RANDOM MUTAGENESIS OF A G-PROTEIN COUPLING DOMAIN

G 蛋白偶联结构域的随机诱变

• 批准号: 2191874
• 负责人: MARK R BRANN
• 金额: $ 21.86万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191874
• 关键词: G protein; chemical models; gene mutation; genetic library; molecular site; muscarinic receptor; phenotype; protein structure; protein structure function; receptor binding; receptor coupling; receptor sensitivity; recombinant proteins

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.

毒蕈碱型乙酰胆碱受体由五种基因亚型组成， 通过与G蛋白偶联介导信号转导。 的结构 G蛋白偶联受体的细节尚不清楚，因为没有高分辨率的 结构信息可从物理测量获得。 这些 已知受体是膜蛋白的一部分， 形成七个跨膜（TM）结构域（TM 1-TM 7）的疏水区域 由三个外环（O 1-O3）和三个胞内环（I1-I3）连接。 对各种突变受体的研究表明， 细胞内表面TM结构域附近的酸是必需的 用于G蛋白偶联。 嵌合受体的研究表明， I2和I3环在定义受体的选择性时， 不同的G蛋白 对于毒蕈碱受体亚型， i3环N-末端侧的氨基酸（Ni 3）是关键的 确定G蛋白Gq与 吉 在α肾上腺素能受体的情况下，c- i3环的末端区域（C13）已经涉及受体 激动剂结合的活化和变构调节。 获得洞察力 进入受体/G蛋白偶联的结构要求，我们将 使m5毒蕈碱受体的i2、N13和C13区经受 随机饱和突变。 具有多种突变型毒蕈碱受体 通过筛选所有重组体， 通过我们开发的快速功能分析。 我们的屏幕 设计用于鉴定氨基酸取代的范围， 保留整个受体功能，激活受体中的受体， 不存在激动剂，改变受体对G蛋白的亲和力，和 改变受体/G蛋白偶联效率。 当结合 分子模型和物理测量的数据，我们预计， 我们的实验将提供确定结构基础的经验数据 配体结合和毒蕈碱受体激活的过程。