Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the ,B2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CDI1a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG 3-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.
We have performed a structure-function analysis of extracellular domain regions of the human IFN-α receptor (hIFNAR1) using mAbs generated by immunizing mice with a soluble hIFNAR1-IgG. Five mAbs described in this study recognize different epitopes as determined by a competitive binding ELISA and by alanine substitution mutant analyses of the hIFNAR1-IgG. Two mAbs, 2E1 and 4A7, are able to block IFN-stimulated gene factor 3 (ISGF3) formation and inhibit the antiviral cytopathic effect induced by several IFN-α (IFN-α2/1, -α1, -α2, -α5, and -α8). None of these anti-IFNAR1 mAbs were able to block activity of IFN-β. mAb 4A7 binds to a domain 1-hIFNAR1-IgG but not to a domain 2-hIFNAR1-IgG, which suggests that its binding region is located in domain 1. The binding of the most potent blocking mAb, 2E1, requires the presence of domain 1 and domain 2. The most critical residue for 2E1 binding is a lysine residue at position 249, which is in domain 2. These findings suggest that both domain 1 and domain 2 are necessary to form a functional receptor and that a region in domain 2 is important. IFN-β recognizes regions of the hIFNAR complex that are distinct from those important for the IFN-α.
The human IFN-α receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-α2/α1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-α2/α1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-α8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-α2, but remained responsive to hIFN-β. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-α2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.
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