will test the hypothesis that arrestins cause uncoupling of m2 mAChR from G-proteins. Over expression of arrestin binding proteins and anti-sense constructs will be used to deplete endogenous arrestins and ascertain their effects on R/G uncoupling. Aim two will define the mechanisms and role of phosphorylation-dependent, arrestin-independent internalization of mAChRs. Potential roles of caveolae and mAChR palmitoylation will be examined A search will be made for interacting proteins that may guide the receptors to the endocytic machinery. Aim 3 will test the hypothesis that S/T-rich clusters analogous to those identified in the m2 mAChR might be newly recognized motifs that have a common regulatory role in regulation of GPRs. The S/T residues in two clusters in the third cytoplasmic domains of the Gq-coupled m3 and the Gi-coupled m4 mAChRs will be mutated and the effects on agonist-dependent phosphorylation and desensitization will be assessed. The results will provide new insights into novel events involved in turning off the signals from mAChRs.

脱敏是一种在关闭的机制中起着重要作用的机制 受体介导的信号转导途径。提议的目标 研究是为了定义与相关的两个过程的分子基础 G蛋白偶联受体（GPRS）脱敏的快速阶段， 即受体/G蛋白（R/G）解偶联和受体内在化。 这些研究将使用毒蕈碱胆碱能受体（MACHR）， 特别是M2 MACHR作为模型，作为增强或减少的受体 通过MACHR子类型发出信号来发挥关键作用 在包括阿尔茨海默氏症和帕金森氏病在内的多种疾病中的角色， 以及与查加斯病有关的心血管病理。这 基于该提议的假设是激动剂诱发 MACHR在定义的丝氨酸/苏氨酸（S/T）上的磷酸化，这 启动几个不同的下游事件，这些事件导致R/G解开和 内部化。在其他系统中，逮捕蛋白与磷酸化的GPR结合 引起r/g解偶联并引导GPRS覆盖的网状坑 内吞作用。在理解方面取得了重大进展 与MR脱敏有关的磷酸化依赖性事件 但是，尚不清楚磷酸化如何导致R/G解偶联 和内在化。引人入胜的数据表明，逮捕蛋白不会发挥 M2 MACHR内在化中的主要作用，使我们质疑 逮捕蛋白在r/g解偶联中的推定作用。 AIM将测试 假设逮捕蛋白会导致G蛋白的M2 MACHR解偶。 过度表达抑制蛋白结合蛋白和抗敏感构建体 将用于耗尽内源性抑制蛋白并确定其作用 在r/g解偶联上。目标两个将定义机制和作用 MACHR的磷酸化依赖性，抑制蛋白独立的内在化。 小屋和棕榈酰化的潜在作用将检查 将对可能引导受体的相互作用蛋白进行搜索 到内吞机械。 AIM 3将检验以下假设： 类似于M2 MACHR中的群集可能是新的 公认的主题，在调节中具有共同的调节作用 GPRS。第三个细胞质结构域的两个簇中的S/T残基 GQ耦合的M3和GI耦合M4 MACHR将被突变，并将 对激动剂依赖性磷酸化和脱敏的影响将是 评估。结果将为涉及的新事件提供新的见解 关闭MACHRS的信号。