Figure 7
From: I8-arachnotocin–an arthropod-derived G protein-biased ligand of the human vasopressin V2 receptor
Characterization of β-arrestin-1 recruitment and ERK 1/2 phosphorylation induced by I8-arachnotocin vs. VP at the human V2R. (a) Kinetic profile of VP- and I8-arachnotocin-mediated β-arrestin-1 recruitment in HEK293 cells co-expressing EGFP-V2R and β-arrestin-1-Nluc. Cells were stimulated by 10 µM of VP or I8-arachnotocin, respectively, 5 min after addition of the luciferase substrate (furimazine). The results are shown as differences in the BRET signals in the presence of ligands and are expressed as the mean value ± SD; n = 2. (b) Concentration-response curves of VP and I8-arachnotocin at V2R using HEK 293 cells co-expressing EGFP-tagged V2R and β-arrestin-1-Nluc. Cells were pretreated with furimazine and measurements were taken 5 min after addition of ligands. Ligand-induced BRET was calculated as: (emission EGFPligand/emission NLucligand) − (emission EGFPHBSS/emission NLucHBSS). Results were normalized to β-arrestin-1 recruitment in response to VP. Data points were fitted by nonlinear regression curves (sigmoidal, slope = 1); error bars indicate SEM; n = 3. (c) Representative Western blot images of ERK 1/2 phosphorylation by stimulation of V2R with I8-arachnotocin vs. VP and (d) quantification of I8-arachnotocin- and VP-induced phosphorylated ERK 1/2 (pERK) relative to total ERK 1/2 (tERK) from four independent experiments (±SEM). Cells were transiently transfected with EGFP-V2R encoding plasmid and treated with 1 µM I8-arachnotocin or 1 µM VP at 37 °C for indicated time periods. Immunoblots were prepared from the same membranes using the same exposure method. Regions of interest were cropped from the full image (see Supplementary Information). Statistical significance was determined by Student’s t test (*P < 0.05; **P < 0.01; ns non-significant).
