The Role of the Nerve Growth Factor Carboxyl Terminus in Receptor Binding and Conformational Stability

Abstract

The role of the nerve growth factor (NGF) carboxyl terminus in the function of NGF is not well understood. Previous work showed that deletion of residues 112-120 abolished NGF bioactivity. Several mutagenesis studies, however, have localized the binding sites of the two NGF receptors, p75 and TrkA, to other regions of the NGF molecule. To investigate the role of the NGF COOH terminus, we performed a detailed structure-function analysis of this region by deleting stepwise each of the nine COOH-terminal residues as well as constructing six point mutants. We found that point mutations within the 111-115 region, but not deletion of residues 116 -120, significantly decreased NGF bioactivity, as determined by TrkA tyrosine phosphorylation and neurite outgrowth from PC12 cells. Mutation of the absolutely conserved Leu 112 led to severely disrupted p75 binding on A875 cells but had only a modest effect on TrkA binding to MG87-TrkA fibroblasts. This suggests that the p75 binding surface is more extended than previously believed and includes not only charged residues within loops 1 and 5 but also spatially discontinuous, uncharged residues in a region where the NH 2 and COOH termini are in close proximity. Unexpectedly, deletion of COOH-terminal residues beyond Ala 116 led to significantly decreased stability. These results demonstrate that residues 111-115, but not residues 116 -120, are important for both the structural stability and biological activity of NGF.The neurotrophins are a family of survival and differentiation factors that exert a profound influence on development and maintenance of the nervous system (1). This feature makes neurotrophins promising candidates as therapeutic agents for the treatment of neurological disorders (2, 3). The crystal structure of the prototype neurotrophin, NGF, 1 revealed that it belongs to the cystine knot superfamily of structurally related growth factors (4). The well conserved "core" of the NGF molecule consists of two pairs of anti-parallel ␤-strands that form an elongated dimer interface and are held together by three disulfide bridges uniquely arranged in a characteristic cystine knot motif. The ␤-strands are connected to the "north" of the protomer by three ␤-hairpin loops, whereas the NH 2 and COOH termini appear to be located closer to the "south" of the protomers (see Fig. 7 for details). The loops and termini were not well resolved by x-ray crystallography and are thus likely to be highly flexible (5, 6). Moreover, these domains are regions of highest sequence divergence among neurotrophins. There is ample evidence for an involvement of these neurotrophin regions in specific receptor binding (7).Two NGF receptors have been identified: the p75 neurotrophin receptor and the tyrosine kinase TrkA (8 -10). Although TrkA is able to elicit NGF responses by itself in some systems, p75 can co-operate with TrkA to increase NGF binding to and activation of TrkA at low ligand concentration (11,12). Mutagenesis studies have previously demonstrated that the binding of NGF to p75 is ...

A TrkA-selective, Fast Internalizing Nerve Growth Factor-Antibody Complex Induces Trophic but Not Neuritogenic Signals

“…This is consistent with previous observations (16,46,47). Also, important NGF-p75 interactions are mediated by NGF amino acids 30 -35 within the AЈ-AЉ ␤-turn (39 -41); and these residues in one NGF protomer seem to pack closely with the C terminus of the other protomer within the NGF dimer (42,43).…”

“…The TrkA receptor mediates all of the trophic and neuritogenic activities of NGF whether or not p75 is expressed (13)(14)(15) or bound by the ligand (16,17). However, co-expression of p75 affects ligand affinity and TrkA efficacy (11,13) and also affects ligand-induced internalization (18).…”

Solution Structure and Internal Motion of a Bioactive Peptide Derived from Nerve Growth Factor

“…Deletion studies have also identified the N terminus (22)(23)(24) and C terminus (25)(26)(27) as necessary for TrkA binding. NGF binding to p75 is mainly mediated by the positively charged residues Lys 32 , Lys 34 , and Lys 95 (28) and the reverse-turn residues Asp 72 , Lys 74 , and His 75 (29).…”

Nerve Growth Factor (NGF) Loop 4 Dimeric Mimetics Activate ERK and AKT and Promote NGF-like Neurotrophic Effects

“…Loop 1 consists of the previously mapped residues 29 -35, and subsequent studies confirmed that region 29 -35 synthetic peptides inhibited NGF activity and NGF p75 receptor binding (16). Recombinant substitution studies indicated that residues Lys 32 and Lys 34 (17) and other residues (18) are likely to interact with p75 receptors. The NGF sites interacting with TrkA have also been derived via chemical modification (11,19,20), recombinant protein (18, 20 -23), and NGF-TrkA co-crystallization approaches (24).…”