Mapping human interferon-alpha (IFN-alpha 2) binding determinants of the type I interferon receptor subunit IFNAR-1 with human/bovine IFNAR-1 chimeras.
Type I interferons bind to a common receptor (IFNAR), composed of two transmembrane polypeptides, IFNAR-1 and IFNAR-2. Although human IFNAR-1 has a weak intrinsic affinity for human Type I interferons (IFNs), bovine IFNAR-1 binds human Type I IFNs with moderate (nM) affinity, and can be conveniently used to investigate the regions of IFNAR-1 involved in ligand binding. We have constructed 14 bovine/human IFNAR-1 chimeras by exchanging homologous subdomains in the extracellular portion of the receptor. These chimeras were expressed at very high levels on COS cells, and their ability to bind HuIFN-alpha2 was measured. No single bovine subdomain substituted into human IFNAR-1 could confer moderate-affinity ligand binding on the resulting chimera. Simultaneous substitution of bovine IFNAR-1 subdomains 2 and 3 for the homologous human subdomains resulted in a dramatic increase in the binding of IFN-alpha2, suggesting that critical determinants for moderate-affinity ligand binding by BoIFNAR-1 reside in these two subdomains. Bovine subdomains 1 and/or 4 each further enhanced IFN-alpha2 binding in the presence of bovine subdomains 2 and 3. Thus, the binding interactions of BoIFNAR-1 with IFNs appears to be more complex than that of other class II cytokine receptors with their ligands.
Goldman
L A
LA
Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Cutrone
E C
EC
Dang
A
A
Hao
X
X
Lim
J K
JK
Langer
J A
JA
D000818:Animals; D019556:COS Cells; D002417:Cattle; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism*
C507964:IFNAR1 protein, human; D016898:Interferon-alpha; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D017471:Receptors, Interferon; D011993:Recombinant Fusion Proteins; D053633:Receptor, Interferon alpha-beta