Analysis of the role of the interleukin-2 receptor gamma chain in ligand binding.
Interleukin-2 is the primary T cell growth factor secreted by activated T cells. IL-2 is an alpha-helical cytokine that binds to a multisubunit receptor expressed on the surface of a variety of cell types. IL-2Ralpha, IL-2Rbeta, and IL-2Rgammac receptor subunits expressed on the surface of cells may aggregate to form distinct binding sites of differing affinities. IL-2Rgammac was the last receptor subunit to be identified. It has since been shown to be shared by at least five other cytokine receptors. In this study, we have probed the role of IL-2Rgammac in the assembly of IL-2R complexes and in ligand binding. We demonstrate that in the absence of ligand IL-2Rgammac does not possess detectable affinity for IL-2Ralpha, IL-2Rbeta, or the pseudo-high-affinity binding site composed of preformed IL-2Ralpha/beta. We also demonstrate that IL-2Rgammac possesses an IL-2-dependent affinity for IL-2Rbeta and IL-2Ralpha/beta. We performed a detailed biosensor analysis to examine the interaction of soluble IL-2Rgammac with IL-2-bound IL-2Rbeta and IL-2-bound IL-2Ralpha/beta. The kinetic and equilibrium constants for sIL-2Rgammac binding to these two different liganded complexes were similar, indicating that IL-2Ralpha does not play a role in recruitment of IL-2Rgammac. We also determined that the binding of IL-2 to the isolated IL-2Rgammac was very weak (approximate K(D) = 0.7 mM). The experimental methodologies and principles derived from these studies can be extended to at least five other cytokines that share IL-2Rgammac as a receptor subunit.
Liparoto
Stefano F
SF
Department of Biological Chemistry, The University of Michigan Medical School, Medical Sciences Building I, Ann Arbor, Michigan 48109-4581, USA.
Myszka
David G
DG
Wu
Zining
Z
Goldstein
Byron
B
Laue
Thomas M
TM
Ciardelli
Thomas L
TL
D015374:Biosensing Techniques; D006801:Humans; D007376:Interleukin-2 / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011869:Radioligand Assay; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation
D007376:Interleukin-2; D008024:Ligands; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins