Two Restricted Sites on the Surface of the Nerve Growth Factor Molecule Independently Determine Specific TrkA Receptor Binding and Activation

Abstract

Nerve growth factor (NGF) and neurotrophin-3 (NT-3) mediate activities such as survival, differentiation, and proliferation in various subsets of neurons. In this report, we define precisely the residues in human NGF responsible for NGF biological activity and binding specificity to the neurotrophin receptor TrkA. In earlier studies we defined five amino acid residues of NGF which confer NGF-like activity to NT-3 when replacing corresponding residues in the 120-amino acid long NT-3 molecule. Using this gain-of-function strategy we report the further dissection of this functional epitope. We also define another motif separated topographically in the NGF dimer and determined to be independently responsible for NGF specificity. The first of the two motifs determined to elicit NGF specificity is defined by the residues Val-48, Pro-49, and Gln-96, which are situated in the two top ␤-loops of NGF. The second motif is represented by residues Pro-5 and Phe-7 situated in the proximal part of the NH 2 terminus. Both motifs contain structurally important residues revealing a novel principle, where specificity for neurotrophin ligand-receptor interactions could be determined by variable residues modifying the conformation of the neurotrophin backbone. These findings will enhance further the possibility of mimicking NGF with low molecular weight compounds.

Selectivity of Cell Signaling in the Neuronal Response Based on NGF Mutations and Peptidomimetics in the Treatment of Alzheimers Disease

“…However, a chimeric molecule that additionally replaced residues 3-9 of the N-terminus reduced TrkA binding, activation, and biological activity to <1% (Ibanez et al, 1993). Other substitution and deletion studies confirmed the importance of the N-terminus in specificity (Kullander et al, 1997;Woo et al, 1995;Kahle et al, 1992;Shih et al, 1994). Data from the crystal structure of human recombinant NGF complexed with the TrkA-d5 domains showed that the amino terminal residues 6-9, which were not well-defined in the original NGF crystal structure, adopted a helical conformation upon complex formation with their side chains almost completely buried in the interface (McDonald et al, 1991;Wiesmann et al, 1999) (Fig.…”

“…Scott et al, 1983;Walsh et al, 1999;Wiesmann et al, 1999;Yeo et al, 1997) . Sequence alignments of NGF, BDNF, and NT-3 (Ibanez et al, 1992;Ibanez et al, 1991;Ibanez et al, 1993;Ilag et al, 1994;Kullander & Ebendal, 1994;Kullander et al, 1997) demonstrate that each neurotrophin possesses seven variable regions that include the same residue positions. These are the N-terminus (residues 1-9), variable region I ( -hairpin loop 1, residues 23-35), variable region II ( -hairpin loop 2, residues 40-49), variable region III ( -reverse turn loop 3, residues 59-66), variable region IV ( -strand, residues 79-88), variable region V ( -hairpin loop 4, residues 95-99), and the C-terminus (residues 111-118) (Fig.…”

“…1B), in general, correspond to loops and surface residues in NGF and the other neurotrophins. Generation of NGF-BDNF chimeras identified variable region II as holding important receptor binding determinants (Ibanez et al, 1991;Ibanez et al, 1993;Ilag et al, 1994;Kullander & Ebendal, 1994;Kullander et al, 1997). The variable region was further dissected into IIa (residues 40-44) and IIb (residues 45-49) (Ibanez et al, 1993).…”

“…Region IIb was shown to play a more pivotal role than IIa when replacement of only the latter led to a dramatic reduction in receptor activation and biological activity (Ibanez et al, 1993). The variable region II, with help from variable region V (loop 4), was also shown to confer specificity between NT-3 and NGF (Ilag et al, 1994;Kullander & Ebendal, 1994;Kullander et al, 1997). The C-terminal linker region of TrkA could serve as a possible binding partner of this region (Wiesmann et al, 1999) (Fig.…”

“…Additional residues, V22, F53, H75, and R100, may not occupy the conserved patch, per se, but are as highly conserved as r e s i d u e s r e s i d i n g w i t h i n o r n e a r t h e p a t c h . S i m i l a r considerations have been made for BDNF and NT-3 (Ibanez et al, 1992;Ibanez et al, 1991;Ibanez et al, 1993;Ilag et al, 1994;Kullander & Ebendal, 1994;Kullander et al, 1997;Suter et al, 1992;Urfer et al, 1997). These regions of the NGF molecule were targeted to obtain the signal selective muteins discussed in Section 5.2.…”