The role of protein tyrosine phosphatase δ in axon guidance

研究了信号素- 3a （Sema3A）信号通路与LAR类酪氨酸蛋白磷酸酶LAR、PTPδ和PTPσ的关系。我发现PTPδ和PTPσ的外结构域与Neuropilin-1 （NRP1）结合，而不是LAR。使用碱性磷酸酶（AP）融合蛋白，我确定了外结构域PTPδ/σ的结合常数。AP-PTPδ和AP-PTPσ结合NRP1的Kd值分别为1.1nM和1.9nM。AP-Sema3A在相同条件下与NRP1结合，Kd为0.19nM。PTPδ/σ的第一个免疫球蛋白结构域与NRP1的CUB结构域结合，这是Sema3A的结合位点。PIPδ/σ外域抑制sema3a诱导的背根神经节神经元生长锥塌陷。在神经元中异位表达PTPδ的细胞质缺失突变体可抑制这种反应。令人惊讶的是，过度表达磷酸酶失活的PTPδ突变体，而非野生型PIPδ突变体，会干扰神经元中的sema3a反应。在Sema3A刺激下，共表达NRP1和PIPδ的COS-7细胞减少了粘附面积。这种反应被PTPδ的磷酸酶失活突变体完全消除。LAR和PTPσ都不是中介。响应。此外，NRP1与PTPδ在小鼠胚胎脑中共免疫沉淀。这些结果表明，PTPδ/σ的胞外结构域将NRP1与Sema3A的结合隔离开来，而全长PTPδ通过其磷酸酶结构域介导Sema3A信号转导。

The relation between Semaphorin-3A (Sema3A) signaling and LAR class protein tyrosine phosphatases, LAR, PTPδ and PTPσ, was investigated. I found that the ectodomains of PTPδ and PTPσ but not LAR bind to Neuropilin-1 (NRP1). Using alkaline phosphatase (AP) fusion proteins, I have determined the binding constant of the ectodomains PTPδ/σ. AP-PTPδ and AP-PTPσ bind NRP1 with Kd values of 1.1nM and 1.9nM, respectively. AP-Sema3A binds to NRP1 with a Kd of 0.19nM at the same condition. The first immunoglobulin domain of PTPδ/σ binds to the CUB domain of NRP1, which is the binding site for Sema3A. The ecdodomains of PIPδ/σ suppress Sema3A-induced growth cone collapse of dorsal root ganglion neurons. Ectopically expressed a cytoplasmic deletion mutant of PTPδ in the neurons suppresses the response. Surprisingly, overexpression of a phosphatase inactive mutant of PTPδ, but not of wild-type PIPδ, interferes Sema3A-response in the neurons. COS-7 cells co-expressing NRP1 and PIPδ reduce the adhered area upon Sema3A stimulation. This response is completely abolished with the phosphatase inactive mutant of PTPδ. Neither LAR nor PTPσ mediate. the response. Furthermore, NRP1 is co-immunoprecipitated with PTPδ from mouse embryonic brain. These results suggest that the extracellular domains of PTPδ/σ insulate NRP1 from Sema3A binding while full-length PTPδ mediates Sema3A signaling via its phosphatase domains.