Fig. 4.

Role of β-arrestin-2 for HCA₃ and GPR84 signaling and effect of methyl-β-cyclodextrin (MβCD). a, b CHO-K1 cells were transiently transfected with HCA₃ or GPR84. a MβCD inhibited both, the 3HO- and 3HDec-induced reduction of forskolin (fsk)-induced cAMP levels in HCA₃-transfected cells. For GPR84, only the C10-induced but not the 3HDec-induced decrease in cAMP was inhibited. Barbardin (100 µM) inhibited only the 3HO-induced HCA₃-mediated reduction of cAMP levels. cAMP levels of HCA₃- or GPR84-transfected cells in absence of agonist are set 100%, respectively. b 3 mM MβCD did not affect HCA₃-mediated activation of ERK by 3HO, but caused a decrease of the signal induced by 100 μM 3HDec. Presence of MβCD caused a decrease in C10-induced GPR84-mediated ERK activation and had no effect on the 3HDec-induced ERK activation. The HCA₃-mediated activation of ERK by 3HO, but not 3HDec, was inhibited in presence of barbardin. Barbardin had no effect on the GPR84-mediated activation of ERK by 3HDec and C10. pERK/total ERK of HCA₃- or GPR84-transfected cells in absence of agonist is set 1, respectively. c Live-cell images of HEK293-T cells co-expressing HCA₃-mRuby (red) and β-arrestin-2-YFP (green) were acquired before stimulation and 30 min post-stimulation with 100 μM 3HO or 100 μM 3HDec. d HEK293-T cells stably expressing β-arrestin-2-EA cells transiently transfected with HCA₃ were stimulated with 3HO and 3HDec. Quantification of β-arrestin-2 recruitment using the PathHunter β-arrestin assay (Eurofins DiscoverX) showed recruitment of β-arrestin-2 by HCA₃ following 3HO but not 3HDec stimulation. Luminescence of HCA₃ or empty vector transfected cells in absence of agonist is set 1, respectively. a, b, d Data is given as mean ± SEM of at least three independent experiments each carried out in triplicates. ^#P ≤ 0.1* P ≤ 0.05; ** P ≤ 0.01