FUNCTIONAL-ANALYSIS OF MUTANT NEUROTROPHINS DEFICIENT IN LOW-AFFINITY BINDING REVEALS A ROLE FOR P75(LNGFR) IN NT-4 SIGNALING

The neurotrophins mediate their effects through binding to two classes of receptors, a tyrosine kinase receptor, member of the Trk family, and the low-affinity neurotrophin receptor, p75(LNGFR), of as yet undefined signalling capacity. The need for a two-component receptor system in neurotrophin signalling is still not understood. Using site-directed mutagenesis, we have identified positively charged surfaces in BDNF, NT-3 and NT-4 that mediate binding to p75(LNGFR). Arg31 and His33 in NT-3, and Arg34 and Arg36 in NT-4, located in an exposed hairpin loop, were found to be essential for binding to p75(LNGFR). In BDNF, however, positively charged residues critical for p75(LNGFR) binding (Lys95, Lys96 and Arg97) were found in a spatially close but distinct loop region. Models of each neurotrophin were built using the coordinates of NGF. Analysis of their respective electrostatic surface potentials revealed similar clusters of positively charged residues in each neurotrophin but with differences in their precise spatial locations. Disruption of this positively charged interface abolished binding to p75(LNGFR) but not activation of cognate Trk receptors or biological activity in Trk-expressing fibroblasts. Unexpectedly, loss of low-affinity binding in NT-4, but not in BDNF or NT-3, affected receptor activation and biological activity in neuronal cells co-expressing p75(LNGFR) and TrkB, suggesting a role for p75(LNGFR) in regulating biological responsiveness to NT-4. These findings reveal a possible mechanism of ligand discrimination by p75(LNGFR) and suggest this receptor may selectively modulate the biological actions of specific neurotrophin family members.