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A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

White, Carl W.; Kilpatrick, Laura E.; Pfleger, Kevin D.G.; Hill, Stephen J.

A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding Thumbnail


Authors

Carl W. White

Kevin D.G. Pfleger

STEPHEN HILL STEVE.HILL@NOTTINGHAM.AC.UK
Professor of Molecular Pharmacology



Abstract

© 2020 The Author(s) Secreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time.

Journal Article Type Article
Acceptance Date Dec 28, 2020
Online Publication Date Dec 30, 2020
Publication Date 2021-01
Deposit Date Apr 13, 2021
Publicly Available Date Apr 20, 2021
Journal iScience
Electronic ISSN 2589-0042
Publisher Cell Press
Peer Reviewed Peer Reviewed
Volume 24
Issue 1
Article Number 102011
DOI https://doi.org/10.1016/j.isci.2020.102011
Keywords Multidisciplinary
Public URL https://nottingham-repository.worktribe.com/output/5251722
Publisher URL https://www.sciencedirect.com/science/article/pii/S2589004220312086
Additional Information This article is maintained by: Elsevier; Article Title: A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding; Journal Title: iScience; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.isci.2020.102011; Content Type: article; Copyright: © 2020 The Author(s).

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