IMMUNOLOGICAL DETERMINANTS OF NERVE GROWTH-FACTOR INVOLVED IN P140(TRK) (TRK) RECEPTOR-BINDING

Monoclonal anti-NGF antibodies that specifically inhibit the biologica l activity of mouse beta-NGF were used to study the structural determi nants involved in the interaction of NGF with its receptors gp75(LNGFR ) and Trk. None of the three antibodies-N60, M15, and 27/21-showed any reactivity toward denatured NGF. Three experimental methods-radioimmu noassay (RIA), enzyme-linked immunoassay (ELISA), and slot blots-detec ted no significant cross reactivity between the antibodies and BDNF or NT-3. RIA showed that M15 and N60 recognize the same or an overlappin g antigenic site, but 27/21 recognizes a different epitope. Only 27/21 , and not N60 or M15, immunoprecipitated beta-NGF crosslinked to LNGFR receptor. Thus, the epitope recognized by 27/21 does not overlap the LNGFR receptor binding site. N60, M15, and 27/21 all block binding of NGF to Trk in a manner consistent with competitive inhibition. Purifie d Fab fragments of N60 and M15 gave similar results to the intact anti bodies. The other subunits present in the 7S complex of NGF, i.e. the alpha and gamma subunits, competitively inhibited binding of antibodie s to beta-NGF. Only the gamma subunit inhibited phosphorylation of Trk and biological activity of beta-NGF. These findings suggest that the M15, N60, and 27/21 antibodies bind to a specific site on the surface of NGF where they competitively inhibit binding to the Trk NGF recepto r. The region encompassing the N-terminus, the C-terminus, and the loo p on the surface of beta-NGF containing residues 60-80 is proposed as important for binding to the Trk receptor. (C) 1994 Wiley-Liss, Inc.