Human interferon-gamma receptor. Mapping of epitopes recognized by neutralizing antibodies using native and recombinant receptor proteins.
Monoclonal antibodies produced against native interferon-gamma receptor (IFN gamma-R) have been characterized for their capacity to react with purified receptor and receptor-positive cells, to inhibit the binding of IFN gamma to cellular receptor, to precipitate the receptor protein when cross-linked to IFN-gamma, and to recognize the recombinant interferon-gamma receptor and 19 overlapping fragments of this protein expressed in Escherichia coli. The results of this analysis showed that: (i) the extracellular portion of human IFN gamma-R is located between the N terminus and the transmembrane region (amino acids 18-246). (ii) The intracellular domain is between the transmembrane region and the C terminus (amino acids 269-489). (iii) The monoclonal antibodies that react with the IFN gamma-R intracellular domain recognize small linear epitopes. (iv) The human IFN gamma-R binding site is located between the N terminus and the transmembrane region. (v) The monoclonal antibodies that react with IFN gamma-R extracellular domain and inhibit the binding of IFN gamma recognize two different epitopes. One of these epitopes (included between amino acids 26 and 133) is very close to the binding site for IFN gamma. The second (included between amino acids 70 and 210) is related to the binding site for IFN gamma without including it. (vi) These two functional epitopes are conformational and need S-S bridges to maintain their architecture. (vii) These conformational epitopes are formed in receptor fragments expressed in E. coli.
Garotta
G
G
Central Research Units, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Ozmen
L
L
Fountoulakis
M
M
Dembic
Z
Z
van Loon
A P
AP
Stüber
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010957:Plasmids; D011971:Receptors, Immunologic / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D017471:Receptors, Interferon; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D007371:Interferon-gamma