Purification of TrkA intracellular domain and the characterization of novel intracellular proteins : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Biology at Massey University

Abstract

Nerve growth factor (NGF) binds to its receptor, TrkA, at the tips of nerve cell axons to inhibit apoptosis, causing survival and differentiation. Some factors within this process are largely unknown, such as the role of the p75 receptor and the molecular mechanisms that occur within the cell. NGF binding causes dimerization of TrkA, which activates the intracellular kinase domain. Autophosphorylation on tyrosine residues stimulates binding to the receptor of several intracellular proteins that mediate the NGF response. This receptor complex has been demonstrated to be retrogradely transported to the cell body. Retrograde transport is hypothesized to occur in small vesicles that have been isolated in our lab using a cell fractionation protocol using in vitro reactions with an ATP regenerating system. Discovering the initial molecular interactions that occur upon NGF binding could further our knowledge of NGF's inhibition of apoptosis, providing us with a possible tool for treatment of diseases that occur when the regulation of apoptosis no longer exists. Novel proteins that were not previously identified were associated with TrkA in small vesicles after NGF activation. To isolate these proteins for further characterization, TrkA's intracellular domain (TrkAID) was expressed in E. Coli. This protein was found to be constitutively tyrosine-phosphorylated and therefore presumably active. In E.Coli, TrkAID protein was localized to the soluble fraction but smaller amounts were detected in the insoluble fraction. TrkAID was partially purified from the soluble fraction using a combination of salt disruption and denaturing techniques. The unpurified TrkAID was immunoprecipitated from the bacterial soluble fraction with an antibody to the C-terminus of TrkA, and some results suggest that immunoprecipitated TrkAID was able to stimulate ERK activation in untreated PC12 cells, but unfortunately this was not reproducible. If the protein could be purified with a combination of techniques, then it would provide a useful tool for studying the initial events in NGF stimulation, that is, the recruitment of several intracellular proteins to the tyrosine-phosphorylated intracellular domain of TrkA.