Figure 6. The C482R mutation mimics the effect of bound ligand by promoting a structural change in the EC+TM VEGFR-2 dimer.
(A) FRET efficiencies determined in individual plasma-membrane-derived vesicles. (B) Corrected FRET as a function of receptor concentration. The corrected FRET for the mutant does not depend on receptor concentration, demonstrating that the mutant is a constitutive dimer in the presence and absence of ligand. (C) Measured donor versus acceptor concentrations in each vesicle. (D) Histograms of measured FRET efficiencies for the mutant, yielding Intrinsic FRET values of ~0.42. (E) Western blots showing an increase in phosphorylation due to the C482R mutation, in the absence of ligand, and no further increase upon ligand treatment. The top bands correspond to the mature fully glycosylated form of VEGFR-2. (F) Confocal images of VEGF-A121-AF594 binding to EC+TM VEGFR-2 C482R in CHO membrane vesicles, demonstrating that the C482R mutant is capable of ligand binding.