SNAP-Tagged Nanobodies Enable Reversible Optical Control of a G Protein-Coupled Receptor via a Remotely Tethered Photoswitchable Ligand
SNAP-Tagged Nanobodies Enable Reversible Optical Control of a G Protein-Coupled Receptor via a Remotely Tethered Photoswitchable Ligand
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DOI：
10.1021/acschembio.8b00628
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发表时间：
2018-09-01
影响因子：
4
通讯作者：
Broichhagen, Johannes
中科院分区：
文献类型：
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作者：
Farrants, Helen;Gutzeit, Vanessa A.;Broichhagen, Johannes
G 蛋白偶联受体 (GPCR) 介导细胞外信号转导为复杂的细胞内反应。尽管 GPCR 在生理过程中发挥着普遍的作用，并且是多种疾病的药物靶点，但 GPCR 在分子、细胞和系统水平上的精确功能机制仍不清楚。为了以高时空精度剖析个体受体亚型的功能，已经报道了利用光的力量来激活和失活受体的各种光遗传学和光药理学方法。在这里，我们介绍了一种新颖且迄今为止最远程的方法，通过使用 SNAP 标签融合纳米体来应用光可切换正交远程束缚配体。我们的纳米体-光开关缀合物可用于通过纯化复合物的无基因应用或基因编码纳米体的共表达来靶向绿色荧光蛋白融合代谢型谷氨酸受体，以产生对激动剂结合和随后的下游激活的稳健、可逆控制。通过隐藏和结合纳米抗体和自标记蛋白（或自杀酶）的选择性和灵活性，我们为体内靶向内源性受体奠定了基础。
G protein-coupled receptors (GPCRs) mediate the transduction of extracellular signals into complex intracellular responses. Despite their ubiquitous roles in physiological processes and as drug targets for a wide range of disorders, the precise mechanisms of GPCR function at the molecular, cellular, and systems levels remain partially understood. To dissect the function of individual receptor subtypes with high spatiotemporal precision, various optogenetic and photopharmacological approaches have been reported that use the power of light for receptor activation and deactivation. Here, we introduce a novel and, to date, most remote way of applying photoswitchable orthogonally remotely tethered ligands by using a SNAP-tag fused nanobody. Our nanobody-photoswitch conjugates can be used to target a green fluorescent protein-fused metabotropic glutamate receptor by either gene-free application of purified complexes or coexpression of genetically encoded nanobodies to yield robust, reversible control of agonist binding and subsequent downstream activation. By harboring and combining the selectivity and flexibility of both nanobodies and self-labeling proteins (or suicide enzymes), we set the stage for targeting endogenous receptors in vivo.