title: Kinetic analysis of cytokine-mediated receptor assembly using engineered FC heterodimers.
abstract: 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 IFNα2a/IFNAR interactions confirmed prior affinity measurements, while the affinity of the IFNα2a/IFNAR1/IFNAR2-FChk interaction reproduced the affinity of IFNα2a binding to living cells. In cellular assays, IFNAR1/IFNAR2-FChk potently neutralized IFNα2a bioactivity with an inhibitory concentration equivalent to the KD 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.
authors.affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, 35294, USA.
authors.forename: Ashlesha
authors.initials: A
authors.lastname: Deshpande
authors.forename: Balananda-Dhurjati Kumar
authors.initials: BD
authors.lastname: Putcha
authors.forename: Srilalitha
authors.initials: S
authors.lastname: Kuruganti
authors.forename: Mark R
authors.initials: MR
authors.lastname: Walter
mesh_terms: D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D055503:Protein Multimerization; D053633:Receptor, Interferon alpha-beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D007141:Immunoglobulin Fc Fragments; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D008024:Ligands; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D053633:Receptor, Interferon alpha-beta
keywords: IFNAR1; IFNAR2; cytokine; engineered FC; heterodimer; interferon; surface plasmon resonance