A profile of the residues in the second extracellular loop that are critical for ligand recognition of human prostacyclin receptor.
A profile of the residues in the second extracellular loop that are critical for ligand recognition of human prostacyclin receptor.
复制标题
第二个胞外环中残基的分布图，这些残基对于人前列环素受体的配体识别至关重要。
DOI：
10.1111/j.1742-4658.2007.06183.x
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Ruan,Ke-He
中科院分区：
文献类型：
--
作者：
Ni,Feng;So,Shui-Ping;Cervantes,Vanessa;Ruan,Ke-He
前列腺素受体的第二胞外环（eLP 2）中的残基对于特异性配体识别是重要的，在我们早期的血栓素A2受体（TP）的研究中使用NMR光谱和重组蛋白方法的组合预测了这些残基。 为了进一步验证这一假设，另一种前列腺素受体，前列环素受体（IP），它具有相反的生物学特性的TP，被用作这些研究的模型。使用Ala扫描方法构建了一组在eLP 2非保守残基处具有定点突变的重组人IP，然后在HEK 293和COS-7细胞中表达。HEK 293细胞中重组受体的表达水平比COS-7细胞高6倍。通过诱变分析鉴定了IP eLP 2的N末端片段（G159、Q162和C165）和C末端片段（L172、R173、M174和P179）内对配体识别和结合重要的残基。通过使用关键残基Q162、L172、R173和M174具有独特化学性质的不同氨基酸残基进行特异性定点诱变，进一步证明了IP特异性配体识别的分子机制。与TP eLP 2中鉴定的相应功能残基的比较显示，四个残基中的三个（Q162、R173和M174）是非保守的，并且这些残基被提议参与特异性配体识别。 通过环构象的构型讨论了G159和P179在配体识别中的重要性。这些研究进一步表明，对所有8种前列腺素受体的eLP 2区域中的残基进行表征可能是揭示G蛋白偶联受体配体选择性的分子机制的有效方法。
The residues in the second extracellular loop (eLP2) of the prostanoid receptors, which are important for specific ligand recognition, were previously predicted in our earlier studies of the thromboxane A2receptor (TP) using a combination of NMR spectroscopy and recombinant protein approaches. To further test this hypothesis, another prostanoid receptor, the prostacyclin receptor (IP), which has opposite biological characteristics to that of TP, was used as a model for these studies. A set of recombinant human IPs with site‐directed mutations at the nonconserved eLP2 residues were constructed using an Ala‐scanning approach, and then expressed in HEK293 and COS‐7 cells. The expression levels of the recombinant receptors were six‐fold higher in HEK293 cells than in COS‐7 cells. The residues important for ligand recognition and binding within the N‐terminal segment (G159, Q162, and C165) and the C‐terminal segment (L172, R173, M174, and P179) of IP eLP2 were identified by mutagenesis analyses. The molecular mechanisms for the specific ligand recognition of IP were further demonstrated by specific site‐directed mutagenesis using different amino acid residues with unique chemical properties for the key residues Q162, L172, R173, and M174. A comparison with the corresponding functional residues identified in TP eLP2 revealed that three (Q162, R173, and M174) of the four residues are nonconserved, and these are proposed to be involved in specific ligand recognition. We discuss the importance of G159 and P179 in ligand recognition through configuration of the loop conformation is discussed. These studies have further indicated that characterization of the residues in the eLP2 regions for all eight prostanoid receptors could be an effective approach for uncovering the molecular mechanisms of the ligand selectivities of the G‐protein‐coupled receptors.