Molecular characterization of a purified 5-HT4 receptor -: A structural basis for drug efficacy
Molecular characterization of a purified 5-HT4 receptor -: A structural basis for drug efficacy
复制标题
DOI：
10.1074/jbc.m412009200
复制
发表时间：
2005-05-27
影响因子：
4.8
通讯作者：
Bockaert, J
中科院分区：
文献类型：
--
作者：
Banères, JL;Mesnier, D;Bockaert, J
血清素 5-HT4(a) 受体是一种 G 蛋白偶联受体 (GPCR)，是使用大肠杆菌作为表达系统生产的功能性分离蛋白。分离的受体在分子水平上通过圆二色性（CD）和稳态荧光进行表征。在激动剂与纯化的受体结合后，观察到与连接第三跨膜结构域和第二细胞外环 (e2) 的 GPCR 二硫键相关的近 UV CD 带的特定变化。这是受体激活后 e2 环构象发生变化的直接实验证据。根据配体是激动剂（部分或全部）还是反向激动剂，获得不同的变化。相反，拮抗剂结合不会引起任何变异。这些观察结果为以下事实提供了第一个直接证据：受体的游离（或拮抗剂占据）、活跃（部分或完全激动剂占据）和沉默（反向激动剂占据）状态涉及e2环的不同排列。最后，配体诱导的纯化受体荧光发射谱的变化证实部分激动剂稳定了单一的、明确的构象状态，而不是不同状态的混合物。这一结果在药理学角度特别令人感兴趣，因为它直接证明药物的功效可能是由于配体特异性状态的稳定而不是受体不同构象状态混合物的选择。
Serotonin 5-HT4(a) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist ( partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free ( or antagonist-occupied), active (partial- or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, well-defined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.