Neuropilin-1 directs PDGFRa-entry into lung fibroblasts and signaling from very early endosomes

Abstract

Platelet-derived growth factor receptor-a (PDGFRa) is absolutely required for the development of secondary pulmonary alveolar septa. Our earlier observations indicated that PDGFRa 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 PDGFRa in LF obtained from mice, some bearing a targeted deletion of Nrp1 in myofibroblasts. Using the proximity ligation assay, we observed that PDGFRa and Nrp1 colocalized 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 PDGFRa was observed in Rab5-containing vesicles residing within 100nm of the plasma membrane. Nrp1 deletion reduced the phosphorylation of Akt (protein kinase B), the major downstream target of PDGFRa, and limited accumulation of inositol-3 phosphates in APPL1-containing endosomes after exposure to PDGFA. PDGFRa co-immunoprecipitated with APPL1, indicating that PDGFRa enters VEE. Targeted deletion of Nrp1 or APPL1-depletion in control LF reduced the activity of an Akt1 biosensor following stimulation with PDGFA. Our findings demonstrate that Nrp1 enhances the entry of PDGFRa into APPL1 containing VEE and that APPL1 enhances PDGFRa signaling. Therefore, Nrp1 promotes endosomal signaling by PDGFRa offering a potential mechanism to explain our prior observation that Nrp1 supports the formation of alveolar ducts and alveoli during secondary septation in mice.