Characterization Of Ptprg In Knockdown And Phosphatase-Inactive Mutant Mice And Substrate Trapping Analysis Of Ptprg In Mammalian Cells

Receptor tyrosine phosphatase gamma (PTPRG, or RPTP gamma) is a mammalian receptor-like tyrosine phosphatase which is highly expressed in the nervous system as well as other tissues. Its function and biochemical characteristics remain largely unknown. We created a knockdown (KD) line of this gene in mouse by retroviral insertion that led to 98-99% reduction of RPTP gamma gene expression. The knockdown mice displayed antidepressive-like behaviors in the tail-suspension test, confirming observations by Lamprianou et al. 2006. We investigated this phenotype in detail using multiple behavioral assays. To see if the antidepressive-like phenotype was due to the loss of phosphatase activity, we made a knock-in (KI) mouse in which a mutant, RPTP gamma C1060S, replaced the wild type. We showed that human wild type RPTP gamma protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTP gamma C1060S mutant was completely inactive. Phenotypic analysis showed that the KI mice also displayed some antidepressive-like phenotype. These results lead to a hypothesis that an RPTPc inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPc for use in an assay for identifying inhibitors, "substrate trapping" mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTP gamma s retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTP gamma did not. This suggested that wild type RPTP gamma might auto-dephosphorylate which was confirmed by an in vitro dephosphorylation experiment. Using truncation and mutagenesis studies, we mapped the auto-dephosphorylation to the Y1307 residue in the D2 domain. This novel discovery provides a potential natural substrate peptide for drug screening assays, and also reveals a potential functional regulatory site for RPTP gamma. Additional investigation of RPTP gamma activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.