Kinetic analysis of cytokine‐mediated receptor assembly using engineered FC heterodimers |
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Authors: | Ashlesha Deshpande Balananda‐Dhurjati Kumar Putcha Srilalitha Kuruganti Mark R Walter |
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Institution: | Department of Microbiology, University of Alabama at Birmingham, , Birmingham, Alabama, 35294 |
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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. |
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Keywords: | surface plasmon resonance engineered FC cytokine interferon IFNAR1 IFNAR2 heterodimer |
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