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.
abstract: 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.
authors.affiliation: Cell Signalling and Pharmacology Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Laura E
authors.initials: LE
authors.lastname: Kilpatrick
authors.affiliation: Promega Corporation, Madison, WI, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Friedman-Ohana
authors.affiliation: Cell Signalling and Pharmacology Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Diana C
authors.initials: DC
authors.lastname: Alcobia
authors.affiliation: Promega Corporation, Madison, WI, USA.
authors.forename: Kristin
authors.initials: K
authors.lastname: Riching
authors.affiliation: Cell Signalling and Pharmacology Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Chloe J
authors.initials: CJ
authors.lastname: Peach
authors.affiliation: Cell Signalling and Pharmacology Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Wheal
authors.affiliation: Cell Signalling and Pharmacology Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Briddon
authors.affiliation: Promega Corporation, Madison, WI, USA.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Robers
authors.affiliation: Promega Corporation, Madison, WI, USA.
authors.forename: Kris
authors.initials: K
authors.lastname: Zimmerman
authors.affiliation: Promega Corporation, Madison, WI, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Machleidt
authors.affiliation: Promega Corporation, Madison, WI, USA.
authors.forename: Keith V
authors.initials: KV
authors.lastname: Wood
authors.affiliation: Cell Signalling and Pharmacology Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom. Electronic address: jeanette.woolard@nottingham.ac.uk.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Woolard
authors.affiliation: Cell Signalling and Pharmacology Research Group, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom. Electronic address: stephen.hill@nottingham.ac.uk.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Hill
mesh_terms: D000595:Amino Acid Sequence; D003199:Computer Systems; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes / Q000378:metabolism*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: BRET; Cediranib (Pubchem CIC 9933475); Furimazine (Pubchem CIC 219083); Ligand binding; Pazopanib (Pubchem CIC 10113978); Receptor tyrosine kinase inhibitors; Sorafenib (Pubchem CIC 216239); VEGF; VEGFR2; Vandetanib (Pubchem CIC 3081361)