Kinetic analysis of cytokine-mediated receptor assembly using engineered FC heterodimers

A method for analyzing ligand-receptor binding kinetics is described, which is based on an engineered FC domain (FChk) that forms a covalent heterodimer. To validate the system, the type I IFN receptors (IFNAR1 and IFNAR2) were expressed as IFNAR1-FChk, IFNAR2-FCkh, and IFNAR1/IFNAR2-FChk fusion proteins. Surface plasmon resonance (SPR) analysis of binary IFN alpha 2a/IFNAR interactions confirmed prior affinity measurements, while the affinity of the IFN alpha 2a/IFNAR1/IFNAR2-FChk interaction reproduced the affinity of IFN alpha 2a binding to living cells. In cellular assays, IFNAR1/IFNAR2-FChk potently neutralized IFN alpha 2a bioactivity with an inhibitory concentration equivalent to the K-D measured by SPR. These studies suggest that FChk provides a simple reagent to evaluate the binding kinetics of multiple ligand-receptor signaling systems that control cell growth, development, and immunity. (C) 2013 The Protein Society