Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells
Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, single-site (N-terminal cysteine) labelled versions of VEGF165a, VEGF165b and VEGF121a were synthesised. These were...
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| Format: | Article |
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Elsevier
2018
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| Online Access: | https://eprints.nottingham.ac.uk/52730/ |
| _version_ | 1848798794959814656 |
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| author | Peach, Chloe J. Kilpatrick, Laura E. Friedman-Ohana, Rachel Zimmerman, Kris Robers, Matthew B. Wood, Keith V. Woolard, Jeanette Hill, Stephen J. |
| author_facet | Peach, Chloe J. Kilpatrick, Laura E. Friedman-Ohana, Rachel Zimmerman, Kris Robers, Matthew B. Wood, Keith V. Woolard, Jeanette Hill, Stephen J. |
| author_sort | Peach, Chloe J. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, single-site (N-terminal cysteine) labelled versions of VEGF165a, VEGF165b and VEGF121a were synthesised. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc- NRP1 with a similar high affinity (4.4nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasise the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signalling. |
| first_indexed | 2025-11-14T20:25:26Z |
| format | Article |
| id | nottingham-52730 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| last_indexed | 2025-11-14T20:25:26Z |
| publishDate | 2018 |
| publisher | Elsevier |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-527302020-05-04T19:42:56Z https://eprints.nottingham.ac.uk/52730/ Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells Peach, Chloe J. Kilpatrick, Laura E. Friedman-Ohana, Rachel Zimmerman, Kris Robers, Matthew B. Wood, Keith V. Woolard, Jeanette Hill, Stephen J. Fluorescent VEGF-A isoforms have been evaluated for their ability to discriminate between VEGFR2 and NRP1 in real-time ligand binding studies in live cells using BRET. To enable this, single-site (N-terminal cysteine) labelled versions of VEGF165a, VEGF165b and VEGF121a were synthesised. These were used in combination with N-terminal NanoLuc-tagged VEGFR2 or NRP1 to evaluate the selectivity of VEGF isoforms for these two membrane proteins. All fluorescent VEGF-A isoforms displayed high affinity for VEGFR2. Only VEGF165a-TMR bound to NanoLuc- NRP1 with a similar high affinity (4.4nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF165a > VEGF189a > VEGF145a. VEGF165b, VEGF-Ax, VEGF121a and VEGF111a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF165a-TMR for NRP1 and VEGFR2. These data emphasise the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signalling. Elsevier 2018-06-29 Article PeerReviewed Peach, Chloe J., Kilpatrick, Laura E., Friedman-Ohana, Rachel, Zimmerman, Kris, Robers, Matthew B., Wood, Keith V., Woolard, Jeanette and Hill, Stephen J. (2018) Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells. Cell Chemical Biology . ISSN 2451-9456 (In Press) VEGFR2; Neuropilin-1; NanoBRET; Ligand binding kinetics; VEGF isoforms; Receptor mechanisms |
| spellingShingle | VEGFR2; Neuropilin-1; NanoBRET; Ligand binding kinetics; VEGF isoforms; Receptor mechanisms Peach, Chloe J. Kilpatrick, Laura E. Friedman-Ohana, Rachel Zimmerman, Kris Robers, Matthew B. Wood, Keith V. Woolard, Jeanette Hill, Stephen J. Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells |
| title | Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells |
| title_full | Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells |
| title_fullStr | Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells |
| title_full_unstemmed | Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells |
| title_short | Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells |
| title_sort | real-time ligand binding of fluorescent vegf-a isoforms that discriminate between vegfr2 and nrp1 in living cells |
| topic | VEGFR2; Neuropilin-1; NanoBRET; Ligand binding kinetics; VEGF isoforms; Receptor mechanisms |
| url | https://eprints.nottingham.ac.uk/52730/ |