MUTATIONS OF MUSCARINIC CHOLINERGIC RECEPTOR GENES

该项目的总体目标是确定分子 激活、信号转导和调节的机制。 原型G蛋白偶联受体（GPCR），即，人类毒蕈碱 胆碱能Hm 1受体。 胆碱能缺乏是老年痴呆症的一个标志 痴呆症，如阿尔茨海默氏症，以及对 胆碱能神经传递是开发治疗药物所必需的 战略布局 该项目采用遗传学方法来确定 Hm 1受体的功能结构域通过广泛的突变分析， 重点关注Hm 1的三个关键过程，这些过程仍然知之甚少 特别是对于一般的GPCR。 第一个包括分子 G蛋白介导的受体激活和信号转导机制 和第二信使途径。 其次，由于激活， 受体经历快速的细胞运输，即，隔离， 内化和循环利用。 第三个较慢的过程涉及到 功能性受体的破坏（下调）。 快速感受器 在几个测试的组织中检测不到Hm 1的脱敏作用， 因此，在此不作研究。 这个实验室建造了 许多Hm 1突变体，允许新的第一次解剖这些 途径，并确定几个必要的受体结构域的位置。 这些包括第二胞内环的中心部分（i2） 这是出乎意料地发现，发挥主要作用，在G蛋白激活 和内化，在i3环中间的S/T丰富的结构域 调节内化和随后的下调，以及 i3环的结构域，其介导不同的第二途径， 下调 由于这些域尚未确定为任何 GPCR，它们的完整表征将大大增加我们的 了解GPCR法规。 相似的受体结构域存在于大多数 因此，GPCR和这些新结构域的一般意义将 通过对所选其它受体进行突变分析来确定 在这个大家庭中的基因。 因为调节性i3环结构域 包括几个丝氨酸和苏氨酸残基， 蛋白激酶介导的内化过程磷酸化 会得到考验 这些研究将扩展到分析 Hm 2、Hm 3、β 2肾上腺素受体和促甲状腺激素中的相似结构域 释放激素（TRH）受体，以测试的一般有效性， GPCRS的功能域。 这项研究的结果将澄清 Hm 1的分子机制及其功能意义 激活和细胞贩运。 负责的受体结构域 然后将作为一种工具来分离靶蛋白介导 受体功能 Hm 1受体调节的知识将有助于 用毒蕈碱激动剂治疗阿尔茨海默病

The overall objective of this project is to determine the molecular mechanism of activation, signal transduction, and regulation of a prototype G protein coupled receptor (GPCR), i.e., the human muscarinic cholinergic Hm1 receptor. Cholinergic deficits are a hallmark of senile dementias such as Alzheimer, and a fundamental understanding of cholinergic neurotransmission is needed in development of therapeutic strategies. This project employees a genetic approach to determine the functional domains of the Hm1 receptor by extensive mutational analysis, whit focus on three key processes that remain poorly understood for Hm1 specifically and for GPCRs in general. The first includes the molecular mechanisms of receptor activation and signal transduction via G proteins and second messenger pathways. Second, as a result of activation, the receptors undergo rapid cellular trafficking, i.e., sequestration, internalization, and recycling. The third and slower process involves the destruction of functional receptor (down-regulation_. Rapid receptor desensitization is not detectable for Hm1 in several tissue tested, and it is therefore not studied here. This laboratory has constructed numerous Hm1 mutants that permit ne for the first time to dissect these pathways and define the location of several requisite receptor domains. These include the central portion of the second intracellular loop (i2) which was unexpectedly found to play a major role in G protein activation and internalization, an S/T rich domain in the middle of the i3 loop which regulation internalization and subsequent downregulation, and a domain of the i3 loop which mediates a distinct second pathway of downregulation. As such domains have not been identified for any of the GPCRs, their complete characterization will add significantly to our understanding of GPCR regulation. Similar receptor domains exist in most GPCRs, and the general significance of these novel domains will therefore be determine d by mutational analysis of selected additional receptor genes within this large family. Because the regulatory i3 loop domain includes several serine and threonine residues, the involvement of protein kinase mediated phosphorylation in the internalization process will be tested. These studies will be extended to the analysis of similar domains in Hm2, Hm3, beta-2 adrenoceptor, and the thyrotropin releasing hormone (TRH) receptor to test the general validity of the functional domains for GPCRS. The results from this study will clarify the molecular mechanisms and the functional significance of Hm1 activation and cellular trafficking. The responsible receptor domains will then serve as a tool to isolate the target protein mediating receptor functions. Knowledge of Hm1 receptor regulation will benefit therapy of Alzheimer disease with muscarinic agonist.