A Nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

Abstract

Secreted chemokines are critical mediators of cellular communication that elicit intracellular signalling 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 analysed 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.

CXCL17 is an endogenous inhibitor of CXCR4 via a novel mechanism of action

“…In our initial experiments we also noted that CXCL17 inhibits basal/constitutive CXCR4 signalling, suggesting inverse agonist activity. However, CXCL12 is endogenously expressed in HEK293 cells at levels sufficient to initiate signalling 29 . To investigate if CXCL17 was disrupting signalling mediated by endogenous CXCL12, we took advantage of HEK293 cells engineered using CRISPR/Cas9 genomeediting to express CXCL12 tagged with a small 11 amino acid self-complementing fragment of NLuc (HiBiT).…”

“…Nluc was then sub-cloned into the vector from existing constructs using the restriction enzymes XhoI and ApaI. Generation of pcDNA3.1 (+) neo expression constructs encoding NLuc/CXCR4, HiBiT/CXCR4 49 , SNAP/CXCR4 29 , NLuc/VEGFR2 36 and NLuc/NRP 35 have been described previously, as have pcDNA3 expression constructs encoding CXCR4/Rluc8 50 , CXCR4/N luc 51 , β-arrestin2/Venus 52 . Venus/G γ2 was a kind gift from Dr. Martina Kocan.…”

“…Stable transfections were performed using FuGENE (Promega, USA) according to the manufacturer’s instructions. Generation of HEK293 cell lines stably-expressing Nluc/CXCR4, HiBiT/CXCR4 or SNAP/CXCR4, or generation of HEK293 or HeLa cells that were genome-edited to express NLuc/CXCR4 or CXCL12-HiBiT under endogenous promotion, have been described previously 29,49 . To generate cells for use in the GloSensor cAMP assay, HEK293G cells stably-expressing the GloSensor cAMP biosensor were transfected with a pcDNA3.1 (+) neo expression vector encoding SNAP/CXCR4 and subsequently selected for incorporation of the transgene using G418 (ThermoFisher).…”

“…Expression cDNA constructs encoding untagged chemokine receptors, β2-adrenoceptor or GPR35 were purchased from the cDNA Resource Center. To generate the pcDNA3.1(+) mammalian expression vector encoding for GPR35 tagged on the C-terminus with NLuc, the stop codon was first removed by NLuc/CXCR4 or CXCL12-HiBiT under endogenous promotion, have been described previously 29,49 .…”

Receptor levels determine binding affinity of WNT-3A to Frizzled 7 in a colorectal cancer model

“…This technology has been employed with success in a number of studies designed to understand the biology of GPCRs [25] as well as a study on the co-receptor for Wnt/β-catenin signalling, LRP6 [26]. Among these, several studies have reported on NanoBRET ligand binding to endogenous class A GPCRs, mostly in HEK293 cells [27][28][29][30][31][32]. Here, as a continuation of our studies on ligand binding in the class F GPCRs [13,14,33], we measured the binding of the only available functional fluorescent WNT -eGFP-tagged mouse WNT-3A [14,34] -to endogenous FZD7 in SW480 cells typifying colorectal cancer, aiming to apply a more relevant cell model.…”