Spatial approximation between secretin residue five and the third extracellular loop of its receptor provides new insight into the molecular basis of natural agonist binding
Spatial approximation between secretin residue five and the third extracellular loop of its receptor provides new insight into the molecular basis of natural agonist binding
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DOI：
10.1124/mol.108.047209
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发表时间：
2008-08-01
影响因子：
3.6
通讯作者：
Miller, Laurence J.
中科院分区：
文献类型：
--
作者：
Dong, Maoqing;Lam, Polo C. -H.;Miller, Laurence J.
II类G蛋白偶联受体的氨基末端在配体结合和受体活化中起重要作用。随着促肾上腺皮质激素释放因子、垂体腺苷酸环化酶激活多肽和胃抑制多肽受体这一区域的核磁共振和晶体结构的解决方案，对这些受体的氨基末端结构域构象的理解已经大大推进。然而，氨基末端相对于受体核心的取向以及受体如何在配体结合后被激活仍不清楚。在这项工作中，我们已经使用光亲和标记，以确定一个关键的空间近似的残基5的分泌素和建议的第三胞外环的分泌素受体内的一个残基。这是通过纯化，去糖基化，溴化氰切割，和标记的野生型和突变体促胰液素受体的测序。这种限制已被用来完善我们不断发展的分泌素停靠在完整的受体，这是第一次包括完善的螺旋束和环区，并反映了受体氨基末端内的肽结合槽，指导氨基末端的肽对受体体的分子模型。该模型与II类G蛋白偶联受体激活的内源性激动剂机制完全一致，其中配体结合促进受体氨基末端的一部分与受体体的相互作用以激活它。
The amino terminus of class II G protein-coupled receptors plays an important role in ligand binding and receptor activation. Understanding of the conformation of the amino-terminal domain of these receptors has been substantially advanced with the solution of nuclear magnetic resonance and crystal structures of this region of receptors for corticotrophin-releasing factor, pituitary adenylate cyclase-activating polypeptide, and gastric inhibitory polypeptide. However, the orientation of the amino terminus relative to the receptor core and how the receptor gets activated upon ligand binding remain unclear. In this work, we have used photoaffinity labeling to identify a critical spatial approximation between residue five of secretin and a residue within the proposed third extracellular loop of the secretin receptor. This was achieved by purification, deglycosylation, cyanogen bromide cleavage, and sequencing of labeled wild-type and mutant secretin receptors. This constraint has been used to refine our evolving molecular model of secretin docked at the intact receptor, which for the first time includes refined helical bundle and loop regions and reflects a peptide-binding groove within the receptor amino terminus that directs the amino terminus of the peptide toward the receptor body. This model is fully consistent with the endogenous agonist mechanism for class II G protein-coupled receptor activation, where ligand binding promotes the interaction of a portion of the receptor amino terminus with the receptor body to activate it.