Interactions of neurotrophins and their receptors : a dissertation presented in partial satisfaction of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular Biosciences at Massey University, New Zealand
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In order to investigate the interactions of neurotrophins with their receptors, a number of different domains of the extra-cellular regions of the TrkA, TrkB and TrkC receptors were expressed and the interactions of neurotrophins with these domains were investigated by biosensor. The entire extracellular domains of all three receptors were expressed in the yeast Pichia pastoris, while the leucine-rich regions and the immunoglobulin-like domains were expressed as MBP-fusion proteins in E. coli. Peptides representing the second leucine-rich regions and purported neurotrophin-binding domain of TrkA, TrkB and TrkC were synthesized. Proteins expressed in Pichia pastoris were purified by anion, cation and metal chelating columns; proteins expressed in E. coli., were purified on amylose columns. All recombinant Trk proteins were covalently attached, using EDC/NHS chemistry, to the methyldextran surface of a biosensor cuvette. Extensive kinetics measurements of the interactions of the neurotrophins with immobilized recombinant proteins established a difference in the binding interactions of NGF with TrkA compared with the interactions of BDNF with TrkB and NT-3 with TrkC. All NGF interactions with TrkA proteins showed biphasic kinetics. Interactions of BDNF and NT-3 with TrkB and TrkC showed monophasic kinetics. No interaction of NGF with the immunoglobulin-like domain of TrkA was observed for those proteins expressed in E. coli, however the interaction of BDNF was observed with the immunoglobulin-like domain of TrkB when expressed in E coli. Interaction of NGF and BDNF was observed with the leucine-rich domain of TrkA and TrkB respectively. These results differ from previously reported studies, both in vivo and in vitro, of the interactions of the extra-cellular domains of the Trk receptors with neurotrophins. Previous studies have claimed to establish exclusive interaction of the neurotrophins with either the leucine-rich or immunoglobulin-like domains of the Trk receptors. The interaction studies reported here show a clear interaction of neurotrophins with both leucine-rich and immunoglobulin-like domains of the Trk receptors. These interactions have similar affinity. This result suggests that the interactions of neurotrophin and receptor may be more complex than previously suggested. It is conceivable that neurotrophins bind initially to the leucine-rich domain of the receptor, followed by movement to the Immunoglobulin-like domain and the initiation of phosphorylation of the intra-cellular domains or internalization of receptor and bound neurotrophin. Kinetics studies of the synthetic peptides failed to show that these represent the exclusive neurotrophin-receptor interaction domain as previously reported. These results suggest that the development of small molecule mimetics of the neurotrophins as a therapy for Alzheimer's and other neurological diseases may be more complicated than previously envisioned.
Neurotrophins, Neurotrophin receptors, Trk receptors