Screening of ligand binding sites of a G-protein coupled receptors by using photocrosslinking

大多数药物都以G蛋白偶联受体(GPCRs)为靶点，GPCRs通常可以激活不同的信号事件。最近发展起来的促肾上腺皮质激素释放因子(CRF)受体的多肽配体既是激动剂，又是不同G蛋白激活的拮抗剂。这一发现表明，当与不同的G蛋白偶联时，受体的活性构象和可能不同的配体结合位置之间存在显著差异。基于结构的信号选择性配体的开发可以产生一种新型的候选药物，但由于缺乏高分辨率的GPCRs结构而受到阻碍。我们将使用定点掺入方法将可光激活的氨基酸(BPA)掺入蛋白质中，以制备一组在与配体结合相关的不同位置上携带BPA的CRF受体突变体。这将允许比较配体根据其不同的结合位置对激活途径显示不同的选择性。在此过程中，我们将了解BPA标记受体是否能证实以前用BPA标记配体获得的结果，并验证CRF受体的推测配体结合模型，该模型被认为是B类GPCRs的常见模型。

Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs), which can generally activate different signaling events. Recently developed polypeptide ligands of the corticotropinreleasing factor (CRF) receptor behaved as agonists and, at the same time, antagonists for the activation of different G-proteins. This finding implies significant differences between active conformations of the receptor and probably different ligand binding sites when coupled to different Gproteins. The structure-based development of signaling-selective ligands can result in a new type of drug candidates, but is hampered by a lack of high-resolution structures of GPCRs. We will use methods for site-directed incorporation of a photo-activatable amino acid (Bpa) into proteins to prepare a set of CRF receptor mutants bearing Bpa at various positions relevant for ligand binding. This will allow comparing ligands showing different selectivity for activation pathways with respect to their distinct binding sites. In course of this, we will find out whether Bpa-labeling of the receptor can confirm former results obtained with Bpa-labeled ligands, and we will verify the speculative ligand binding model for the CRF receptor, which is considered as common model for class B GPCRs.