Real-Time Ligand Binding of Fluorescent VEGF-A Isoforms that Discriminate between VEGFR2 and NRP1 in Living Cells

被引：27
|
作者：
Peach, Chloe J. [1 ,2 ,3 ]
Kilpatrick, Laura E. [1 ,2 ,3 ]
Friedman-Ohana, Rachel [4 ]
Zimmerman, Kris [4 ]
Robers, Matthew B. [4 ]
Wood, Keith, V [4 ]
Woolard, Jeanette [1 ,2 ,3 ]
Hill, Stephen J. [1 ,2 ,3 ]
机构：
[1] Univ Nottingham, Sch Life Sci, Div Physiol Pharmacol & Neurosci, Nottingham NG7 2UH, England
[2] Univ Birmingham, Ctr Membrane Prot & Receptors, Nottingham NG7 2UH, England
[3] Univ Nottingham, Nottingham NG7 2UH, England
[4] Promega Corp, 2800 Woods Hollow Rd, Madison, WI 53711 USA
来源：
CELL CHEMICAL BIOLOGY | 2018年 / 25卷 / 10期
基金：
英国生物技术与生命科学研究理事会;
关键词：
PATHOLOGICAL ANGIOGENESIS; CYTOPLASMIC DOMAIN; RECEPTOR COMPLEX; SPLICE VARIANT; NEUROPILIN-1; PROTEIN; EXPRESSION; INTERNALIZATION; CORECEPTOR; VEGF(121);
D O I：
10.1016/j.chembiol.2018.06.012
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
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.
引用
收藏
页码：1208 / +
页数：16
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