Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.
The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.
Myszka
D G
DG
Oncological Sciences Department, Huntsman Cancer Institute, University of Utah, Salt Lake City 84112, USA.
Arulanantham
P R
PR
Sana
T
T
Wu
Z
Z
Morton
T A
TA
Ciardelli
T L
TL
D000818:Animals; D015374:Biosensing Techniques; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D015375:Receptors, Interleukin-2 / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D015375:Receptors, Interleukin-2