A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

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

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Article number102011
JournalIscience
Volume24
Issue number1
DOIs
Publication statusPublished - 22 Jan 2021

Fingerprint Dive into the research topics of 'A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding'. Together they form a unique fingerprint.

Cite this