VEGFR-2 conformational switch in response to ligand binding
- Johns Hopkins University, United States
- Paul Scherrer Institute, Switzerland
Figures
Figure 1
The plasmid constructs used in this study.
(A) The constructs used in the FRET experiments. The full-length receptors had fluorescent proteins attached to their C-termini via a flexible GGS linker. The truncated receptors had the …
Figure 2 with 3 supplements
FRET measurements of VEGFR-2 dimerization in CHO plasma membranes.
(A) FRET efficiencies as a function of acceptor concentration for full length VEGFR-2 (solid red diamonds), EC+TM VEGFR-2 (solid black diamonds), and TM VEGFR-2 (open olive circles). Two hundred to …
Figure 2—figure supplement 1
(A) CHO cells do not express VEGFR-2 endogenously.
(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.
Figure 2—figure supplement 2
A single vesicle imaged and analyzed in the FRET, acceptor, and donor channels.
Images were acquired with a Nikon laser scanning confocal microscope. The intensity across the membrane (open blue symbols) is fit to a Gaussian (solid line) after background correction. Shown also …
Figure 2—figure supplement 3
Cross-linking of CHO cells expressing full-length wild-type VEGFR-2.
Cells were starved for 24 hr to ensure that there was no ligand present. Staining with anti-VEGFR-2 antibodies shows the presence of a glycosylated monomer band at MW ~240 kDa and glycosylated dimer …
Figure 3
A conformational change in the TM domain dimer upon ligand binding increases VEGFR-2 phosphorylation.
(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-A121 and VEGF-A165a. Two hundred to 500 …
Figure 4 with 2 supplements
The published NMR structure of the isolated VEGFR-2 TM domain (Manni et al., 2014b) is consistent with the unliganded TM dimer structure observed in the FRET experiments.
(A) Amino acid sequence of wild-type VEGFR-2 TM domain. The amino acids that mediate helix-helix contacts in the NMR dimer structure (Manni et al., 2014b) are shown in red. (B) The NMR structure, …
Figure 4—figure supplement 1
FRET data, reporting on the effects of the E764I-T771I-F778I and N762I-V769I-G770I sets of mutations, engineered in the EC+TM VEGFR-2 plasmids.
https://doi.org/10.7554/eLife.13876.011
Figure 4—figure supplement 2
All mutations in the TM domain decrease VEGFR-2 phosphorylation.
https://doi.org/10.7554/eLife.13876.012
Figure 5 with 1 supplement
The D4 and D7 mutants do not respond to ligand.
(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 …
Figure 5—figure supplement 1
Dimerization curves for the wild-type VEGFR-2 in the absence of ligand, the D4→D5 mutant in the absence of ligand, and the D4→D5 mutant in the presence of VEGF-A121.
https://doi.org/10.7554/eLife.13876.014
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 …
Figure 7 with 1 supplement
The N762I-V769I-G770I set of mutations in the TM domain of the C482R mutant receptor alters dimer structure and activity, as in the case of the ligand-bound wild-type (see Figure 4).
(A) Corrected FRET efficiencies for the EC+TM C482R-N762I-V769I-G770I mutant as a function of receptor concentration, indicative of constitutive dimers. (B) Histograms of Intrinsic FRET measured for …
Figure 7—figure supplement 1
Left: Western blots under reducing conditions, for full length C482R VEGFR-2 and for the C482R EC+TM-YFP VEGFR-2 construct.
Right: Western blots under non-reducing conditions. No dimeric bands were observed under any conditions. The constitutive dimerization of the C482R mutant is not due to cysteine-induced …
Figure 8
Proposed model for VEGFR-2 activation.
VEGFR-2 pre-dimerizes in the absence of ligand. Under physiological conditions, corresponding to 10 to 100 VEGFR-2 molcules per square micron, 30 to 60% of the receptors are dimeric. The dimers are …
Tables
Table 1
Dimerization free energies, ΔG, and Intrinsic FRET efficiencies , obtained from least-square parameter fits to the single-vesicle FRET data for the different VEGFR-2 constructs studied here. d is …
| VEGFR-2 variant | Dissociation constant, Kdiss (rec.μm-2) | △G (kcal.mol-1) | I-FRET | d (Å) |
|---|---|---|---|---|
| full length | 35 (16 to 53) | -6.1 (-5.8 to -6.5) | 0.82 (0.78 to 0.85) | 41.3 (39.8 to 43) |
| EC+TM | 3200 (2712 to 3750) | -3.4 (-3.3 to -3.5) | 0.61 (0.55 to 0.68) | 49.3 (46.8 to 51.4) |
| TM | 310 (220 to 415) | -4.8 (-4.6 to -5.0) | 0.61 (0.59 to 0.65) | 49.3 (47.9 to 50.0) |
| EC+TM +VEGF-A121 | 100% dimer | 100% dimer | 0.45 ± 0.02 | 55 ± 1 |
| EC+TM +VEGF-A165a | 100% dimer | 100% dimer | 0.43 ± 0.02 | 56 ± 1 |
| EC+TM +VEGF-A165b | 100% dimer | 100% dimer | 0.42 ± 0.02 | 56 ± 1 |
| EC+TM +VEGF-C | 100% dimer | 100% dimer | 0.45 ± 0.02 | 55 ± 1 |
| EC+TM +VEGF-D | 100% dimer | 100% dimer | 0.43 ± 0.02 | 56 ± 1 |
| EC+TM +VEGF-E | 100% dimer | 100% dimer | 0.44 ± 0.02 | 55 ± 1 |
| EC+TM(E764I-T771I-F778I) | 100% dimer | 100% dimer | 0.31 ± 0.02 | 61 ± 1 |
| EC+TM(E764I-T771I-F778I) + VEGF-A121 | 100% dimer | 100% dimer | 0.38 ± 0.02 | 58 ± 1 |
| EC+TM(N762I-V769I-G770I) | 3100 (2090 to 3600) | -3.4 (-3.3 to -3.7) | 0.61 (0.58 to 0.65) | 49.3 (47.9 to 50.3) |
| EC+TM(N762I-V769I-G770I) + VEGF-A121 | 100% dimer | 100% dimer | 0.24 ± 0.02 | 64 ± 1 |
| EC(D4→D5) +TM | 390 (271 to 480) | -4.7 (-4.5 to -4.9) | 0.82 (0.78 to 0.89) | 41.4 (37.5 to 43.0) |
| EC(D4→D5) + TM+VEGF-A121 | 210 (130 to 350) | -5.0 (-4.7 to 5.3) | 0.83 (0.77 to 0.89) | 41.0 (37.5 to 43.4) |
| EC(C482R)+TM | 100% dimer | 100% dimer | 0.45 ± 0.02 | 55 ± 1 |
| EC(C482R)+TM+VEGF-A121 | 100% dimer | 100% dimer | 0.47 ± 0.02 | 54 ± 1 |
| EC(C482R)+TM(N762I-V769I-G770I) | 100% dimer | 100% dimer | 0.31 ± 0.02 | 61 ± 1 |
