NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing

Carl W. White, Elizabeth K. M. Johnstone, Heng B. See, Kevin D. G. Pfleger

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)27-34
Number of pages8
JournalCellular Signalling
Volume54
DOIs
Publication statusPublished - Feb 2019

Cite this

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title = "NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing",
abstract = "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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author = "White, {Carl W.} and Johnstone, {Elizabeth K. M.} and See, {Heng B.} and Pfleger, {Kevin D. G.}",
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pages = "27--34",
journal = "Cellular Signalling",
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NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing. / White, Carl W.; Johnstone, Elizabeth K. M.; See, Heng B.; Pfleger, Kevin D. G.

In: Cellular Signalling, Vol. 54, 02.2019, p. 27-34.

Research output: Contribution to journalArticle

TY - JOUR

T1 - NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing

AU - White, Carl W.

AU - Johnstone, Elizabeth K. M.

AU - See, Heng B.

AU - Pfleger, Kevin D. G.

PY - 2019/2

Y1 - 2019/2

N2 - 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.

AB - 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.

KW - CRISPR/Cas9

KW - Fluorescent ligands

KW - G protein-coupled receptor

KW - Methods

KW - NanoBRET

KW - Ligand binding

KW - Nluc

KW - LIVING CELLS

KW - RECEPTOR ANTAGONISTS

KW - PROTEIN INTERACTIONS

KW - BETA-ARRESTIN

KW - POTENT

KW - BRET

KW - ACTIVATION

KW - A(2A)

U2 - 10.1016/j.cellsig.2018.11.018

DO - 10.1016/j.cellsig.2018.11.018

M3 - Article

VL - 54

SP - 27

EP - 34

JO - Cellular Signalling

JF - Cellular Signalling

SN - 0898-6568

ER -