Tchetchelnitski, V.; (2011) Regulation of neurotrophin receptors by receptor‐type protein tyrosine phosphatases. Doctoral thesis , UCL (University College London).

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Abstract

Reversible protein phosphorylation plays a key role in cell signalling during neural development and thus controls cell proliferation, survival, differentiation and function. Kinases and their counter-partners the phosphatases tightly regulate protein phosphorylation. In the developing nervous system the neurotrophin receptor family of protein tyrosine kinases (TrkA, B and C) are major players in this signalling network during normal neuron development and also in several diseases such as neuropathies, degenerative disorders and cancers. Recently, receptor-type protein tyrosine phosphatases (RPTPs) were suggested to be possible regulators of Trks. Thus understanding the relationships between RPTPs and Trks may help to develop new therapeutics to control aberrant neurotrophin signalling in disease. In this study I investigated the relationship between RPTPs and Trks in murine embryonic sensory neurons from dorsal root ganglia (DRGs), a primary cell model. The expression and coexpression of RPTPs and Trks was extensively studied during critical stages of DRG maturation using qPCR arrays at Merck-Serono, Geneva, and fluorescent in-situ hybridization and immunohistochemical techniques. This revealed a relatively high expression of several candidate RPTPs, which were expressed in particular TrkA+, TrkB+ and/or TrkC+ subpopulations of sensory neurons, indicating a potential relationship in their signalling functions. To further analyze a potential direct interaction between candidate RPTPs with Trk proteins, a bimolecular-fluorescent complementation assay (BiFc) was tested. However, this particular assay, when used with type I transmembrane proteins, suffered from high, unspecific protein interactions. In the main experimental approach, a lentiviral-mediated shRNAi-induced knockdown system in primary cell cultures was set-up and the effects of the knockdowns of Ptprf, Ptprs and Ptpro on endogenous Trk gene expression and Trk phosphorylation and activation were analysed. These results suggest a potential role of the encoded proteins LAR and RPTPσ in Trk function and of RPTP-BK in the differentiation and specification of Trk+ neurons.

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