Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France.
Functional Genomics Laboratory and Biomolecular Simulations Laboratory, Institut Pasteur de Montevideo, Montevideo, Uruguay.
J Lipid Res. 2021;62:100059. doi: 10.1016/j.jlr.2021.100059. Epub 2021 Feb 26.
Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.
胆固醇是哺乳动物质膜的主要成分，不仅影响脂双层的物理性质，而且还是许多膜蛋白（包括 G 蛋白偶联受体）的功能。催产素受体（OXTR）参与哺乳动物的分娩和泌乳，以及它们的情绪和社会行为。胆固醇作为别构调节剂作用于 OXTR，通过尚未确定的分子机制诱导对变构配体的高亲和力状态。我们使用离子通道偶联受体技术，开发了一种独立于细胞内信号通路且在活细胞中运行的 G 蛋白偶联受体胆固醇调节的功能测定法。使用该测定法，我们发现当受体采用变构配体结合状态时，胆固醇分子与受体稳定结合。这种稳定的相互作用在胆固醇耗尽的膜中保留了受体的胆固醇依赖性活性。通过在 CHO 细胞中表达的 WT OXTR 的时间分辨 FRET 实验证实了这一机制。因此，在 OXTR 中，胆固醇和变构配体之间会发生正向交叉调节。
