Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes.
To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was 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 s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex 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 beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Jeffery J
JJ
Scibek
Emma
E
Evergren
Stefan
S
Zahn
Gabriela A
GA
Canziani
Donald
D
Van Ryk
Irwin M
IM
Chaiken
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D002850:Chromatography, Gel; D015848:Interleukin-5 / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018123:Receptors, Interleukin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053649:Receptors, Interleukin-5; D018411:Spodoptera; D013997:Time Factors
D015848:Interleukin-5; D046911:Macromolecular Substances; D021122:Protein Subunits; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5