Abstract
Hedgehog (HH) signaling critically regulates embryonic and postnatal development as well as adult tissue homeostasis, and its perturbation can lead to developmental disorders, birth defects, and cancers. Neuropilins (NRPs), which have well-defined roles in Semaphorin and VEGF signaling, positively regulate HH pathway function, although their mechanism of action in HH signaling remains unclear. Here, using luciferase-based reporter assays, we provide evidence that NRP1 regulates HH signaling specifically at the level of GLI transcriptional activator function. Moreover, we show that NRP1 localization to the primary cilium, a key platform for HH signal transduction, does not correlate with HH signal promotion. Rather, a structure-function analysis suggests that the NRP1 cytoplasmic and transmembrane domains are necessary and sufficient to regulate HH pathway activity. Furthermore, we identify a previously uncharacterized, 12-amino acid region within the NRP1 cytoplasmic domain that mediates HH signal promotion. Overall, our results provide mechanistic insight into NRP1 function within and potentially beyond the HH signaling pathway. These insights have implications for the development of novel modulators of HH-driven developmental disorders and diseases.
Highlights
Hedgehog (HH) signaling critically regulates embryonic and postnatal development as well as adult tissue homeostasis, and its perturbation can lead to developmental disorders, birth defects, and cancers
The addition of HH ligand is sufficient to induce a transcriptional response, we found that NRP1 and NRP2 both significantly increase ligand-activated HH pathway activity, as detected by glioma-associated oncogene homolog (GLI)-dependent luciferase output (Fig. 1A and supplemental Fig. S1), consistent with a previous report [35]
Western blot analysis confirmed that HA-tagged NRP1 and NRP2 are expressed at similar levels in NIH-3T3 cells (Fig. 1B)
Summary
A previous study showed that NRP1 overexpression increases ligand-stimulated HH pathway activity in HH-responsive fibroblasts [35]. Western blot analyses in NIH-3T3 cells confirmed equal expression of NRP1 and NRP1⌬CD (Fig. 2C) These data are consistent with recent results suggesting that NRP1 utilizes its cytoplasmic domain to promote HH pathway activity [36]. In contrast to previous work, a version of NRP1 that lacks all functional extracellular domains (NRP1⌬ECD) is sufficient to promote HH signaling (Fig. 2, A and B). Transfection of Nrp1CD is not sufficient to promote HH signaling in NIH-3T3 cells (supplemental Fig. S3), suggesting a role for the NRP1 transmembrane (TM) domain in HH signal promotion. Together, these data suggest that the membrane-anchored NRP1 CD is both necessary and sufficient to promote HH signaling. Observed a reliable increase in activity with Nrp co-expression Surprisingly, Nrp still promotes HH signaling when co-ex-
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