title: Zone mapping of the binding domain of the rat low affinity nerve growth factor receptor by the introduction of novel N-glycosylation sites.
abstract: The binding of NGF (nerve growth factor) to the rat low affinity nerve growth factor receptor (p75NGFR) has been studied by site-directed mutagenesis of the receptor. Introduction of non-native N-glycosylation sites within the binding domain indicates that the second of the characteristic cysteine-rich repeats may be particularly important to NGF binding. Two mutants of the second repeat, S42N and S66N, are glycosylated and bind NGF at a drastically reduced level, while still maintaining a conformation recognized by the monoclonal antibody against p75, MC192. Alanine substitution at these sites does not affect NGF binding. Two other mutations that result in local structural changes in the second repeat also greatly decrease binding. One of these altered residues, Ser50, appears to play an essential structural role, since it cannot be replaced by Asn, Ala, or Thr without loss of both NGF binding and MC192 recognition on a Western. Glycosylation of selected sites in the other repeats has little effect on NGF binding or antibody recognition. The introduction of non-native N-glycosylation sites may provide a generally useful scanning technique for the study of protein-protein interactions.
authors.affiliation: Department of Neurobiology, Stanford University School of Medicine, California 94305.
authors.forename: A N
authors.initials: AN
authors.lastname: Baldwin
authors.forename: E M
authors.initials: EM
authors.lastname: Shooter
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D006031:Glycosylation; D006358:Hot Temperature; D010449:Peptide Mapping; D017354:Point Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D051381:Rats; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D017475:Receptors, Nerve Growth Factor; D003545:Cysteine