(A) FRET measurements for the D4 and D7 mutants, in the absence of ligand and in the presence of VEGF-A121. The two mutants exhibit much higher FRET efficiencies, compared to the wild-type. The ligand, at 3 μg.ml-1, has no effect on receptor dimerization. (B) VEGF-A121 does not bind to the D4→D5 mutant, when it binds to the wild-type and the D7→D6 mutant. Here, a single vesicle containing YFP-tagged mutant receptors (two top rows) or wild-type receptors (bottom row) is shown after incubation with 3 μg.ml-1 AlexaFluor 594-labeled VEGF-A (VEGF-A121-AF594) (SibTech Inc., CT). Note the differences in the middle column, showing the fluorescence of the bound ligand. (C) Western blots comparing the phosphorylation of the full-length D7→D6 mutant in the absence and presence of VEGF-A121. The top band corresponds to the mature fully glycosylated form of VEGFR-2. The ligand does not increase the phosphorylation.
DOI:
http://dx.doi.org/10.7554/eLife.13876.013