Lee, S.F.K.; (2007) Understanding protein tyrosine phosphatase sigma function: Dimer formation and interacting proteins. Doctoral thesis , University of London.

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Abstract

Cytoplasmic and transmembrane protein tyrosine phosphatases (PTPs) provide the enzymatic counterbalance to protein tyrosine kinase activity. PTP sigma (PTPa) is an adhesion molecule-like receptor PTP (RPTP) that is expressed on the growth cones of developing axons. PTPa binds ligands located in the basement membrane (heparan sulphate proteoglycans: agrin and collagen 18) and developing muscle (Nucleolin). Disruption of ligand-PTPa interactions affects axon guidance, although neither the role of PTPa in neurons nor the effect of ligand binding on PTPa activity is well understood. Further characterisation of PTPa function remains difficult in the absence of an understanding of PTPa biochemistry that would allow a functional assay to measure the effects of an experimental manipulation on PTPa function. PTPa was shown to be dimeric using a combination of disulphide cross-linking and co-immunoprecipitation techniques. This dimeric form of PTPa was principally cell surface localised according to its accessibility to trypsinisation. However, neither co-immunoprecipitation nor glutathione S-transferase (GST) pull-down techniques allowed the identification of proteins that interact with wild type or recombinant substrate-trapping PTPa. Secondly, H202 treatment of PTPa-expressing cells induced the formation of reduction-sensitive, high molecular weight species. In contrast to the formation of PTPa dimers under the same conditions, this did not require the tyrosine phosphatase domain catalytic site cysteines. It may be possible to utilise the formation of high molecular weight species on non-reducing SDS-PAGE analysis as a proxy measure of PTPa oligomerisation. Moreover, unlike co-immunoprecipitation it can be used on wild type and even endogenously expressed proteins. Finally, the type Ila RPTP family consists of PTPa, PTPS and LAR. To allow the simultaneous disruption of type Ila RPTPs, chicken LAR was cloned from embryonic chick whole body mRNA and characterised.

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