Binding site on the murine IFN-gamma receptor for IFN-gamma has been identified using the synthetic peptide approach.
We have studied the structural parameters involved in the binding of murine IFN-gamma (MuIFN-gamma) to its receptor. Ten synthetic overlapping peptides corresponding to the extracellular domain of the MuIFN-gamma receptor (MuIFN-gamma R) were synthesized. In direct binding studies, biotinylated MuIFN-gamma bound specifically to receptor peptide (95-120). Further, the NH2-terminal IFN-gamma peptide, MuIFN-gamma (1-39), also specifically bound to receptor peptide (95-120). Binding of both labeled MuIFN-gamma and MuIFN-gamma (1-39) to MuIFN-gamma R peptide (95-120) was inhibited by either unlabeled molecule. The COOH-terminal receptor binding peptide, MuIFN-gamma (95-133), neither bound to any receptor peptides nor blocked the binding of intact MuIFN-gamma or MuIFN-gamma (1-39) to receptor peptide (95-120). Polyclonal antibodies to each of the peptides were then produced. Each of the anti-peptide antisera recognized its corresponding peptide and bound denatured cloned soluble receptor by Western blotting. Furthermore, the antisera to peptides representing the inner region of the extracellular domain of the receptor bound to nondenatured soluble MuIFN-gamma R. Specifically, antisera to the receptor peptides (73-97), (95-120), (118-143), (142-163), and (161-182) bound to soluble MuIFN-gamma R, whereas antisera to peptides (1-21), (20-49), (46-74), (178-203), and (202-227) did not bind. Most important, antisera to peptides (95-120) and (118-143) competed with [125I]MuIFN-gamma for binding to soluble receptor. These results show that the region of the MuIFN-gamma R encompassing amino acid residues (95-120) is a binding site on the receptor for the NH2-terminal of MuIFN-gamma by direct binding, and that the larger region (95-143) on the receptor may play a role in binding of intact MuIFN-gamma based on blocking of binding by site-specific antibodies.
VanVolkenburg
M A
MA
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
Griggs
N D
ND
Jarpe
M A
MA
Pace
J L
JL
Russell
S W
SW
Johnson
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005260:Female; D007371:Interferon-gamma / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D017471:Receptors, Interferon / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D017471:Receptors, Interferon; D007371:Interferon-gamma