Specificity determinants in neurotrophin-3 and design of nerve growth factor-based trkC agonists by changing central beta-strand bundle residues to their neurotrophin-3 analogs

Neurotrophic factors mediate their signal by binding to specific cell surface receptors of the trk family. The binding sites of neurotrophin-3 (NT-3) and nerve growth factor (NGF) to their preferred receptors trkC and trkA, respectively, were previously determined by mutational analyses. These and other studies showed that trkA can discriminate between NGF and NT-3 primarily by recognition of their N-terminal residues. The mechanism of trkC discrimination, however, remained unclear, especially since the most important residue in NT-3 involved in binding to trkC, R103, is conserved in all neurotrophins. In this study residues that are part of the central beta-strand bundle of NT-3 and are not conserved among the neurotrophins were grafted onto NGF and tested for recruitment of trkC affinity. Exchange of NGF residues at positions 18, 20, 23, 29, 84, and 86 by their NT-3 counterparts resulted in NGF variants that bound to trkC, while maintaining their affinity to trkA, and were able to induce autophosphorylation and differentiation of PC12 cells expressing trkC. These variants show that the amino acid at position 23 (glycine in NGF, threonine in NT-3) is critical for trkC recognition while other residues fine tune the specificity of NT-3 for trkC. The results demonstrate the importance of nonconserved residues of the central beta-strand bundle region for the interaction of NT-3 with trkC and emphasize the different mechanism of specificity determination that is employed in the NT-3/trkC and NGF/trkA ligand/receptor pairs.