Journal: eLife

Article Title: VEGFR-2 conformational switch in response to ligand binding

doi: 10.7554/eLife.13876

Figure Lengend Snippet: ( B ) CHO cells do not express VEGF endogenously. Here, CHO cells, HEK293T cells, and MECs (microvascular endothelial) cells) were stained for VEGF-A. Lysates were reduced before loading. DOI: http://dx.doi.org/10.7554/eLife.13876.005

Article Snippet: 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-A 121 -AF594) (SibTech Inc., CT).

Techniques: Staining

Journal: eLife

Article Title: VEGFR-2 conformational switch in response to ligand binding

doi: 10.7554/eLife.13876

Figure Lengend Snippet: ( A ) FRET efficiency measured as a function of acceptor concentration for EC+TM VEGFR-2, in the absence of ligand and in the presence of 3 μg.ml -1 VEGF-A 121 and VEGF-A 165 a. Two hundred to 500 individual vesicles were imaged in at least three independent experiments. Each data point corresponds to a single vesicle. The stochastic FRET contribution is shown as a solid line. Black stars: FRET data without ligand. ( B ) FRET efficiencies for individual vesicles, corrected for the stochastic FRET contribution. There is no dependence on receptor concentration, indicative of constitutive dimerization. ( C ) Donor concentrations versus acceptor concentrations. ( D ) Intrinsic FRET values, measured for VEGFR-2 EC+TM in the presence of 3 μg.ml -1 of VEGF-A 121 , VEGF-A 165 a, VEGF-A 165 b, VEGF-C, VEGF-E, and VEGF-D. ( E ) Graphical representation of the ligand-induced changes in distance between fluorescent proteins, and the inferred changes in TM domain structures. ( F ) The six VEGF ligands increase VEGFR-2 phosphorylation, to the same extent. A representative Western Blot comparing the phosphorylation of Tyr 1175 in the absence of ligand, and in the presence of six VEGF ligands, at concentrations of 3 μg.ml -1 . Only the top bands, corresponding to mature fully glycosylated receptors, are considered here. Phosphorylation is significantly increased upon ligand addition, with the increase being as high as 10 times. ( G ) Quantification of Western blot results for the fully glycosylated receptors (top bands), in the presence of the six ligands. VEGFR-2 phosphorylation in the presence of the six ligands is very similar. DOI: http://dx.doi.org/10.7554/eLife.13876.009

Article Snippet: 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-A 121 -AF594) (SibTech Inc., CT).

Techniques: Concentration Assay, Western Blot

Journal: eLife

Article Title: VEGFR-2 conformational switch in response to ligand binding

doi: 10.7554/eLife.13876

Figure Lengend Snippet: ( A ) FRET measurements for the D4 and D7 mutants, in the absence of ligand and in the presence of VEGF-A 121 . 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-A 121 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-A 121 -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-A 121 . 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

Article Snippet: 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-A 121 -AF594) (SibTech Inc., CT).

Techniques: Mutagenesis, Incubation, Labeling, Fluorescence, Western Blot

Journal: eLife

Article Title: VEGFR-2 conformational switch in response to ligand binding

doi: 10.7554/eLife.13876

Figure Lengend Snippet: ( 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-A 121 -AF594 binding to EC+TM VEGFR-2 C482R in CHO membrane vesicles, demonstrating that the C482R mutant is capable of ligand binding. DOI: http://dx.doi.org/10.7554/eLife.13876.015

Article Snippet: 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-A 121 -AF594) (SibTech Inc., CT).

Techniques: Derivative Assay, Concentration Assay, Mutagenesis, Western Blot, Binding Assay, Ligand Binding Assay