SNAP-Tagged Nanobodies Enable Reversible Optical Control of a G Protein-Coupled Receptor via a Remotely Tethered Photoswitchable Ligand,ACS Chemical Biology

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SNAP-Tagged Nanobodies Enable Reversible Optical Control of a G Protein-Coupled Receptor via a Remotely Tethered Photoswitchable Ligand
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-08-24 00:00:00 , DOI: 10.1021/acschembio.8b00628
Helen Farrants _{1,

2} , Vanessa A. Gutzeit ₃ , Amanda Acosta-Ruiz ₃ , Dirk Trauner ₄ , Kai Johnsson _{1,

2} , Joshua Levitz ₃ , Johannes Broichhagen _{1,

2}

Affiliation

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.

中文翻译：

SNAP标记的纳米抗体可通过远程束缚的光开关配体对G蛋白偶联受体进行可逆的光学控制。

G蛋白偶联受体（GPCR）介导将细胞外信号转导为复杂的细胞内反应。尽管它们在生理过程中无处不在，并且作为多种疾病的药物靶标，但GPCR在分子，细胞和系统水平上的确切机制仍被部分了解。为了以高时空精度解剖单个受体亚型的功能，已报道了各种利用光的力量激活和失活受体的光遗传学和光药理学方法。在这里，我们介绍一种新颖的，迄今为止最遥远的方法，即通过使用SNAP标签融合的纳米抗体来应用可光转换的正交远程束缚的配体。我们的纳米抗体-光开关缀合物可通过无基因应用纯化的复合物或共表达遗传编码的纳米抗体来靶向绿色荧光蛋白融合的代谢型谷氨酸受体，以产生对激动剂结合和后续下游激活的强大，可逆的控制。通过包含和结合纳米抗体和自我标记蛋白（或自杀酶）的选择性和灵活性，我们为靶向内源性受体奠定了基础体内。

更新日期：2018-08-24

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