The relation between LAR class protein tyrosine phosphates and Semaphorin-Plexin signaling

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We have investigated whether LAR class Protein Tyrosine Phosphatase (PTP) is involved in Semaphorin-3A (Sema3A) signaling. In Drosophila, DLAR mutant exhibits 'bypass' phenotype of ISNb motoneuron projection that is observed in Dsemal and DPlexA mutants, suggesting the relation between the PTP and Semaphorin signaling. In mammals, LAR class PTP consists of LAR, PTP delta, and PTP sigma. We expressed a series of truncated mutant of these PTPs in chick dorsal root ganglions and tested Sema3A-induced growth cone collapse. We found that PTP delta mutants attenuated Sema3A signaling. To explore the role of PTP delta in Sema3A signaling and in axon guidance, we performed following experiments:1) Seeking tyrosine phosphorylated proteins interacting with PTP delta and Plexins, 2) Utilizing yeast hybrid system for isolation of PTPdelta substrates, 3) Evaluation of extracellular dornain of PTPdelta, and 4) Screening the genes up-regulated by Sema3A stimulation.1) PTPdelta from mouse embryonic ce … More rebral neurons was co-immunoprecipitated with a 100kDa phosphotyrosine containing protein. The phosphorylation level transiently decreased after Sema3A stimulation. Ectopically expressed Plexin-A4 in rat primary neurons also associated with a 100kDa protein that was rapidly phosphorylated after Sema3A stimulation. The identities of these two 100kDa proteins are currently unknown.2) Among 3X10^7 clones of mouse brain library screened, 47 clones of AIRC were isolated. AIRC is ADE1 and ADE2 enzyme complex that is involved in purine de novo synthesis. GST-pull down assay confirmed the interaction between PTP delta and AIRC.3) A fusion protein of alkaline Phosphatase and PTP delta extracellular domain bound to several specific regions in mouse embryonic brain such as subcortical layer of cerebrum, fascicules retroflex us and oblongata. The protein promoted the outgrowth of embryonic cerebral neurons.4) Using rat brain cDNA arrays, we compared the RNAs from embryonic brain dissociated cultures with or without Sema3A stimulation. We found that PKA regulatory subunit, stannin, and HAR1R-62 were up-regulated by Sema3A. Less