FUNCTIONAL-ANALYSIS OF MUTANT NEUROTROPHINS DEFICIENT IN LOW-AFFINITYBINDING 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, a nd the low-affinity neurotrophin receptor, p75(LNGFR), of as yet undef ined signalling capacity. The need for a two-component receptor system in neurotrophin signalling is still not understood. Using site-direct ed 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, howev er, positively charged residues critical for p75(LNGFR) binding (Lys95 , Lys96 and Arg97) were found in a spatially close but distinct loop r egion. Models of each neurotrophin were built using the coordinates of NGF. Analysis of their respective electrostatic surface potentials re vealed similar clusters of positively charged residues in each neurotr ophin but with differences in their precise spatial locations. Disrupt ion of this positively charged interface abolished binding to p75(LNGF R) but not activation of cognate Trk receptors or biological activity in Trk-expressing fibroblasts. Unexpectedly, loss of low-affinity bind ing in NT-4, but not in BDNF or NT-3, affected receptor activation and biological activity in neuronal cells co-expressing p75(LNGFR) and Tr kB, suggesting a role for p75(LNGFR) in regulating biological responsi veness to NT-4. These findings reveal a possible mechanism of ligand d iscrimination by p75(LNGFR) and suggest this receptor may selectively modulate the biological actions of specific neurotrophin family member s.