Identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a Protein Receptor for Botulinum Neurotoxin Serotype A (BoNT/A)

Abstract

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25 ₂₀₆) to yield SNAP25 ₁₉₇. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, H _C/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. H _C/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Author Summary

Botulinum neurotoxin serotype A (BoNT/A) is one of seven neurotoxins (BoNT/A-G), produced by the bacteria Clostridium botulinum that are both poisons and versatile therapeutics. These toxins enter motor neurons where they prevent the release of acetylcholine at the neuromuscular junction. The specific uptake of BoNT/A across the neuronal cell membrane is dependent on specific receptor interactions. Binding to high density ganglioside GT1b mediates the initial binding step and via a low affinity interaction concentrates BoNT/A on the cell surface. Once anchored in the membrane, lateral movements within the plasma membrane facilitate intermolecular interactions of BoNT/A with additional lower density but higher affinity protein receptors. Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A. We show that BoNT/A binds to FGFR3 with high affinity and functions as an agonist ligand for FGFR3. The identification of this novel receptor for BoNT/A represents an important advance in the understanding of the mechanism of action of BoNT/A, especially on the initial steps of neuronal uptake, and can be the basis for the development of new specific countermeasures and new BoNT/A-based therapeutics.

Highlights

▸ Recombinant H _C/A binds to the two extra-cellular loops of FGFR3b with a K _D∼15 nM ▸ Recombinant H _C/A acts as an agonist ligand for FGFR3 ▸The level of BoNT/A uptake is dependent on FGFR3 expression ▸ FGFR3 is expressed in motor nerve terminals