Pro-NGF is a novel ligand that initiates apoptosis through a complex withp75 and sortilin

Nerve growth factor (NGF) has the capacity to transduce cell survival or apoptotic signals by binding to either the TrkA or p75 receptor. Although the signaling mechanisms and binding interaction between NGF and TrkA have been well characterized, there are several inconsistencies concerning the interaction between NGF and p75 which remain elusive to date. Neurotrophins are synthesized as prepro-proteins that are cleaved in the TGN by furin to release mature peptides. Based on highly conserved regions in the pro-domain across all neurotrophins and significant endogenous levels of secreted pro-NGF, the biological activities of these pro-forms were investigated. To this end, we generated cleavage-resistant pro-NGF by mutating the dibasic furin site. Utilizing a histidine tag and nickel-column chromatography, we purified both pro-NGF and mature NGF from the media of stably transfected 293 cells, and compared their ability to activate Trk and p75 in both binding and biological assays. Pro-NGF, as compared to mature NGF, has a five-fold enhanced affinity of binding to p75. In biological assays, pro-NGF is ten-fold more effective in inducing apoptosis than its mature counterpart. In contrast, pro-NGF fails to bind or activate TrkA, suggesting that pro-NGF is the preferred ligand for p75 in mediating apoptotic responses.; To further investigate the interaction between pro-NGF and p75, we have identified sortilin as a co-receptor to p75 that is necessary for the transduction of apoptotic signaling responses. By affinity crosslinking, we demonstrated that pro-NGF binds cells expressing both sortilin and p75 receptors, but not cells expressing these receptors individually. Furthermore, only cells co-expressing p75 and sortilin undergo apoptosis in response to pro-NGF binding. Both the biochemical detection of this pro-NGF:sortilin complex as well as the pro-NGF induced apoptotic response can be competitively inhibited by the pro-domain of NGF. The disruption of this multimeric complex may lead to the impairment of the pro-apoptotic actions of pro-NGF. Taken together, these results suggest that pro-NGF induces apoptosis by its participation in a multimeric complex consisting of p75 and sortilin receptors.