Identification of a direct interaction between interleukin 2 and the p64 interleukin 2 receptor y chain (interleukin 2-binding site/ligand dissociation/interleukin 2 receptor 13 chain/two-dimensional gel electrophoresis)

The interleukin 2 receptor (IL-2R) consists of at least two subunits, a and (3, both of which can bind interleukin 2 (IL-2). Recent studies have demonstrated the existence of a third subunit, a 64-kDa molecule termed IL-2R y chain, and have suggested that y chain functions to regulate the rate of IL-2 dissociation from the receptor. In the present report we have addressed whether the Vchain modulates IL-2R affinity by contributing contact sites for IL-2 binding. Using reagents that allow the IL-2R complex to be immunoprecipi- tated through the IL-2 molecule itself, we demonstrate the existence of a stable IL-2-IL-2R y-chain complex. These studies thus establish that the IL-2R y chain directly contrib- utes to the IL-2-binding site, consistent with the hypothesis that y chain influences IL-2R affmity through its direct interaction with IL-2. Interleukin 2 (IL-2) plays a central role in the activation and maintenance of both specific and nonspecific immune re- sponses. The receptor for IL-2 exists in low-, intermediate-, and high-affinity forms (1-9), consisting of various combina- tions of at least three distinct protein subunits. Two of the subunits, a and (3, have been cloned and shown to directly bind IL-2 (10-14), whereas the third subunit, a 64-kDa molecule termed y, which was recently identified in lymphoid cells (15-17), is largely uncharacterized. A role for y chain in influencing ligand-binding affinity, however, has been in- ferred from receptor reconstitution experiments (12-14). Recombinant IL-2 receptor (IL-2R) (3chain and recombinant a(3 complexes, expressed by cDNA transfection in fibro- blasts, displayed rapid rates of IL-2 dissociation, as com- pared with the same proteins reconstituted in lymphoid cells (14). This finding suggested that p64 y chain present in lymphoid cells regulated receptor affinity through its effects on ligand dissociation. The possible contribution of this protein to modulation of IL-2R affinity was further empha- sized in two separate studies showing that the amount of p64 ychain relative to 3chain in anti-,8-chain immunoprecipitates closely correlated with the fraction of cell-surface (8 chain involved in intermediate-affinity IL-2 binding (16, 17). Recent cloning of the cDNA encoding the IL-2R 'y chain (18) and the demonstration that cotransfection of ( and y-chain cDNAs into fibroblast cells allowed reconstitution ofan intermediate- affinity IL-2-binding site confirmed these earlier studies, suggesting a requirement for the y chain in formation of the IL-2-binding site. These studies did not, however, address whether the y chain modulates receptor affinity by directly contributing contact sites for IL-2 binding, as opposed to indirectly influencing receptor formation by modifying con- formation of the (3 chain. The present report documents the existence ofa stable interaction between IL-2 and the 'y chain in the absence of the , chain, thus substantiating one mech- anism by which the y chain may modulate IL-2R affinity.