Capturing a rhodopsin receptor signalling cascade across a native membrane.
Capturing a rhodopsin receptor signalling cascade across a native membrane.
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DOI：
10.1038/s41586-022-04547-x
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发表时间：
2022-04
期刊：
影响因子：
64.8
通讯作者：
中科院分区：
文献类型：
--
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G蛋白偶联受体（GPCR）是细胞表面受体，其响应于各种刺激以诱导跨细胞膜的信号传导途径。最近的进展已经产生了关键中间体的原子结构和脂质在信号传导中的作用。然而，在真实的时间内捕获野生型受体的信号事件，穿过天然膜到达其下游效应物，仍然是难以捉摸的。在这里，我们探测原型A类GPCR，视紫红质，直接从片段的原生光盘膜使用质谱。我们监测实时光转换暗适应视紫红质视蛋白，描绘视网膜异构化和水解步骤，并进一步表明，该反应是显着较慢，在其原生膜比在洗涤剂胶束。考虑到与视紫红质喷出的脂质，我们表明，视蛋白可以再生膜通过光异构化的视黄酸-脂质共轭物，我们提供的证据增加视紫红质与不饱和长链磷脂酰胆碱在信号。捕获信号级联反应的二级步骤，我们通过GDP的损失监测转导素（Gt）的光活化，以产生中间脱辅基三聚体G蛋白，并观察Gαt·GTP亚基与PDE 6相互作用以水解环GMP。我们还展示了视紫红质靶向化合物如何通过视紫红质-视蛋白和转导蛋白信号通路刺激或抑制信号传导。我们的研究结果不仅揭示了天然脂质对视紫红质信号传导和再生的影响，而且使我们能够提出一个在天然膜环境中发现GPCR药物的范例。由光引发的视紫红质信号级联反应使用天然膜的质谱法捕获。
G protein-coupled receptors (GPCRs) are cell-surface receptors that respond to various stimuli to induce signalling pathways across cell membranes. Recent progress has yielded atomic structures of key intermediates and roles for lipids in signalling. However, capturing signalling events of a wild-type receptor in real time, across a native membrane to its downstream effectors, has remained elusive. Here we probe the archetypal class A GPCR, rhodopsin, directly from fragments of native disc membranes using mass spectrometry. We monitor real-time photoconversion of dark-adapted rhodopsin to opsin, delineating retinal isomerization and hydrolysis steps, and further showing that the reaction is significantly slower in its native membrane than in detergent micelles. Considering the lipids ejected with rhodopsin, we demonstrate that opsin can be regenerated in membranes through photoisomerized retinal–lipid conjugates, and we provide evidence for increased association of rhodopsin with unsaturated long-chain phosphatidylcholine during signalling. Capturing the secondary steps of the signalling cascade, we monitor light activation of transducin (Gt) through loss of GDP to generate an intermediate apo-trimeric G protein, and observe Gαt•GTP subunits interacting with PDE6 to hydrolyse cyclic GMP. We also show how rhodopsin-targeting compounds either stimulate or dampen signalling through rhodopsin–opsin and transducin signalling pathways. Our results not only reveal the effect of native lipids on rhodopsin signalling and regeneration but also enable us to propose a paradigm for GPCR drug discovery in native membrane environments. The rhodopsin signalling cascade, initiated by light, is captured using mass spectrometry of a native membrane.
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DOI：
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