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NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing
Cellular Signalling ( IF 4.8 ) Pub Date : 2018-11-22 , DOI: 10.1016/j.cellsig.2018.11.018
Carl W. White , Elizabeth K.M. Johnstone , Heng B. See , Kevin D.G. Pfleger

Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A2B receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A2B receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A2B. However, in cells expressing gene-edited Nluc/A2B receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (Kd = 21.4 nM). Additionally, at gene-edited Nluc/A2B receptors we derived pharmacological parameters of ligand binding; Kd as well as Kon and Koff for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A2B were used to determine the pKi of unlabelled adenosine receptor ligands in competition ligand binding assays. Utilising CRISPR/Cas9 genome engineering here we show that NanoBRET ligand binding assays can be performed at gene-edited receptors under endogenous promotion in live cells, therefore overcoming a fundamental limitation of NanoBRET ligand assays.



中文翻译:

CRISPR / Cas9基因组编辑促进内源性促进下GPCR上的NanoBRET配体结合

生物发光共振能量转移(BRET)是用于研究膜受体信号传导和功能的多功能工具。我们最近开发了一种均匀的NanoBRET配体结合测定法,以监测G蛋白偶联受体与荧光配体之间的相互作用。然而,该测定法需要与纳米荧光素酶(Nluc)融合的受体的外源表达,因此不适用于天然表达的受体。为了克服HEK293细胞中的这一局限性,我们利用CRISPR / Cas9基因组工程技术将Nluc与内源性ADORA2B基因座插入框内,从而导致HEK293细胞表达腺苷A 2B内源性促进作用下的受体在其N末端标记有Nluc。如预期的那样,与用编码Nluc / A 2B的表达载体瞬时转染的细胞相比,我们发现内源性(基因编辑的)Nluc / A 2B受体表达水平相对较低。但是,在表达基因编辑的Nluc / A 2B受体的细胞中,我们观察到了非特异性荧光腺苷受体拮抗剂XAC-X-BY630(K d  = 21.4 nM)的清楚的饱和配体结合。另外,在基因编辑的Nluc / A 2B受体上,我们推导出了配体结合的药理学参数。K d以及K onK off通过NanoBRET缔合动力学结合测定法检测XAC-X-BY630的结合。最后,在竞争配体结合测定中,使用表达基因编辑的Nluc / A 2B的细胞确定未标记的腺苷受体配体的p K i。在这里利用CRISPR / Cas9基因组工程,我们证明了NanoBRET配体结合测定法可以在活细胞内源性促进下在基因编辑受体上进行,因此克服了NanoBRET配体测定法的基本局限性。

更新日期:2018-11-22
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