A novel substrate of receptor tyrosine phosphatase PTPRO is required for nerve growth factor-induced process outgrowth.

The receptor protein tyrosine phosphatase PTPRO may be involved in axon guidance both as a ligand and as a neuronal receptor. We have begun to characterize signaling by PTPRO as a receptor by screening for proteins interacting with the intracellular domain of PTPRO. In a yeast-two hybrid screen, we identified a novel class of protein, which we named neuronal pentraxin with chromo domain (NPCD), as a PTPRO-interacting protein. We have shown recently that NPCD has multiple cytoplasmic isoforms as a result of alternative splicing and that these proteins are present in many neurons, mainly associated with the inner side of the plasma membrane. Through additional two-hybrid experiments, cotransfection and reciprocal coprecipitation, glutathione S-transferase pulldown, and immunoprecipitation in vivo, we confirm that NPCD isoforms interact with the catalytic phosphatase domain of PTPRO. We also find that at least one NPCD isoform is tyrosine phosphorylated in vivo and can serve as a substrate for PTPRO in vitro. Analysis of PTPRO knock-out mice demonstrates that normal localization of NPCD at the plasma membrane requires PTPRO expression, suggesting a physiological role for the NPCD/PTPRO interaction. NPCD is likely to be relevant to axon growth and/or guidance, because RNA interference mediated knock-down of NPCD expression in pheochromocytoma cells inhibits NGF-induced neuronal process outgrowth without affecting NGF-dependent survival or initial NGF signaling.