Platelet-derived growth factor receptor alpha (PDGFRα) is absolutely required for the development of secondary pulmonary alveolar septa. Our earlier observations indicated that PDGFRα resides intracellularly as well as on the plasma membrane of murine lung fibroblasts (LF). We have examined how neuropilin-1 (Nrp1), a surface receptor without kinase activity, regulates the intracellular trafficking of PDGFRα in LF obtained from mice bearing a targeted deletion of Nrp1 in myofibroblasts. Using the proximity ligation assay we observed that PDGFRα and Nrp1 co-localized in both early antigen-1 (EEA1) containing sorting endosomes and with adaptor protein containing a pleckstrin homology domain and a phosphotyrosine-binding domain-1 (APPL1) in very early endosomes (VEE). These findings were confirmed using live cell imaging, which demonstrated that recently internalized PDGFRα was observed in Rab5-containing vesicles residing within 100 nm of the plasma membrane. Nrp1-deletion reduced the phosphorylation of Akt, (protein kinase B) a downstream target of PDGFRα, indicating that Nrp1 enhances PDGFRα-signaling. PDGFRα co-immunoprecipitated with APPL1 and APPL1-depletion reduced Akt phosphorylation after exposure to PDGF-A. Targeted deletion of Nrp1 or APPL1-depletion in control LF reduced the activity of an Akt1 biosensor following stimulation with PDGF-A. Our findings demonstrate that Nrp1 enhances the entry of PDGFRα into APPL1 containing VEE and that APPL1 enhances PDGFRα-signaling. Therefore, Nrp1 enhances endosomal signaling by PDGFRα offering a potential mechanism to explain our prior observation that Nrp1 supports the formation of alveolar ducts and alveoli during secondary septation in mice.

中文翻译：

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Neuropilin-1引导PDGFRα进入肺成纤维细胞，并从非常早的内体发出信号

继发性肺泡隔的发展绝对需要血小板衍生的生长因子受体α（PDGFRα）。我们较早的观察结果表明，PDGFRα存在于鼠肺成纤维细胞（LF）的细胞内以及质膜上。我们已经检查了神经纤维蛋白-1（Nrp1），一种没有激酶活性的表面受体，如何调节从成纤维细胞中Nrp1靶向缺失的小鼠获得的LF中PDGFRα的细胞内运输。使用邻近结扎法，我们观察到PDGFRα和Nrp1共同定位在早期抗原1（EEA1）的分选内体中，以及在非常早期的内体中包含peckstrin同源域和磷酸酪氨酸结合域1（APPL1）的衔接蛋白（VEE）。这些发现已通过活细胞成像得到证实，结果表明，在质膜100 nm内的含Rab5的囊泡中观察到最近内在的PDGFRα。Nrp1删除减少Akt磷酸化（蛋白激酶B）的PDGFRα的下游目标，表明Nrp1增强PDGFRα信号。与APPL1和APPL1耗尽共同免疫沉淀的PDGFRα减少了暴露于PDGF-A后的Akt磷酸化。对照LF中Nrp1或APPL1耗竭的靶向缺失降低了PDGF-A刺激后Akt1生物传感器的活性。我们的发现表明Nrp1增强了PDGFRα进入含有VEE的APPL1的进入，而APPL1增强了PDGFRα信号传导。因此，