RANDOM MUTAGENESIS OF A G PROTEIN COUPLING DOMAIN

乙酰胆碱受体由五种基因亚型组成， 通过与G蛋白偶联来介导信号转导。的结构 G蛋白偶联受体的细节尚不清楚，因为没有高分辨率。 结构信息可以从物理测量中获得。这些 已知的受体是完整的膜蛋白，有七个 形成七个跨膜(TM)结构域的疏水区(TM1-TM7) 由三个外层(o1-o3)和三个细胞内环(i1-i3)连接。 研究表明，多种突变型受体的氨基 胞内表面TM结构域附近的酸是必不可少的 用于G蛋白偶联。对嵌合受体的研究表明 I2和I3在定义受体的选择性方面都有循环 独特的G蛋白。而在毒碱受体亚型的情况下， I3环(Ni3)N-末端的氨基酸是至关重要的 决定G蛋白Gq亚型偏好的决定因素 大兵。在α肾上腺素能受体的情况下，c-肾上腺素能受体中的氨基酸 I3环的末端区域(Ci3)与受体有关 激动剂结合的激活和变构调节。获得洞察力 关于受体/G蛋白偶联的结构要求，我们将 使M5 M受体的I2、Ni3和Ci3区 随机饱和诱变。突变型M受体与多种 将通过筛选所有重组子来鉴定功能表型 通过我们开发的快速功能分析。我们的屏幕是 旨在识别氨基酸替代的范围，以使 保留整体受体功能，激活脑内受体 缺乏激动剂，改变G蛋白受体的亲和力，以及 改变受体/G蛋白偶联效率。当与 分子模型和物理测量数据，我们预计 我们的实验将提供定义结构基础的经验数据 M受体的配体结合和激活。

Muscarinic acetylcholine receptors consist of five genetic subtypes that mediate signal transduction by coupling with G-proteins. The structures of G-protein-coupled receptors are not known in detail, as no high resolution structural information is available from physical measurements. These receptors are known to be integral membrane proteins, that have seven hydrophobic regions that form seven transmembrane (TM) domains (TM1-TM7) connected by three outer (o1-o3) and three intracellular loops (i1-i3). Studies will a variety of mutant receptors have indicated that the amino acids adjacent to the TM domains on the intracellular face are essential for G-protein coupling. Studies with chimeric receptors have implicated both the i2 and i3 loops in defining the selectivity of receptors for distinct G-proteins. And in the case of muscarinic receptor subtypes, amino acids on the N-terminal side of the i3 loop (Ni3) are critical determinants that define subtype preferences for the G-proteins Gq versus Gi. In the case of the alpha adrenergic receptors, amino acids in the c- terminal region of the i3 loop (Ci3) have been implicated in receptor activation and allosteric regulation of agonist binding. To gain insight into the structural requirements of receptor/G-protein coupling, we will subject the i2, Ni3 and Ci3 regions of the m5 muscarinic receptor to random-saturation mutagenesis. Mutant muscarinic receptors with a variety of functional phenotypes will be identified by screening all recombinants via rapid functional assays that we have developed. Our screens are designed to identify the range of amino acid substitutions that allow retention of overall receptor function, activate the receptor in the absence of agonist, change the affinity of the receptor for G-protein, and change receptor/G-protein coupling efficiency. When combined with molecular modeling and data from physical measurements, we anticipate that our experiments will provide empirical data defining the structural basis of ligand binding and activation of a muscarinic receptor.