Using CRISPR/Cas9 and NanoLuc to investigate “endogenous” CXCR4 ligand binding, internalization and β‐arrestin2 recruitment

Abstract

Ligand binding, receptor internalisation and receptor‐protein interactions are all vital components to receptor signalling that can be investigated using bioluminescence resonance energy transfer (BRET) or fragment complementation assays. However a fundamental limitation of these techniques is the requirement for exogenous expression of fusion proteins, which precludes the application of these methods to study endogenous proteins. Recently, we have demonstrated that BRET can be used to measure protein‐protein interactions when the luciferase NanoLuc was inserted into the genome by CRISPR/Cas9 engineering. Using NanoBRET and the NanoBiT luciferase complementation here we aimed to investigate ligand binding, receptor internalisation and receptor‐β‐arrestin2 interactions when the CXCR4 and/or interacting partners were expressed under endogenous promotion.CRISPR/Cas9‐mediated homology‐directed repair was used to insert Nluc or NanoBiT fragments into the CXCR4, ARRB2 or CXCL12 genomic loci in HEK293 cells. For NanoBRET ligand binding studies, cells expressing HiBiT/ or Nluc/CXCR4 were incubated for 1hr with CXCL12‐AF647 in the absence or presence of AMD3100 (10 μM). Alternatively cells expressing gene‐edited CXCL12/HiBiT were transfected with SNAP/CXCR4, labelled with cell surface SNAP‐AF488 and incubated with or without AMD3100 (10 pM‐10 μM). For internalisation assays cells were incubated with unlabelled ligands only. For β‐arrestin recruitment assays cells expressing both gene‐edited CXCR4/LgBiT and β‐arr2/SmBiT, or cells expressing one gene‐edited protein transfected with the other, were stimulated with CXCL12 (1 pM – 1 μM) and luminescence measured for 1 hr. Luminescence was generated by addition of furimazine and for cells expressing HiBiT 10 nM LgBiT and was measured on a PHERAstar plate reader (BMG).Binding affinities (Kd) of CXCL12‐AF647 were obtained via NanoBRET saturation binding experiments at Nluc/CXCR4 or HiBiT/CXCR4 expressed under endogenous promotion (nM; 22.74 ± 2.05 and 23.47 ± 4.16, n=4 respectively). Binding of gene edited CXCL12/HiBiT to SNAP/CXCR4 was displaced by AMD3100 (pIC50, 7.09 ± 0.16, n=5). In internalization experiments application of CXCL12 to cells expressing gene‐edited HiBiT/CXCR4 resulted in a concentration dependent decrease in luminescence (pEC50, 8.67 ± 0.27, n=5) suggestive of internalization. In cells expressing gene‐edited CXCR4/LgBiT, β‐arr2/SmBiT or both NanoBiT fragments application of CXCL12 resulted in a concentration dependent increase in luminescence respectively that was inhibited by AMD3100, indicative of ligand induced β‐arrestin2 recruitment to CXCR4Using CRISPR/Cas9 mediated genome engineering, NanoBRET and NanoBiT complementation here we have shown that multiple facets of GPCR activation, desensitisation and signalling can be investigated using proteins expressed under endogenous promotion.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.