Ligand-specific conformation determines agonist activation and antagonist blockade in purified human thromboxane A2 receptor.
Ligand-specific conformation determines agonist activation and antagonist blockade in purified human thromboxane A2 receptor.
复制标题
配体特异性构象决定了纯化的人血栓素 A2 受体中的激动剂激活和拮抗剂阻断。
DOI：
10.1021/bi801443g
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发表时间：
2009
期刊：
影响因子：
2.9
通讯作者：
Wu,Jiaxin
中科院分区：
文献类型：
--
作者：
Ruan,Ke-He;Cervantes,Vanessa;Wu,Jiaxin
激动剂与G蛋白偶联受体（GPCR）的结合导致其与介导信号传导的不同G蛋白偶联。然而，拮抗剂与GPCR的相同位点的结合不能诱导偶联。为了解其分子机制，采用光谱方法对纯化的人血栓素A2受体（TP）与激动剂或拮抗剂结合时的结构灵活性进行了表征。圆二色性不仅显示纯化的TP在溶液中采用超过50%的螺旋构象，而且还显示拮抗剂SQ 29，548比激动剂U46619能在TP中诱导更多的β-折叠结构。同时，荧光研究表明，拮抗剂诱导的内源性色氨酸荧光信号的变化比激动剂。此外，三个参与不同的配体为基础的结构变化的9个色氨酸残基，证明了NMR光谱。低pH诱导的受体构象和分子相互作用领域的变化显着增加了激动剂结合，但没有显着影响拮抗剂结合。不同的构象变化也观察到在TP重组成磷脂酰胆碱/磷脂酰丝氨酸/磷脂酰乙醇胺形成的脂质体。这些研究首次显示了GPCR中配体特异性构象依赖性激动剂激活和拮抗剂阻断的可能机制。
The binding of an agonist to a G protein-coupled receptor (GPCR) causes its coupling to different G proteins, which mediate signaling. However, the binding of an antagonist to the same site of the GPCR could not induce coupling. To understand the molecular mechanism involved, the structural flexibility of the purified human thromboxane A2receptor (TP) was characterized by spectroscopic approaches, while bound to an agonist or antagonist. Circular dichroism not only revealed that the purified TP adopted more than 50% helical conformation in solution but also showed that the antagonist, SQ29,548, could induce more of a β-sheet structure in the TP than that of the agonist, U46619. Also, fluorescence studies showed that the antagonist induced the intrinsic Trp fluorescence signal change more than the agonist. Furthermore, three of the nine tryptophan residues involved in the different ligand-based structural changes were demonstrated by NMR spectroscopy. Low pH-induced changes in the receptor conformation and molecular interaction field dramatically increased the agonist binding but did not significantly affect the antagonist binding. Different conformational changes were also observed in the TP reconstituted into phosphatidylcholine/phosphatidylserine/phosphatydylethanolamine-formed liposomes. These studies are the first to show a possible mechanism of the ligand-specific conformation-dependent agonist activation and antagonist blockage in the GPCR.