Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes
Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF₁₆₅a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF₁₆₅a...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
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Elsevier
2017
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| Online Access: | https://eprints.nottingham.ac.uk/42120/ |
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| author | Kilpatrick, Laura E. Friedman-Ohana, Rachel Alcobia, Diana C. Riching, Kristin Peach, Chloe J. Wheal, Amanda J. Briddon, Stephen J. Robers, Matthew B. Zimmerman, Kris Machleidt, Thomas Wood, Keith V. Woolard, Jeanette Hill, Stephen J. |
| author_facet | Kilpatrick, Laura E. Friedman-Ohana, Rachel Alcobia, Diana C. Riching, Kristin Peach, Chloe J. Wheal, Amanda J. Briddon, Stephen J. Robers, Matthew B. Zimmerman, Kris Machleidt, Thomas Wood, Keith V. Woolard, Jeanette Hill, Stephen J. |
| author_sort | Kilpatrick, Laura E. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF₁₆₅a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF₁₆₅a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF₁₆₅a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane. |
| first_indexed | 2025-11-14T19:47:45Z |
| format | Article |
| id | nottingham-42120 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T19:47:45Z |
| publishDate | 2017 |
| publisher | Elsevier |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-421202020-05-04T18:41:16Z https://eprints.nottingham.ac.uk/42120/ Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes Kilpatrick, Laura E. Friedman-Ohana, Rachel Alcobia, Diana C. Riching, Kristin Peach, Chloe J. Wheal, Amanda J. Briddon, Stephen J. Robers, Matthew B. Zimmerman, Kris Machleidt, Thomas Wood, Keith V. Woolard, Jeanette Hill, Stephen J. Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF₁₆₅a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF₁₆₅a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF₁₆₅a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane. Elsevier 2017-04-07 Article PeerReviewed Kilpatrick, Laura E., Friedman-Ohana, Rachel, Alcobia, Diana C., Riching, Kristin, Peach, Chloe J., Wheal, Amanda J., Briddon, Stephen J., Robers, Matthew B., Zimmerman, Kris, Machleidt, Thomas, Wood, Keith V., Woolard, Jeanette and Hill, Stephen J. (2017) Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes. Biochemical Pharmacology . ISSN 1873-2968 (In Press) VEGF; VEGFR2; BRET; Ligand binding; Receptor tyrosine kinase inhibitors http://www.sciencedirect.com/science/article/pii/S0006295217301958 doi:10.1016/j.bcp.2017.04.006 doi:10.1016/j.bcp.2017.04.006 |
| spellingShingle | VEGF; VEGFR2; BRET; Ligand binding; Receptor tyrosine kinase inhibitors Kilpatrick, Laura E. Friedman-Ohana, Rachel Alcobia, Diana C. Riching, Kristin Peach, Chloe J. Wheal, Amanda J. Briddon, Stephen J. Robers, Matthew B. Zimmerman, Kris Machleidt, Thomas Wood, Keith V. Woolard, Jeanette Hill, Stephen J. Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes |
| title | Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes |
| title_full | Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes |
| title_fullStr | Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes |
| title_full_unstemmed | Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes |
| title_short | Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes |
| title_sort | real-time analysis of the binding of fluorescent vegf₁₆₅a to vegfr2 in living cells: effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes |
| topic | VEGF; VEGFR2; BRET; Ligand binding; Receptor tyrosine kinase inhibitors |
| url | https://eprints.nottingham.ac.uk/42120/ https://eprints.nottingham.ac.uk/42120/ https://eprints.nottingham.ac.uk/42120/ |