A disulfide bonding interaction role for cysteines in the extracellular domain of the thyrotropin-releasing hormone receptor
A disulfide bonding interaction role for cysteines in the extracellular domain of the thyrotropin-releasing hormone receptor
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DOI：
10.1210/en.137.7.2851
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发表时间：
1996-07-01
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;McGregor, A;Eidne, KA
研究了二硫键和巯基在TRH与其受体特异性结合中的作用。到目前为止，从不同物种分离的TRH受体中，只有两个胞外半胱氨酸残基(Cys98位于跨膜螺旋2和3之间的胞外环和Cys179位于跨膜螺旋4和5之间的胞外环)与其他G蛋白偶联受体中的半胱氨酸残基处于同源位置。另一个半胱氨酸(Cys100)位于第一细胞外环和第三跨膜结构域之间的交界处，与Cys98非常接近。为了评估这些TRH-R半胱氨酸残基在二硫键相互作用中的作用，将它们突变为Ser或Ala。在COS-1细胞中表达了6种突变型受体(Cys98Ser、Cys98Ala、Cys179Ser、Cys179Ala、Cys100Ser和Cys100Ala)，并检测了它们结合TRH和激活总肌醇磷酸(IP)形成的能力。TRH-R突变体Cys100Ser和Cys100Ala表现出与野生型(WT)相似的TRH结合亲和力和IP激活。相反，突变体Cys98Ser、Cys98Ala、Cys179 Ser和Cys179Ala没有显示高亲和力的TRH结合。经IP刺激后，Cys98Ala和Cys179Ala的电势比WT降低了4个数量级。Cys98Ser效价以活体数量级下降，而Cys179Ser没有IP产生。Northern blotting证实所有突变体TRH-Rs均在信使RNA(MRNA)水平上表达。在TRH-R WT和TRH-R Cys突变体的NH2末端加入了来自流感嗜血杆菌血凝素蛋白的表位标签，以允许独立评估受体蛋白的细胞表面表达。未显示受体结合的TRH-R突变体(Cys98Ser、Cys98Ala、Cys179Ala)显示细胞表面受体表达的WT水平，表明这些突变体中受体结合的丧失不是由于受体表达的丧失。二硫苏糖醇(二硫苏糖醇)和对氯汞苯甲酸(p-CMB)呈剂量依赖性抑制TRH的特异性结合。二硫苏糖醇对结合的抑制意味着二硫键的完整性对于TRH与其受体的结合是重要的。P-CMB对TRH结合的显著抑制表明，自由巯基也与配体与其受体的结合有关。这项研究提供的证据表明，在Cys98和Cys179之间存在一个二硫键，这对于维持受体在正确的配基结合构象中是必不可少的。Cys100被认为不具有二硫键相互作用作用。化学修饰后的结果表明，TRH-R中的游离巯基也可能在配体相互作用中起作用。
The roles of disulfide and sulfhydryl groups in the specific binding of TRH to its receptor have been examined. In all TRH receptors (TRH-Rs) isolated from different species so far, there are only two extracellular cysteine residues (Cys98 in the extracellular loop between transmembrane helices 2 and 3 and Cys179 in the extracellular loop between transmembrane helices 4 and 5) that are in positions homologous to cysteine residues in other G protein-coupled receptors. Another Cys (Cys100) is located in close proximity to Cys98 at the interface between the first extracellular loop and third transmembrane domain. To assess the role of these TRH-R Cys residues in disulfide bonding interactions, they were mutated to either Ser or Ala. Six mutant receptors (Cys98Ser, Cys98Ala, Cys179Ser, Cys179Ala, Cys100Ser, and Cys100Ala) were expressed in COS-1 cells and tested for their ability to bind TRH and to activate total inositol phosphate (IP) formation. TRH-R mutants Cys100Ser and Cys100Ala showed TRH binding affinities and IP activation similar to the wild-type (WT). In contrast, mutants Cys98Ser, Cys98Ala, Cys179Ser, and Cys179Ala showed no high affinity TRH binding. The potencies of Cys98Ala and Cys179Ala as measured by IP stimulation were decreased by four orders of magnitude when compared with WT. Cys98Ser potency decreased by live orders of magnitude, whereas Cys179Ser showed no IP production. Northern blotting confirmed expression of all the mutant TRH-Rs at the messenger RNA (mRNA) level. An epitope tag derived from the Haemophilus influenza hemagglutinin protein was incorporated at the NH2 termini of the TRH-R WT and TRH-R Cys mutants to allow the independent assessment of cell surface expression of receptor protein. TRH-R mutants that failed to show receptor binding (Cys98Ser, Cys98Ala, Cys179Ala) showed WT levels of cell surface receptor expression, indicating that loss of receptor binding in these mutants is not attributable to loss of receptor expression. In contrast, cell surface expression of Cys179Ser, which showed no ligand induced IP stimulation, could not be detected.Dithiothreitol, a disulfide bond reducing agent, and p-chloromercuribenzoic acid (p-CMB), a sulfhydryl blocking compound, reduced specific TRH binding in a dose-dependent manner. The inhibition of binding by dithiothreitol implies that the integrity of a disulfide bond is important for TRH binding to its receptor. The dramatic inhibition of TRH binding by p-CMB indicates that free sulfhydryl groups are also associated with the binding of the ligand to its receptor. This study presents evidence that a disulfide bond exists between Cys98 and Cys179 which is essential for maintaining the receptor in the correct conformation for ligand binding. Cys100 is not thought to have a disulfide bonding interaction role. Results obtained after chemical modification have shown that free sulfhydryl groups within the TRH-R may also have a role in ligand interactions.