Determination of residues involved in ligand binding and signal transmission in the human IFN-alpha receptor 2

The human IFN-alpha receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analys is of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F 3, and 3B7) and several type I human IFNs. These mAbs block receptor activa tion, as determined by IFN-stimulated gene factor 3 formation, and block th e 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-alpha 2/alph a 1. We further demonstrated that residues E78, W101, I104, and D105 are cr ucial for the binding of hIFN-alpha 2/alpha 1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To conf irm that residues important for ligand binding are indeed important for IPN signal transduction, we determined the ability of mouse L929 cells express ing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-alpha 8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2, Transfected cells e xpressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-alpha 2, but remained responsive to hIFN-beta. I n summary, we have identified specific residues of hIFNAR2 important for th e binding to hIFN-alpha 2/1 and demonstrate that specific regions of the IF NAR interact with the subspecies of type I IFN in different manners.