5 research outputs found

    Characterization of PTPRG in Knockdown and Phosphatase-Inactive Mutant Mice and Substrate Trapping Analysis of PTPRG in Mammalian Cells

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    <div><p>Receptor tyrosine phosphatase gamma (PTPRG, or RPTPĪ³) 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 98ā€“99% reduction of RPTPĪ³ 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, RPTPĪ³ C1060S, replaced the wild type. We showed that human wild type RPTPĪ³ protein, expressed and purified, demonstrated tyrosine phosphatase activity, and that the RPTPĪ³ 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 RPTPĪ³ inhibitor could be a potential treatment for human depressive disorders. In an effort to identify a natural substrate of RPTPĪ³ for use in an assay for identifying inhibitors, ā€œsubstrate trappingā€ mutants (C1060S, or D1028A) were studied in binding assays. Expressed in HEK293 cells, these mutant RPTPĪ³s retained a phosphorylated tyrosine residue, whereas similarly expressed wild type RPTPĪ³ did not. This suggested that wild type RPTPĪ³ might auto-dephosphorylate which was confirmed by an <em>in vitro</em> 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 RPTPĪ³. Additional investigation of RPTPĪ³ activity and regulation may lead to a better understanding of the biochemical underpinnings of human depression.</p> </div

    RPTPĪ³ dephosphorylated itself in vitro.

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    <p>Wild type RPTPĪ³ or RPTPĪ³ C1060S plasmids were transiently transfected into HEK293F and isolated from lysates with anti c-myc/protein G sepharoses. RPTPĪ³ wild type or C1060S on the protein G sepharose was denatured in 8 M urea to linearized protein as substrates for reaction following. The Purified RPTPĪ³ wild type, C1060s sepharose beads was then incubated with recombinant, active purified RPTPĪ³ cyto enzymes in assay buffer with urea at a final concentration of 0.375 M. Lanes 1 and 2 are RPTPĪ³ WT and RPTPĪ³ C1060S on protein G sepharose in a mock reaction without phosphatase in the same buffer, and lanes 1ā€²and 2ā€² are RPTPĪ³ wild type and RPTPĪ³ C1060S reacted with RPTPĪ³ cytoplasmic region as phosphatase. Samples were subjected to western blot analysis using anti-phosphotyrosine 4G10 mAb. The densitometry of each band was determined to estimate the extent of removal of phosphotyrosine on the protein. The reacted IgG heavy chain bands were used as internal control to ensure equal loading of samples. It is estimated by densitometry that 75% of phosphotyrosine from RPTPC1060S was removed by addition of RPTPĪ³ enzyme.</p

    Behavioral analysis on the wild type (WT) and knockdown mutant mice (MT).

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    <p>See details in the text. A and B. Repeated open field (A) and tail suspension tests (B) on the same animals. * - P<0.05, *** - P<0.001, compared to WT on the same day. C. Immobility time in the forced swim test. * - P<0.05, *** - P<0.001, compared to WT for the same measure.</p

    Determination of Km of phosphatase RPTPĪ³ on peptide substrate ATQDD(pY)VLEVR (derived from peptide adjacent to Y1307).

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    <p>Figure here shows Pi released (microM per minute) was plotted against phospho-peptide in a reaction in which Vmax was 2.1 ĀµM/mim/14 nM RPTPĪ³ and Km is 49.6 ĀµM, using one site nonlinear binding algorithm in Graphpad Prism.</p

    RPTPĪ³ substrate-trapping mutants in HEK293 cells.

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    <p>Phosphotyrosine-containing protein of about 190 kDa size ā€œtrappedā€ by RPTPĪ³ C1060S or RPTPĪ³ D1028A is RPTPĪ³. Recombinant wild type RPTPĪ³ and substrate-trapped mutants were immunoprecipitated from transiently transfected HEK293F lysate with anti-myc mAb. Lanes 1 and 1ā€²were IP product from pCDNA3 transfected cells, 2 and 2ā€² were from wild type RPTPĪ³ transfected cells, 3 and 3ā€² were from RPTPĪ³ C1060S transfected cells and 4 and 4ā€² were from RPTPĪ³ D1060A transfected cells. Lane 1ā€“4 were western reacted with anti-phosphotyrosine 4G10 mAb and lanes 1ā€²-4ā€² were with anti-myc mAb.</p
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