Biosensor analysis of dynamics of interleukin 5 receptor subunit βcinteraction with IL5:IL5Rα complexes

Abstract

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit βc, we constructed a soluble βcectodomain (sβc) and developed an optical biosensor assay to measure its binding kinetics. Functionally active sβcwas anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of sβcto IL5Rα complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either Rα or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 μM) and relatively fast off rate for the sβcinteraction with IL5:Rα complexes. The data support a model in which βcrecruitment occurs with preformed IL5:Rα complex. Dissociation kinetics analysis suggests that the IL5-α-βccomplex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of βcrecruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within Rα, βc, and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation. © 2002 Elsevier Science (USA). All rights reserved.