Upon activation by NGF, TrkA is internalized, trafficked and sorted through different endosomal compartments. Proper TrkA trafficking and sorting are crucial events since alteration of these processes hinders NGF-mediated functions. However, it is not fully known which proteins are involved in the trafficking and sorting of TrkA. Here we report that Nedd4-2 regulates the trafficking of TrkA and NGF functions in sensory neurons. Depletion of Nedd4-2 disrupts the correct sorting of activated TrkA at the early and late endosome stage, resulting in an accumulation of TrkA in these compartments and, as a result of the reduced trafficking to the degradative pathway, TrkA is either reverted to the cell surface through the recycling pathway or retrogradely transported to the cell body. In addition, Nedd4-2 depletion enhances TrkA signaling and the survival of NGF-dependent DRG neurons, but not those of BDNF-dependent neurons. Furthermore, neurons from a knock-in mouse expressing a TrkA mutant that does not bind Nedd4-2 protein exhibits increased NGF-mediated signaling and cell survival. Our data indicate that TrkA trafficking and sorting are regulated by Nedd4-2 protein.

Using the TrkA KI mouse model, we delineated a novel neuro-immune pathway and suggest that NGF-induced production of PGD in joint mast cells is critical for referred mechanical hypersensitivity in OA, probably through the activation of PGD receptor 1 in nociceptors: TrkA blockade in mast cells constitutes a potential target for OA pain.

Trk neurotrophin receptor ubiquitination in response to ligand activation regulates signaling, trafficking, and degradation of the receptors. However, the in vivo consequences of Trk ubiquitination remain to be addressed. We have developed a mouse model with a mutation in the TrkA neurotrophin receptor (P782S) that results in reduced ubiquitination due to a lack of binding to the E3 ubiquitin ligase, Nedd4-2. In vivo analyses of TrkAP782S indicate that defective ubiquitination of the TrkA mutant results in an altered trafficking and degradation of the receptor that affects the survival of sensory neurons. The dorsal root ganglia from the TrkAP782S knock-in mice display an increased number of neurons expressing CGRP and substance P. Moreover, the mutant mice show enhanced sensitivity to thermal and inflammatory pain. Our results indicate that the ubiquitination of the TrkA neurotrophin receptor plays a critical role in NGF-mediated functions, such as neuronal survival and sensitivity to pain.

The development of the nervous system is a temporally and spatially coordinated process that relies on the proper regulation of the genes involved. Neurotrophins and their receptors are directly responsible for the survival and differentiation of sensory and sympathetic neurons; however, it is not fully understood how genes encoding Trk neurotrophin receptors are regulated. Here, we show that rat Bex3 protein specifically regulates TrkA expression by acting at the trkA gene promoter level. Bex3 dimerization and shuttling to the nucleus regulate the transcription of the trkA promoter under basal conditions and also enhance nerve growth factor (NGF)-mediated trkA promoter activation. Moreover, qChIP assays indicate that Bex3 associates with the trkA promoter within a 150 bp sequence, immediately upstream from the transcription start site, which is sufficient to mediate the effects of Bex3. Consequently, the downregulation of Bex3 using shRNA increases neuronal apoptosis in NGF-dependent sensory neurons deprived of NGF and compromises PC12 cell differentiation in response to NGF. Our results support an important role for Bex3 in the regulation of TrkA expression and in NGF-mediated functions through modulation of the trkA promoter.