Author:
James Nicholas G.,Malik Shiazah,Sanstrum Bethany J.,Rhéaume Catherine,Broide Ron S.,Jameson David M.,Brideau-Andersen Amy,Jacky Birgitte S.
Abstract
AbstractClostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an effective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (HC/A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant HC/A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand-receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rHC/A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rHC/A dimerized FGFR subtypes in the rank order FGFR3c (EC50 ≈ 27 nM) > FGFR2b (EC50 ≈ 70 nM) > FGFR1c (EC50 ≈ 163 nM); rHC/A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC50 ≈ 18 nM). Mutating the ganglioside binding site in HC/A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein HC/A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization.
Publisher
Springer Science and Business Media LLC
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