SPECIFICITY DETERMINANTS IN NEUROTROPHIN-3 AND DESIGN OF NERVE GROWTHFACTOR-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 t rkC and trkA, respectively, were previously determined by mutational a nalyses. These and other studies showed that trkA can discriminate bet ween NGF and NT-3 primarily by recognition of their N-terminal residue s. The mechanism of trkC discrimination, however, remained unclear, es pecially since the most important residue in NT-3 involved in binding to trkC, R103, is conserved in all neurotrophins. In this study residu es 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 fo r 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 PC 12 cells expressing trkC. These variants show that the amino acid at p osition 23 (glycine in NGF, threonine in NT-3) is critical for trkC re cognition while other residues fine tune the specificity of NT-3 for t rkC. The results demonstrate the importance of nonconserved residues o f the central beta-strand bundle region for the interaction of NT-3 wi th trkC and emphasize the different mechanism of specificity determina tion that is employed in the NT-3/trkC and NGF/trkA ligand/receptor pa irs.