A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding
Carl W. White,
Laura E. Kilpatrick,
Kevin D.G. Pfleger,
Stephen J. Hill
Affiliations
Carl W. White
Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK; Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, QEII Medical Centre, Nedlands, WA 6009, Australia; Australian Research Council Centre for Personalised Therapeutics Technologies, Australia; Corresponding author
Laura E. Kilpatrick
Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK; School of Pharmacy, Biodiscovery Institute, University of Nottingham, Nottingham, NG7 2RD, UK
Kevin D.G. Pfleger
Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, QEII Medical Centre, Nedlands, WA 6009, Australia; Australian Research Council Centre for Personalised Therapeutics Technologies, Australia; Dimerix Limited, Nedlands, WA 6009, Australia
Stephen J. Hill
Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK; Corresponding author
Summary: 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.
