Fig. 5
From: Agonist-induced dimer dissociation as a macromolecular step in G protein-coupled receptor signaling
Agonist-dependent dynamics of FZD6 dimerization. a dcFRAP in HEK293 cells expressing V5-FZD6-mCherry and FZD6-GFP allowed kinetic monitoring of receptor–receptor interactions in the absence of agonist and after 5, 10, 15, 20 min of WNT-5A (300 ng ml−1) stimulation. P = 0.0219; F (5, 109) = 2.757. Before CL (20 ROIs); after CL (unstimulated = 38 ROIs, 5 min = 12 ROIs, 10 min = 18 ROIs, 15 min = 12 ROIs, 20 min = 15 ROIs). N = 10. Unstimulated (before and after CL) are from Fig. 1d and were performed in parallel with stimulation experiments. b Curves depict the recovery of GFP after CL comparing unstimulated (red), 5 min (green) and 20 min (magenta) after WNT-5A stimulation. c Dynamic FCCS experiments in HEK293 cells expressing V5-FZD6-mCherry and FZD6-GFP from 6 different cells are summarized by normalizing the agonist-induced minimum fraction of dimers from individual cells to 0 min (N gr/N r represents ratio of FZD6-GFP/V5-FZD6-mCherry dimers (green/red, gr) over the total number of red molecules (red, r), representing the fraction of dimers present in the detection volume). Box plot presents data from the 25 to 75th percentile and whiskers provide min/max. P = 0.0093; F (7, 39) = 3.178. **P < 0.01 (one-way ANOVA, Fisher’s LSD post hoc analysis). Total number of N r (red) and N g (green) particles in the detection volume over time. Values of N r or N g at the first time point were set to 100. d FCCS curves present cross-correlation of V5-FZD6-mCherry and FZD6-GFP from a representative cell prior to WNT-5A stimulation (red), at maximal dimer dissociation (green) and upon dimer re-association (magenta). When normalized to the green auto-correlation amplitude, the amplitudes of the three G cc curves revealed that 11, 6, and 21% of red molecules were also green, respectively. e Representative example of N gr/N r after WNT-5A stimulation for a cell expressing wt FZD6-GFP and wt FZD6-mCherry. t = 0 refers to first measurement after the addition of WNT-5A. f HEK293 cells expressing SNAP-FZD6 or SNAP-β2-AR were pulsed with a cell-impermeable SNAP substrate to monitor internalization after WNT-5A (300 ng ml−1, 15 min) or isoproterenol (10 µM). Arrows indicate internalized receptors. Scale bar = 5 µm. g Model of FZD6 de- and re-dimerization upon WNT-5A stimulation. Error bar provides s.e.m
