STRUCTURE-FUNCTION OF MUSCARINIC RECEPTOR COUPLING TO G-PROTEINS - RANDOM SATURATION MUTAGENESIS IDENTIFIES A CRITICAL DETERMINANT OF RECEPTOR AFFINITY FOR G-PROTEINS
STRUCTURE-FUNCTION OF MUSCARINIC RECEPTOR COUPLING TO G-PROTEINS - RANDOM SATURATION MUTAGENESIS IDENTIFIES A CRITICAL DETERMINANT OF RECEPTOR AFFINITY FOR G-PROTEINS
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DOI：
10.1074/jbc.270.7.3141
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发表时间：
1995-02-17
影响因子：
4.8
通讯作者：
BRANN, MR
中科院分区：
文献类型：
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作者：
BURSTEIN, ES;SPALDING, TA;BRANN, MR
为了获得受体-G蛋白偶联的结构-功能关系，创建了在第三胞内环的C-末端区域随机突变的人m5毒蕈碱乙酰胆碱受体（m5）的文库。基于其以配体依赖性方式扩增NIH 3 T3细胞的能力来鉴定功能性受体。这些受体具有野生型表型（组1）或功能受损（组2）。未发现“活化受体”，第2组受体中的耐受置换随机分布，通常包括脯氨酸和甘氨酸。相比之下，在第1组受体中的耐受性取代表现出接近跨膜结构域6的周期性，其中未观察到脯氨酸和甘氨酸取代。这些观察结果与来自跨膜结构域6的第三胞内环的C-末端区域的短α-螺旋延伸一致。Ala-441突变最常与第2组受体功能受损相关。在Ala-441的12个点突变进行了测试，所有引起EC（50）值的显着增加，对最大反应或激动剂结合亲和力的影响不大。这些结果表明，Ala-441是G蛋白的m5受体亲和力的关键决定因素，并存在于短α-螺旋的结构范围内。
To derive structure-function relationships for receptor-G protein coupling, libraries were created of human m5 muscarinic acetylcholine receptors (m5) randomly mutated in the C-terminal region of the third intracellular loop. Functional receptors were identified based on their ability to amplify NIH 3T3 cells in a ligand-dependent manner, These receptors either had wildtype phenotypes (Group 1) or were functionally impaired (Group 2). No ''activated receptors'' were identified, Tolerated substitutions in Group 2 receptors were randomly distributed and frequently included prolines and glycines. In contrast, tolerated substitutions in Group 1 receptors exhibited a periodicity proximal to transmembrane domain 6 where proline and glycine substitutions were not observed. These observations are consistent with a short alpha-helical extension of the C-terminal region of the third intracellular loop from transmembrane domain 6. Mutations at Ala-441 were most commonly associated with impaired function of Group 2 receptors. Twelve point mutations at Ala-441 were tested, and all caused marked increases in EC(50) values with little effect on maximal response or agonist binding affinity. These results indicate that Ala-441 is a key determinant of m5 receptor affinity for G proteins and exists within the structural context of a short alpha-helix.