Zone Mapping of the Binding Domain of the Rat Low Affinity Nerve Growth Factor Receptor by the Introduction of Novel N-Glycosylation Sites

doi:10.1074/jbc.270.9.4594

Zone Mapping of the Binding Domain of the Rat Low Affinity Nerve Growth Factor Receptor by the Introduction of Novel N-Glycosylation Sites (*)

Anne N. Baldwin(§) and
Eric M. Shooter

From the Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305

^§ To whom correspondence should be addressed. Tel.: 415-723-5194; Fax: 415-723-0388.

Abstract

The binding of NGF (nerve growth factor) to the rat low affinity nerve growth factor receptor (p75) 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, Ser, 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.

Footnotes

↵* This work was supported in part by Grant NS04270 from the National Institutes of Health, Grant IIRG-92-138 from the Alzheimer's Association, and by the State of California, Department of Health Services, under Contracts 92-15942 and 93-18647. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

p75, or p75

the low affinity nerve growth factor receptor

NGF

nerve growth factor

TNF

tumor necrosis factor

p55

tumor necrosis factor receptor I.
- Received August 5, 1994.
- Revision received December 22, 1994.