In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors.
In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors.
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DOI：
10.1042/bj2830001
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发表时间：
1992-04
期刊：
影响因子：
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通讯作者：
T. Savarese;Claire-M. FRASERt
中科院分区：
文献类型：
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作者：
T. Savarese;Claire-M. FRASERt
大量神经递质、肽类激素、神经调质、自分泌和旁分泌因子通过与细胞膜受体相互作用引起细胞代谢变化，细胞膜受体通过鸟嘌呤核苷酸结合调节蛋白（G蛋白）与细胞内效应酶偶联（见表1）。虽然与G蛋白偶联受体结合的内源性信号传导剂的数量相当大，但介导其作用的不同受体的数量甚至更大。在神经递质受体中，存在至少十种类型的肾上腺素能受体（a1 A、a1 B、a1 C、a2 A、a2 B、a2 C、a2 D、a1 A2和a13），至少五种类型的毒蕈碱乙酰胆碱（m1-m5）和多巴胺（D1-D5）受体，以及几种肾上腺素能、嘌呤能、光（视紫红质）和嗅觉受体。类似地，在多肽激素受体中，受体亚型的存在是有据可查的。G蛋白介导的跨膜信号转导通路已经引起了极大的关注，因为许多生理和药理学事件是由这些机制调制。G蛋白是由α、β和γ亚基组成的异源三聚体蛋白，是一个大基因超家族的成员（Gilman，1987; Iyengar & Birnbaumer，1990）。在基础状态下，G蛋白寡聚体与GDP形成复合物; GDP从G蛋白中解离的速率非常慢。激动剂结合后，G蛋白偶联受体经历一个或多个构象变化，触发受体-G蛋白相互作用，促进GTP在G蛋白a亚基内的位点交换结合的GDP。GTP与G蛋白a亚基的结合促进该亚基与、和y亚基的解离。GTP配体的α亚基（在某些情况下，fy亚基）负责调节不同效应子系统的活性，包括腺苷酸环化酶、磷脂酶、环GMP磷酸二酯酶和离子通道，导致细胞内的代谢和/或离子变化。该反应通过α亚基固有的GTP酶水解结合的GTP而终止，导致Ga与G1 γ重新结合。受体在该系统中的作用是作为G蛋白活化的催化剂。在过去的5年中，超过100个G蛋白偶联受体亚型已被克隆和测序。这种快速的进展在很大程度上是基于G蛋白偶联受体的一级结构的保守性，特别是在家族内，允许通过交叉杂交分离新的cDNA和基因组克隆。此外，新的分子克隆技术的应用，如PCR，对G蛋白偶联受体克隆的分离产生了重大影响。编码跨膜结构域的受体基因区域中的序列同一性已经允许产生与该家族的其他成员杂交的探针。PCR首先由Libert等人（1989）利用来分离G蛋白偶联受体家族的几个新成员，并且随后用于分离NKI（Hershey & Krause，1990）、NK-2（Gerard等人，1990）、D1多巴胺（Zhou等人，1990）和组胺H2（Zeroz等人，1991）受体，以及气味受体的新亚家族（Buck &阿克塞尔，1991）。视色素是第一个获得序列数据的G蛋白相关受体。在20世纪80年代早期，牛的完整氨基酸序列（Ovchinnikov等人，1982; Hargrave等人，1983）和绵羊（Pappin等，1984年）报道了视紫红质，并发现显示出明显的相似性。这些序列数据用于设计寡核苷酸探针并克隆编码牛和人视蛋白的基因（Nathans和Hogness，1983，1984）。当编码哺乳动物β-肾上腺素能受体的基因（狄克逊等人，1986），从推导的序列可以明显看出，该受体表现出类似于视紫红质的结构，并提示存在一个信号受体家族。G蛋白连接受体的二级和三级结构的当前模型在很大程度上基于古老的视网膜连接视觉色素细菌视紫红质的已知折叠模式，其在盐生盐杆菌的紫色膜内的天然存在的晶格中发现。这种色素，作为质子泵，不与任何G蛋白连接（Khorana，1988）。当通过电子显微镜和高分辨率电子衍射分析时，细菌视紫红质被视为七个a-螺旋，垂直于脂双层平面排列成束（亨德森和昂温，1975年;恩格尔曼等人，1980;亨德森等人，1990年）。该模型的基本特征如图1所示。诱变数据支持这样的观点，即螺旋的疏水面指向膜脂，亲水面指向分子上的活性位点（见下文）。G蛋白偶联受体的推导氨基酸序列的比较揭示了相似的二级结构：一个单一的多肽链包含六个相对疏水的结构域和第七个疏水性较低的区域。这些结构域在大多数受体类别之间显示序列相似性，并且在受体亚型之间显示显著的相似性。由于每种受体中的七个疏水结构域大小相似（20-28个氨基酸），且长度足以跨越脂质双层，因此假定它们形成跨膜结构域。
A large number of neurotransmitters, peptide hormones, neuromodulators, and autocrine and paracrine factors elicit changes in cellular metabolism by interaction with cell membrane receptors that are coupled to intracellular effector enzymes by guanine-nucleotide-binding regulatory proteins (G proteins) (see Table 1). While the number of endogenous signalling agents that bind to G protein-coupled receptors is quite large, the number of distinct receptors that mediate their actions is even larger. Among neurotransmitter receptors there exist at least ten types of adrenergic receptors (alA, aclB, alC9 X2A, a2B, a2C, a2D, 1 A2 and /13), at least five types of muscarinic acetylcholine (ml-m5) and dopamine (D1-D5) receptors, and several serotonergic, purinergic, light (rhodopsin) and olfactory receptors. Similarly, among polypeptide hormone receptors the existence of receptor subtypes is well documented. G protein-mediated transmembrane signalling pathways have generated a great deal of attention because of the many physiological and pharmacological events that are modulated by these mechanisms. G proteins, which are heterotrimeric proteins composed of a, /3, and y subunits, are members of a large gene superfamily (Gilman, 1987; Iyengar & Birnbaumer, 1990). In the basal state, the G protein oligomer exists in a complex with GDP; the rate of GDP dissociation from the G protein is extremely slow. Following agonist binding, G protein-coupled receptors undergo one or more conformational changes that trigger receptor-G-protein interactions, facilitating an exchange of GTP for bound GDP at a site within the a subunit of the G protein. The binding of GTP to the a subunit of the G protein promotes