Date Published: May 16, 2013
Publisher: Public Library of Science
Author(s): Birgitte P. S. Jacky, Patton E. Garay, Jérôme Dupuy, Jeremy B. Nelson, Brian Cai, Yanira Molina, Joanne Wang, Lance E. Steward, Ron S. Broide, Joseph Francis, K. Roger Aoki, Raymond C. Stevens, Ester Fernández-Salas, Steven R. Blanke.
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 (SNAP25206) to yield SNAP25197. 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, HC/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. HC/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.
Botulinum neurotoxin serotype A (BoNT/A) is produced by Clostridium botulinum and is a member of the Clostridial neurotoxin family that includes BoNT/A-G and Tetanus neurotoxin (TeNT). BoNT/A causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves nine amino acids from the C-terminus of the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein SNAP25 (SNAP25206) to yield SNAP25197. Intact SNAP25 is required for neurotransmitter release and cleavage of SNAP25 disrupts exocytosis, which blocks neurotransmitter release –. BoNT/A has become a useful pharmacological and biological tool. Because of its high potency and specificity for pre-synaptic nerve terminals, BoNT/A at picomolar concentrations, is used to treat a wide range of neuromuscular disorders –, pain disorders including migraine , and excessive sweating . The key to the exceptional specificity of BoNT/A is believed to be the mechanism of uptake across the presynaptic membrane of neurons that involves a combination of low and high affinity interactions known as the double receptor model –.
In this study we identified FGFR3 as a high affinity protein receptor for BoNT/A. Pull-down experiments with neuronal cells resulted in the identification of a protein complex containing BoNT/A and FGFR3. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete with rHC/A for binding to the receptor and binding of rHC/A results in phosphorylation of FGFR3, demonstrating that BoNT/A acts as an agonist ligand for FGFR3. Since ligand binding and activation of FGFRs are known to result in receptor-mediated endocytosis of both receptor and ligand , we propose that binding of BoNT/A to FGFR3 also results in endocytosis and that FGFR3 may mediate BoNT/A uptake in both stimulation independent and stimulation dependent manners. This hypothesis is supported by the fact that depolarization of nerve cells increases uptake (stimulation dependent), while at the same time BoNT/A uptake can take place in resting neurons (stimulation independent) , , –. This is also supported by the observation that the uptake, but not the initial binding step, is altered by nerve stimulation .