Department of Biochemistry, Institute of Pharmacy and Biochemistry, Gutenberg-University Mainz, Johann-Joachim Becherweg 30, D-55128 Mainz, Germany.
Biochem J. 2011 Aug 1;437(3):541-53. doi: 10.1042/BJ20101795.
Recent studies suggest that cholesterol binding is widespread among GPCRs (G-protein-coupled receptors). In the present study, we analysed putative cholesterol-induced changes in the OTR [OT (oxytocin) receptor], a prototype of cholesterol-interacting GPCRs. For this purpose, we have created recombinant OTRs that are able to bind two small-sized fluorescence-labelled ligands simultaneously. An OTR antagonist was chosen as one of the ligands. To create a second ligand-binding site, a small-sized α-BTB (bungarotoxin binding) site was inserted at the N-terminus or within the third extracellular loop of the OTR. All receptor constructs were functionally active and bound both ligands with high affinity in the nanomolar range. Measurements of the quenching behaviour, fluorescence anisotropy and energy transfer of both receptor-bound ligands were performed to monitor receptor states at various cholesterol concentrations. The quenching studies suggested no major changes in the molecular environment of the fluorophores in response to cholesterol. The fluorescence anisotropy data indicated that cholesterol affects the dynamics or orientation of the antagonist. The energy transfer efficiency between both ligands clearly increased with increasing cholesterol. Overall, cholesterol induced both a changed orientation and a decreased distance of the receptor-bound ligands, suggesting a more compact receptor state in association with cholesterol.
最近的研究表明胆固醇结合在 G 蛋白偶联受体（GPCR）中广泛存在。在本研究中，我们分析了假定的胆固醇诱导的 OTR [OT（催产素）受体]变化，OTR 是与胆固醇相互作用的 GPCR 的典型代表。为此，我们创建了能够同时结合两种小型荧光标记配体的重组 OTR。我们选择了一种 OTR 拮抗剂作为一种配体。为了创建第二个配体结合位点，在 OTR 的 N 末端或第三细胞外环内插入了一个小型的α-BTB（斑蝥素结合）位点。所有受体构建体均具有功能活性，并且以纳摩尔范围内的高亲和力结合两种配体。测量两种受体结合配体的猝灭行为、荧光各向异性和能量转移，以监测各种胆固醇浓度下的受体状态。猝灭研究表明，胆固醇对荧光团的分子环境没有重大变化。荧光各向异性数据表明，胆固醇影响拮抗剂的动力学或取向。两种配体之间的能量转移效率随胆固醇的增加而明显增加。总体而言，胆固醇诱导受体结合配体的取向和距离发生变化，表明与胆固醇结合时受体状态更加紧凑。
