The current signaling hypothesis is that PTN-induced/enforced dimerization of RPTPβζ results in loss of intracellular RPTPβζ phosphatase activity. In the absence of PTN signaling, RPTPβζ is shown to dephosphorylate beta-catenin (plus other important intracellular proteins), resulting in the normal formation of a β-catenin/E-cadherin molecular complex, which tethers actin filaments to the cell membrane. This is necessary for normal cell-cell adhesion. PTN signaling though the RPTPβζ receptor thus leads to decay of β-catenin/E-cadherin complex formation, due to loss of tyrosine dephosphorylation of β-catenin and disruption of the actin cytoskeleton and loss of cell-cell adhesion. Methods: We are using in vitro biophysical techniques (X-ray crystallography, calorimetry, Surface Plasmon Resonance) on the components of the entire signaling network of PTN/RPTPβζ to validate the current hypothesis.
Results: We have expressed PTN, and several individual domains of the RPTPβζ receptor in Escherichia coli. Biophysical characterization of these proteins is underway. Conclusions: It is possible to express and purify the extracellular PTN and RPTPβζ carbonic anhydrase and active phosphatase domains in E.coli, and this protein can now be used for X-ray crystallography and biophysical characterizations.