Abstract

Knowledge of the kinetic binding characteristics is often critical to the development of ligand/receptor structure-activity relationships. To better understand the contribution of each of the subunits to ligand binding in the multimeric interleukin-2 receptor system, we have previously prepared stable solution complexes of the alpha- and beta-subunits. In this study, we have employed surface plasmon resonance biosensor methodology (BIAcore) to evaluate both the kinetic and equilibrium binding constants for these complexes. The structural nature of the complexes facilitated immobilization on the sensor surfaces in a manner that minimized interference with ligand interactions. The interleukin-2 receptor complex surfaces displayed excellent binding capacity and stability toward regeneration. In all cases where the binding constants were measurable, the values determined for interleukin-2 were in good agreement with those previously determined by other methods. When interleukin-2 analogs with receptor subunit specific mutations were employed, the binding parameters were consistent with the nature of the mutations. The combination of coiled-coil-mediated solution assembly and surface plasmon resonance analysis of ligand binding provides a powerful approach to the study of multimeric cytokine receptor systems.

Highlights

  • Knowledge of the kinetic binding characteristics is often critical to the development of ligand/receptor structure-activity relationships

  • The combination ofcoiled-coil-mediated solution assembly and surface plasmon resonance analysis of ligand binding provides a powerful approach to the study of multimeric cytokine receptor systems

  • The determination of the kinetic constants for IL-2 binding to cell surface IL-2 receptors proved to be of key importance in understanding the nature of the various physiological forms of the receptor [3, 4, 5]

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Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 270, No 27, Issue of July 7, pp. 16045-16051, 1995 Printed in U.S.A. Ligand Binding Analysis of Soluble Interleukin-2 Receptor Complexes by Surface Plasmon Resonance*. In order to determine the kinetic ligand binding properties of the soluble IL-2R·cc complexes and verify their equilibrium dissociation constants, we have employed surface plasmon resonance (SPR) biosensor technology. The specificity and stoichiometry of receptor subunit association are the critical parameters that determine both the ligand binding and signaling properties for the hematopoietin receptors In systems where both homomeric and heteromeric subunit association may occur via ligand-dependent or independent mechanisms and where multiple ligands may share a single common receptor subunit, understanding the nature of receptor aggregation is crucial to developing ligand/receptor structure-activity relationships. As a general approach to the stable solution assembly of cytokine receptors, we have employed coiled-coil molecular recognition to generate both homomeric [1, 2] and heteromeric [2] interleukin-2 receptor complexes (lL-2R1·cc) These complexes bound IL-2 with characteristic cell surface affinities in competitive radioligand binding assays.

MATERIALS AND METHODS
RESULTS AND DISCUSSION
TABLE I Kinetic rate constants
ND ND
Equilibrium dissociation constants
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