Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis.
Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteine-rich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The Mr 32,000 papain-resistant fragment (P32) and the Mr 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
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Vissavajjhala
J D
JD
Leszyk
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J
Lin-Goerke
A H
AH
Ross
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D010206:Papain