NanoLuX: a network-wide Nanoluciferase-based platform to monitor activation, modulation and bias of classical and atypical chemokine receptors
The activity of chemokine receptors is dependent on G proteins that, upon chemokine binding, trigger various intracellular signaling cascades, as well as on β-arrestins that, following receptor phosphorylation by GRK, orchestrate its desensitization, endocytosis and trafficking. In addition to the 19 classical chemokine receptors, 4 receptors form a subfamily of atypical chemokine receptors (ACKR1-4) with ligand-scavenging functions. These receptors are unable to couple to G proteins but their activity can be monitored via β-arrestin recruitment.
Here, we present the NanoLux platform, a network-wide profiling platform for chemokines and chemokines receptors based on the complementation of the Nanoluciferase (NanoBiT). This platform allows to monitor the activation, modulation or bias of receptors or ligands by measuring the binding or the recruitment of effectors, regulators or partners such as G proteins, GRK or β-arrestin isoforms to the classical and atypical chemokine receptors. For that purpose, the LgBiT portion of Nanoluciferase was N-terminally fused to the miniG proteins, GRK2 or human β-arrestin 1 and 2, while the SmBiT was fused to the C terminus of all of the 23 human chemokine receptors. This approach enables to assess and compare the activity of molecules at the chemokine-receptor network level but it can also be applied to investigate the interactions of ligands and intracellular effectors of many other membrane proteins, including GPCRs, receptor tyrosine kinases or ion channels.