Disruption of cysteine-rich repeats of the p75 nerve growth factor receptor leads to loss of ligand binding.
Nerve growth factor (NGF) binds to a low affinity cell surface receptor (p75NGFR) which contains four extracellular repeats, rich in cysteine residues and negatively charged. We have made mutations in the receptor cDNA by inserting linkers in specific domains of the receptor. Nearly all the mutations caused a change in the predicted charge, and resulted in either an insertion or deletion in the primary sequence. Stably transfected fibroblasts were assayed for NGF binding by affinity cross-linking with 125I-NGF. Appropriate expression of the mutated receptors was monitored by rosetting with monoclonal antibodies and by metabolic labeling followed by immunoprecipitation. Although the mutant receptors were recognized by monoclonal antibodies, insertions and deletions in the third and fourth cysteine-rich regions of the receptor had a detrimental effect upon NGF binding. Insertions made outside the cysteine-rich region or in the cytoplasmic domain did not inhibit the ability of 125I-NGF to bind to the receptor, as assessed by affinity cross-linking. A chimeric human-rat NGF receptor transfected into fibroblasts indicates that NGF binding and monoclonal antibody recognition sites are separated but contained within the four cysteine repeats.
Yan
H
H
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021.
Chao
M V
MV
D000345:Affinity Labels; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002678:Chimera; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics*; D006801:Humans; D008024:Ligands; D009154:Mutation; D009414:Nerve Growth Factors / Q000378:metabolism*; D011233:Precipitin Tests; D011956:Receptors, Cell Surface / Q000235:genetics*; D017475:Receptors, Nerve Growth Factor; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D008024:Ligands; D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D003545:Cysteine