Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling.
Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling.
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DOI：
10.1016/s0021-9258(18)41524-4
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发表时间：
1993-10
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影响因子：
--
通讯作者：
O. Moro;J. Lameh;P. Hȍgger;W. Sadee
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文献类型：
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作者：
O. Moro;J. Lameh;P. Hȍgger;W. Sadee
七螺旋G蛋白偶联受体（GPCRs）的信号转导涉及多个受体结构域，但尚未确定用于偶联的通用共有结构域。丙氨酸诱变扫描进行细胞内环和COOH尾的人毒蕈碱胆碱能受体（Hm 1），以确定耦合结构域。将丙氨酸突变体转染入U293人胚肾细胞后，测定磷脂酰肌醇（PI）周转率的刺激。四个区域（环i1、i2和i3的NH 2和COOH连接处）中的丙氨酸取代使偶联效率降低约50%或更多，但最强的降低（> 80%）是由丙氨酸取代单个氨基酸亮氨酸131引起的。该残基位于高度保守的GPCR基序（DRYXXV（I）XXPL）内的第二胞内环（i2）的中间。在几乎所有克隆的GPCR中，与Hm 1中Leu-131相当的位置包含一个庞大的疏水性氨基酸（L、I、V、M或F）。用极性氨基酸（天冬氨酸和天冬酰胺）取代Leu-131也导致强烈的偶联缺陷，而苯丙氨酸（在β 2肾上腺素受体的等效位置发现）可以取代亮氨酸而不失去Hm 1的PI偶联能力。丙氨酸取代相应的氨基酸在Hm 3受体（L174 A）也抑制激动剂刺激的PI营业额，而取代苯丙氨酸-139在β 2肾上腺素受体抑制腺苷酸环化酶的刺激。我们建议，一个庞大的疏水性氨基酸在中间的i2环作为一个一般的网站相关的G蛋白偶联，而耦合的选择性是由其他受体结构域。
Signal transduction of the heptahelical G protein-coupled receptors (GPCRs) involves multiple receptor domains, but a universal consensus domain for coupling has not yet been defined. Alanine mutagenesis scanning was performed on the intracellular loops and the COOH tail of the human muscarinic cholinergic receptor (Hm1) to identify coupling domains. Stimulation of phosphatidylinositol (PI) turnover was determined after transfection of the alanine mutants into U293 human embryonic kidney cells. Alanine substitutions in four regions (loops i1, i2, and NH2 and COOH junctions of i3) impaired coupling efficiency by approximately 50% or more, but the strongest reduction (> 80%) resulted from alanine replacement of a single amino acid, leucine 131. This residue is located in the middle of the second intracellular loop (i2), within the highly conserved GPCR motif (DRYXXV(I)XXPL). The position equivalent to Leu-131 in Hm1 contains a bulky hydrophobic amino acid (L, I, V, M, or F) in nearly all cloned GPCRs. Substitution of Leu-131 with polar amino acids (aspartate and asparagine) also resulted in strongly defective coupling, whereas phenylalanine (found in the equivalent position in the beta 2 adrenoceptor) can replace leucine without losing PI coupling ability of Hm1. Alanine substitution of the corresponding amino acid in the Hm3 receptor (L174A) also inhibited agonist-stimulated PI turnover, while replacing Phe-139 with alanine in the beta 2 adrenoceptor suppressed stimulation of adenylyl cyclase. We propose that a bulky hydrophobic amino acid in the middle of the i2 loop serves as a general site relevant to G protein coupling, whereas coupling selectivity is governed by other receptor domains.