title: Novel Bioluminescent Binding Assays for Ligand-Receptor Interaction Studies of the Fibroblast Growth Factor Family.
abstract: We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand-receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand-receptor interaction studies.
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Ge
authors.initials: G
authors.lastname: Song
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Qing-Ping
authors.initials: QP
authors.lastname: Wu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Zeng-Guang
authors.initials: ZG
authors.lastname: Xu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, Shanghai, China.
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D016222:Fibroblast Growth Factor 2 / Q000032:analysis / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods*; D011485:Protein Binding; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins
chemical_list: D008024:Ligands; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; D005346:Fibroblast Growth Factors