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, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF(165)a, VEGF(165)b, and VEGF(121)a. These w...

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Autores principales: Peach, Chloe J., Kilpatrick, Laura E., Friedman-Ohana, Rachel, Zimmerman, Kris, Robers, Matthew B., Wood, Keith V., Woolard, Jeanette, Hill, Stephen J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cell Press 2018
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200776/
https://www.ncbi.nlm.nih.gov/pubmed/30057299
http://dx.doi.org/10.1016/j.chembiol.2018.06.012
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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.
collection PubMed
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, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF(165)a, VEGF(165)b, and VEGF(121)a. 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 VEGF(165)a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF(165)a > VEGF(189)a > VEGF(145)a. VEGF(165)b, VEGF-Ax, VEGF(121)a, and VEGF(111)a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF(165)a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling.
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spelling pubmed-62007762018-10-26 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. Cell Chem Biol Article 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, we synthesized single-site (N-terminal cysteine) labeled versions of VEGF(165)a, VEGF(165)b, and VEGF(121)a. 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 VEGF(165)a-TMR bound to NanoLuc-NRP1 with a similar high affinity (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF(165)a > VEGF(189)a > VEGF(145)a. VEGF(165)b, VEGF-Ax, VEGF(121)a, and VEGF(111)a were unable to bind to NRP1. There were marked differences in the kinetic binding profiles of VEGF(165)a-TMR for NRP1 and VEGFR2. These data emphasize the importance of the kinetic aspects of ligand binding to VEGFR2 and its co-receptors in the dynamics of VEGF signaling. Cell Press 2018-10-18 /pmc/articles/PMC6200776/ /pubmed/30057299 http://dx.doi.org/10.1016/j.chembiol.2018.06.012 Text en © 2018 The Author(s) http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
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 Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200776/
https://www.ncbi.nlm.nih.gov/pubmed/30057299
http://dx.doi.org/10.1016/j.chembiol.2018.06.012
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