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

ABSTRACT: 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 VEGF₁₆₅a, VEGF₁₆₅b, and VEGF₁₂₁a. 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 (4.4 nM). Competition NRP1 binding experiments yielded a rank order of potency of VEGF₁₆₅a > VEGF₁₈₉a > VEGF₁₄₅a. VEGF₁₆₅b, VEGF-Ax,

SUBMITTER: Peach CJ 

PROVIDER: S-EPMC6200776 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature