An intramolecular disulfide bond between conserved extracellular cysteines in the gonadotropin-releasing hormone receptor is essential for binding and activation
An intramolecular disulfide bond between conserved extracellular cysteines in the gonadotropin-releasing hormone receptor is essential for binding and activation
复制标题
DOI：
10.1210/en.138.7.2800
复制
发表时间：
1997-07-01
期刊：
影响因子：
4.8
通讯作者：
Eidne, KA
中科院分区：
文献类型：
--
作者：
Cook, JVF;Eidne, KA
在这项研究中，使用了位点定向诱变和生化策略来确定细胞外Cys残基之间的二硫键是否有助于GnRH受体（GnRH- r）的结构完整性，如果是这样，则描述所涉及的键合模式的性质。大多数G蛋白偶联受体（gpcr）在第一和第二胞外结构域含有一对保守的Cys残基，这些残基已被证明可以形成受体稳定的二硫桥结构。然而，许多gpcr含有其他非保守的Cys残基，并且在一些gpcr中，这些残基也被证明有助于受体的完整性和稳定性。大鼠GnRH-R含有4个细胞外Cys残基。其中两个在GPCR超家族中是保守的，位于第一和第二细胞外环的Cys114和Cys195位置。分别。另外两个Cys残基位于氨基酸末端的Cys14和第二个细胞外环的Cys199的非保守位置。为了评估细胞外Cys残基在二硫键相互作用中的作用，每个这些残基都突变为Ala。在COS-1细胞中表达，并确定配体结合和第二信使特性。为了监测野生型（WT）和突变型受体细胞表面表达水平，将血凝素（HA）表位标签纳入受体构建物（GnRH-R WT、Cys14Ala、Cys114Ala、Cys195Ala和Cys199Ala）中。Cys199Ala突变体保持了与WT型GnRH-R相当的受体结合和第二信使产生水平，而突变体Cys14Ala表现出一些配体结合和功能受体活性。虽然程度有所降低。突变体Cys114Ala和Cys195Ala的细胞表面表达水平与WT受体相似，但没有表现出功能反应。在二硫弯曲还原剂DTT的存在下，WT和突变体受体Cys14Ala和Cys199Ala的特异性结合受到抑制，这意味着在这些突变体受体中形成了二硫键，并且可以减少二硫键。本研究表明，GnRH-R残基Cys114和Cys195具有维持受体功能所必需的二硫键相互作用。相比之下，Cys14和Cys199不参与配体结合或第二信使产生所需的二硫键。
In this study, site-directed mutagenesis and biochemical strategies have been used to establish whether disulfide bonding between extracellular Cys residues contributes to the structural integrity of the GnRH receptor (GnRH-R) and, if so, to delineate the nature of the bonding patterns involved. The majority of G protein-coupled receptors (GPCRs) contain a pair of conserved Cys residues in the first and second extracellular domains, and these residues have been shown to form a receptor stabilizing disulfide bridge structure. However, many GPCRs contain other nonconserved Cys residues, and in some GPCRs these have also been shown to contribute to receptor integrity and stability. The rat GnRH-R contains four extracellular Cys residues. Two are conserved throughout the GPCR superfamily and lie at positions Cys114 and Cys195 in the first and second extracellular loops. respectively. The other two Cys residues occupy nonconserved positions at Cys14 in the amino terminus and Cys199 in the second extracellular loop. To assess the role of extracellular Cys residues in disulfide bonding interactions, each of these residues were mutated to Ala. expressed in COS-1 cells, and ligand binding and second messenger properties ascertained. To monitor levels of wild-type (WT) and mutant receptor cell surface expression, a hemagglutinin (HA) epitope tag was incorporated into the receptor constructs (GnRH-R WT, Cys14Ala, Cys114Ala, Cys195Ala, and Cys199Ala). Cys199Ala mutant maintained levels of receptor binding and second messenger production comparable with the WT GnRH-R, whereas mutant Cys14Ala exhibited some ligand binding and functional receptor activity. albeit at a reduced level. Mutations Cys114Ala and Cys195Ala showed no functional responses despite displaying levels of cell surface expression similar to the WT receptor, Specific binding of the WT and mutant receptors Cys14Ala and Cys199Ala was inhibited in the presence of the disulfide bend reducing agent, DTT, implying that disulfide bonds are formed and can be reduced in these mutant receptors. This study demonstrates that GnRH-R residues Cys114 and Cys195 have a disulfide bonding interaction role essential for the maintenance of receptor function. In contrast, Cys14 and Cys199 are not involved in disulfide bonding that is required for ligand binding or second messenger production.