Sortilin (approximately 95 kDa) is a member of the recently discovered family of Vps10p-domain receptors, and is expressed in a variety of tissues, notably brain, spinal cord and muscle. It acts as a receptor for neurotensin, but predominates in regions of the nervous system that neither synthesize nor respond to this neuropeptide, suggesting that sortilin has additional roles. Sortilin is expressed during embryogenesis in areas where nerve growth factor (NGF) and its precursor, proNGF, have well-characterized effects. These neurotrophins can be released by neuronal tissues, and they regulate neuronal development through cell survival and cell death signalling. NGF regulates cell survival and cell death via binding to two different receptors, TrkA and p75NTR (ref. 10). In contrast, proNGF selectively induces apoptosis through p75NTR but not TrkA. However, not all p75NTR-expressing cells respond to proNGF, suggesting that additional membrane proteins are required for the induction of cell death. Here we report that proNGF creates a signalling complex by simultaneously binding to p75NTR and sortilin. Thus sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.

The pro-peptide of human nerve growth factor (NGF) functions as an intramolecular chaperone during oxidative renaturation of proNGF in vitro and interacts intramolecularly with the mature part of native proNGF. Here, we analyzed the structure formation and stability of the pro-peptide in the context of proNGF and its intramolecular interaction with the native mature part. Folding and unfolding of the NGF-coupled pro-peptide, as analyzed by fluorescence, were biphasic reactions with both phases depending on the interaction with the mature part. This interaction was characterized by an overall stability of DG ¼ 20.9 kJ/mol that was subdivided into two reactions, native 4 intermediate state (14.8 kJ/mol) and intermediate 4 unfolded state (6.1 kJ/mol). An additional very fast unfolding reaction was observed using circular dichroism (CD), indicating the presence of at least two kinetically populated intermediates in the unfolding of proNGF. The part of the pro-peptide involved in the intramolecular association with mature NGF comprised the peptide Trp À83 -Ala À63 as determined by H/D exchange experiments. Spectroscopic analyses revealed that on the NGF side, a surface area around Trp 21 interacted with the pro-peptide. Trp 21 also participates in binding to TrkA and p75 receptors. These overlapping binding sites of the pro-peptide and the NGF receptors might explain the previously observed lower affinity of proNGF to its receptors as compared to NGF.

Human nerve growth factor (NGF) belongs to the structural family of cystine knot proteins, characterized by a disulfide pattern in which one disulfide bond threads through a ring formed by a pair of two other disulfides connecting two adjacent beta-strands. Oxidative folding of NGF revealed that the pro-peptide of NGF stimulates in vitro structure formation. In order to learn more about this folding assisting protein fragment, a biophysical analysis of the pro-peptide structure has been performed. While proNGF is a non-covalent homodimer, the isolated pro-peptide is monomeric. No tertiary contacts stabilize the pro-peptide in its isolated form. In contrast, the pro-peptide appears to be structured when bound to the mature part. The results presented here demonstrate that the mature part stabilizes the structure in the pro-peptide region. This is the first report that provides a biophysical analysis of a pro-peptide of the cystine knot protein family.

We have previously shown that the pro-peptide of human nerve growth factor (NGF) facilitates oxidative folding of the mature part. For the analysis of functional specificities of the pro-peptides of NGF and the related neurotrophin-3 (NT-3) with respect to structure formation, chimeric proteins with swapped pro-peptides were generated. Neither the structure nor the stability of the mature domains was influenced by the heterologous pro-peptides. For the pro-peptide of NT-3 fused to the mature part of NGF, stabilization of the pro-peptide moiety by the NGF part was observed. Folding kinetics and renaturation yields of this chimeric protein were comparable to those of proNGF. Our results demonstrate functional interchangeability between the pro-peptides of NGF and NT-3 with respect to their role in assisting oxidative folding of the mature part.

Nerve growth factor (NGF), a member of the neurotrophin family, is an all-beta-sheet protein with a characteristic structure motif, the cystine knot. Unfolding of NGF in 6 M GdnHCl has been described previously to involve an initial partial loss of structure and a subsequent very slow conversion to a second, completely unfolded state. This latter conversion was postulated to represent a back-threading of the disulfide bond that passes through the cystine knot (loop threading hypothesis). Here, this hypothesis was questioned with the pro form of the protein (proNGF). In proNGF, the mature part is preceded by the 103-amino acid pro-peptide. Consequently, loop threading of the N-terminally extended protein should be significantly delayed. However, unfolding kinetics of proNGF monitored by RP-HPLC, intrinsic fluorescence, and NMR spectroscopy were comparable to those of mature NGF. Time-resolved (1)H-(15)N HSQC spectra revealed a slow time-dependent loss of residual structure of which the kinetics correlated well with the transition observed by RP-HPLC. Refolding from the completely unfolded state led to a partial recovery of natively folded proNGF. In summary, the sequential unfolding of proNGF only marginally differed from that of mature NGF. Therefore, it is very unlikely that a loop threading mechanism is the cause of the slow unfolding step.