Journal
CELLULAR SIGNALLING
Volume 54, Issue -, Pages 27-34Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.cellsig.2018.11.018
Keywords
CRISPR/Cas9; Fluorescent ligands; G protein-coupled receptor; Methods; NanoBRET; Ligand binding; Nluc
Categories
Funding
- UWA
- ARC Linkage Grant [LP160100857]
- National Health and Medical Research Council (NHMRC) of Australia [1088334, 1085842]
- National Health and Medical Research Council of Australia [1085842, 1088334] Funding Source: NHMRC
- Australian Research Council [LP160100857] Funding Source: Australian Research Council
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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 An receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A(2B) receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A(2B). However, in cells expressing gene-edited Nluc/An receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (K-d = 21.4 nM). Additionally, at gene-edited Nluc/A(2B) receptors we derived pharmacological parameters of ligand binding; K-d as well as K-on and K-off for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A(2B) were used to determine the pK(i) 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.
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