Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710; Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Department of Chemistry, University of Toronto, UTM, Mississauga, Ontario L5L 1C6, Canada.
J Biol Chem. 2019 Sep 6;294(36):13218-13223. doi: 10.1074/jbc.AC119.009848. Epub 2019 Jul 30.
A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the β-adrenergic receptor (βAR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted βAR, the βAR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted βAR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of βAR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that βAR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress βAR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.
G 蛋白偶联受体 (GPCR) 的一个标志是将外部刺激转化为特定的细胞反应。在这个严格调控的过程中，GPCR 与细胞外配体的结合促进了跨膜七螺旋内的特定构象变化，导致细胞内“转导”蛋白（如异三聚体 G 蛋白）的偶联和激活。我们对调控 GPCR 激活的分子机制的大部分理解来自于用重组在去污剂胶束中的纯化受体进行的实验。为了阐明磷脂双层对 GPCR 激活的影响，我们在这里研究了 G 蛋白偶联受体（βAR）在高密度脂蛋白 (HDL) 颗粒中的功能、药理学和生物物理特性。与去污剂重组的βAR 相比，HDL 颗粒中的βAR 具有更高水平的基础（组成型）活性，并且对激动剂激活的敏感性增加，这可以通过异三聚体 G 蛋白的激活和配体结合口袋与转导结合口袋之间的变构偶联来评估。使用 F NMR 光谱学，我们将这些在去污剂和 HDL 中重组的βAR 之间的功能差异直接与无活性和活性受体状态之间平衡的变化联系起来。与细胞中βAR 组成型活性水平低形成对比的是，在分离的磷脂双层中观察到的高组成型活性表明βAR 基础活性取决于重组系统，进一步表明各种细胞机制在生理上抑制βAR 基础活性。我们的研究结果为 GPCR 激活提供了重要的补充见解，并揭示了重组系统对膜蛋白功能的影响有多么显著。
