Objective

Introduction. The chemokine receptor CXCR2 is involved in inflammatory pathologies such as asthma and chronic obstructive pulmonary disease. Small-molecule CXCR2 modulators, such as the diarylurea-based SB265610, block CXCR2 signalling by binding to intracellular sites topographically close to the site of G protein coupling¹. In order to explore the mechanism of action of these modulators further, here we establish live cell NanoBiT luciferase complementation methods² to directly monitor CXCR2 recruitment of mini Galphao³ and βarrestin2 effector proteins.

Methods. HEK293 cells were stably co-transfected with N-terminal SNAP-tagged human CXCR2 cDNA C terminally fused to the LgBit NanoBiT fragment, and either β-Arrestin2 or miniGo proteins tagged with the SmBiT114 peptide. Cells grown to 70 % confluence in 96 well plates were pre-treated with SB265610 at 37°C for 5 minutes prior to furimazine substrate loading. Agonist, antagonist and substrate dilutions were performed in HBSS/0.1% BSA buffer. Luminescence was recorded every 2 min before and after agonist (CXCL8) additions, using a BMG Pherastar2 plate reader. The assays were performed in duplicate and concentration-response curves were fitted from 5 individual experiments using non-linear regression with variable slope. Data were normalised to 100 nM CXCL8 (100%) and the baseline was set as 0%. Pooled pEC₅₀ values are represented as mean ± SEM (n=5).

Results CXCL8 treatment caused the sustained recruitment of β-Arrestin2 and mini Galphao by CXCR2 with similar potencies (pEC₅₀ 9.44 ± 0.07, 9.48 ± 0.12 SEM respectively, n=5, 29 min post-addition). In the presence of SB265610 (100 nM), rightward shifts in the CXCL8 response in both β-Arrestin2 (pEC₅₀ 8.74 ± 0.08, n=5) and mini Galphao (pEC₅₀ 8.69 ± 0.08, n=5) recruitment assays were observed with no significant change in R_max up to 1 µM concentration (one-way ANOVA followed by Dunnet test), and a substantive reduction in R_max in the presence of 10 µM inhibitor.  Furthermore, SB265610 reduced the constitutive recruitment of β-Arrestin2 and Mini Go by CXCR2 in the absence of an agonist.

Conclusion. NanoBiT luciferase complementation provides a real-time reversible readout of CXCR2 engagement with G protein and β-arrestin partners.  Constitutive and agonist-stimulated coupling to both effectors is inhibited by the intracellular modulator SB265610.

References

1. Salchow et al. (2010) A common intracellular binding site for antagonists of the CXCR2 receptor. 159:1429–1439. 2. Dixon et al. (2016) NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. 11:400-408. 3. Wan et al. (2018) Mini G protein probes for active G protein-coupled receptors (GPCRs) in live cells. 293: 7466–7473.