Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes

Jeffery J Scibek, Emma Evergren, Stefan Zahn, Gabriela A Canziani, Donald Van Ryk, Irwin M Chaiken

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)258-65
Number of pages8
JournalAnalytical Biochemistry
Volume307
Issue number2
Publication statusPublished - 15 Aug 2002

Keywords

  • Animals
  • Biosensing Techniques
  • Cell Line
  • Chromatography, Gel
  • Interleukin-5
  • Kinetics
  • Macromolecular Substances
  • Protein Binding
  • Protein Subunits
  • Receptors, Interleukin
  • Receptors, Interleukin-5
  • Spodoptera
  • Time Factors

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