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VEGFR-2 conformational switch in response to ligand binding.

Sarabipour S, Ballmer-Hofer K, Hristova K - Elife (2016)

Bottom Line: Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation.Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation.We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, United States.

ABSTRACT
VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer.

No MeSH data available.


Related in: MedlinePlus

(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.DOI:http://dx.doi.org/10.7554/eLife.13876.005
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fig2s1: (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.DOI:http://dx.doi.org/10.7554/eLife.13876.005

Mentions: The fluorescent proteins YFP and mCherry were attached to the C-terminus of VEGFR-2 via a flexible GGS linker (see Figure 1), to allow for their free rotation (Evers et al., 2006). CHO cells, which do not endogenously express measurable amounts of VEGFR-2 (Figure 2—figure supplement 1) were co-transfected with plasmids encoding VEGFR-2-YFP and VEGFR-2-mCherry. Following VEGFR-2 expression and trafficking to the plasma membrane, the cells were vesiculated by applying osmotic stress. Each vesicle was imaged in the donor, acceptor, and FRET channels (see Figure 2—figure supplement 2). The FRET efficiency, the donor concentration, and the acceptor concentration in each individual vesicle were determined as described in Chen et al. (2010) and in 'Materials and methods'. The measured FRET efficiency for VEGFR-2 is shown as a function of acceptor concentration (VEGFR-2-mCherry) in Figure 2A (red solid symbols). The solid black line shows the so-called 'bystander' or 'stochastic' FRET, which occurs due to random approach of donors and acceptors in the absence of specific interactions (Wolber et al., 1979). The magnitude of stochastic FRET is well understood and characterized, both theoretically and experimentally (King et al., 2014). The measured FRET efficiencies significantly exceed this stochastic FRET contribution, demonstrating the existence of specific interactions between the full-length VEGFR-2 molecules in the cell membrane.


VEGFR-2 conformational switch in response to ligand binding.

Sarabipour S, Ballmer-Hofer K, Hristova K - Elife (2016)

(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.DOI:http://dx.doi.org/10.7554/eLife.13876.005
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829425&req=5

fig2s1: (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.DOI:http://dx.doi.org/10.7554/eLife.13876.005
Mentions: The fluorescent proteins YFP and mCherry were attached to the C-terminus of VEGFR-2 via a flexible GGS linker (see Figure 1), to allow for their free rotation (Evers et al., 2006). CHO cells, which do not endogenously express measurable amounts of VEGFR-2 (Figure 2—figure supplement 1) were co-transfected with plasmids encoding VEGFR-2-YFP and VEGFR-2-mCherry. Following VEGFR-2 expression and trafficking to the plasma membrane, the cells were vesiculated by applying osmotic stress. Each vesicle was imaged in the donor, acceptor, and FRET channels (see Figure 2—figure supplement 2). The FRET efficiency, the donor concentration, and the acceptor concentration in each individual vesicle were determined as described in Chen et al. (2010) and in 'Materials and methods'. The measured FRET efficiency for VEGFR-2 is shown as a function of acceptor concentration (VEGFR-2-mCherry) in Figure 2A (red solid symbols). The solid black line shows the so-called 'bystander' or 'stochastic' FRET, which occurs due to random approach of donors and acceptors in the absence of specific interactions (Wolber et al., 1979). The magnitude of stochastic FRET is well understood and characterized, both theoretically and experimentally (King et al., 2014). The measured FRET efficiencies significantly exceed this stochastic FRET contribution, demonstrating the existence of specific interactions between the full-length VEGFR-2 molecules in the cell membrane.

Bottom Line: Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation.Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation.We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, United States.

ABSTRACT
VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer.

No MeSH data available.


Related in: MedlinePlus