Biophysical characterization of the pleiotrophin/RPTP zeta signaling mechanism

The receptor protein tyrosine phosphatase (RPTP bz) and its extracellular ligand pleiotrophin (PTN or Osteoblast specific factor-1) have been shown to be involved in bone formation but have so far been mostly studied in the brain. Objectives: The long-term aims of our research are to determine the molecular mechanism with which PTN exerts its stimulatory effects of RPTPbz in either bone or the brain. Three-dimensional structural understanding of this signaling system will open up for structure-based drug design of new PTN/RPTP small-molecule binding agents that might be able to treat osteoarthritis, osteopenia/osteoporosis, and other diseases associated with loss of or changes in bone density, in addition to a treatment for cancer.

The current signaling hypothesis is that PTN-induced/enforced dimerization of RPTPbz results in loss of intracellular RPTPbz phosphatase activity. In the absence of PTN signaling, RPTPbz is shown to dephosphorylate beta-catenin (plus other important intracellular proteins), resulting in the normal formation of a b-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 RPTPbz receptor thus leads to decay of b-catenin/E-cadherin complex formation, due to loss of tyrosine dephosphorylation of b-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/RPTPbz to validate the current hypothesis.

Results: We have expressed PTN, and several individual domains of the RPTPbz receptor in Escherichia coli. Biophysical characterization of these proteins is underway. Conclusions: It is possible to express and purify the extracellular PTN and RPTPbz carbonic anhydrase and active phosphatase domains in E.coli, and this protein can now be used for X-ray crystallography and biophysical characterizations.