dissociation of this subunit from the , and y subunits. GTP-liganded a subunits (and in some cases, the fy subunits) are responsible for modulating the activity of distinct effector systems, including adenylate cyclase, phospholipases, cyclicGMP phosphodiesterase and ion channels, leading to metabolic and/or ionic changes within the cell. This reaction is terminated by hydrolysis of bound GTP by a GTPase intrinsic to the a subunit, leading to reassociation ofGa with G,/y. The role of the receptor in this system is to serve as a catalyst for the activation of G proteins. During the past 5 years, more than 100 G protein-coupled receptor subtypes have been cloned and sequenced. This rapid progress has been based in large part on the conservation of primary structure among G protein-coupled receptors, particularly within families, allowing for isolation ofnew cDNA and genomic clones by cross-hybridization. In addition, the application of new molecular cloning techniques, such as the PCR, has had a major impact on the isolation of G protein-coupled receptor clones. Sequence identity in the regions of the receptor genes coding for transmembrane domains has allowed probes to be generated that hybridize to other members of the family. PCR was first exploited by Libert et al. (1989) to isolate several novel members of the G protein-coupled receptor family, and has subsequently been used to isolate cDNAs for the NKI (Hershey & Krause, 1990), NK-2 (Gerard et al., 1990), D1 dopamine (Zhou et al., 1990) and histamine H2 (Gantz et al., 1991) receptors, as well as a new subfamily of odorant receptors (Buck & Axel, 1991). The visual pigments were the first G protein-linked receptors for which sequence data were obtained. In the early 1980s, the complete amino acid sequences for bovine (Ovchinnikov et al., 1982; Hargrave et al., 1983) and ovine (Pappin et al., 1984) rhodopsin were reported and found to display marked similarities. These sequence data were used to design oligonucleotide probes and clone the genes encoding bovine and human opsins (Nathans & Hogness, 1983, 1984). When the gene encoding a mammalian /,-adrenergic receptor (Dixon et al., 1986) was subsequently cloned, it was apparent from the deduced sequence that this receptor exhibited a structure similar to that of the rhodopsins, and suggested the existence of a family of signal receptors. Current models for the secondary and tertiary structure of G protein-linked receptors are based in large part on the known folding patterns of the ancient retinal-linked visual pigment, bacteriorhodopsin, that is found in naturally occurring lattices within the purple membranes of Halobacterium halobium. This pigment, which acts as a proton pump, is not linked to any G protein (Khorana, 1988). When analysed by electron microscopy and high resolution electron diffraction, bacteriorhodopsin is seen as seven a-helices, arranged in a bundle perpendicular to the plane of the lipid bilayer (Henderson & Unwin, 1975; Engelman et al., 1980; Henderson et al., 1990). The basic features of this model are depicted in Fig. 1. Mutagenesis data have supported the idea that the helices are oriented with their hydrophobic faces pointing out into the membrane lipids and their hydrophilic faces point in to the active sites on the molecule (see below). Comparison ofthe deduced amino acid sequences ofG proteincoupled receptors reveals a similar secondary structure: a single polypeptide chain containing six relatively hydrophobic domains plus a seventh region of lower hydrophobicity. These domains display sequence similarity among most receptor classes and marked similarities among receptor subtypes. Since the seven hydrophobic domains in each receptor are similar in size (20-28 amino acids) and of sufficient length to span the lipid bilayer, they have been postulated to form membrane-spanning domains.