LIGAND-BINDING KINETICS OF IL-2 AND IL-15 TO HETEROMERS FORMED BY EXTRACELLULAR DOMAINS OF THE 3 IL-2 RECEPTOR SUBUNITS

Studies on the binding of IL-2 to its receptor (IL-2R) have generally been limited to receptors expressed on cell surfaces. This has hampere d detailed kinetic and mechanistic studies at the molecular level, We have prepared the soluble extracellular domains of all three receptor subunits (called alpha, beta and gamma) by recombinant techniques and have used these to perform detailed kinetic studies of their binding p roperties using the technique of surface plasmon resonance. We describ e a novel approach whereby the receptors are assembled on an antibody surface, being held by an epitope engineered into the C-terminus of ea ch of these domains, Thus the receptors are oriented naturally leading to homogeneous ligand binding kinetics, We have characterized the int eractions of the heteromeric complexes of these subunits with mouse an d human IL-2 and their analogs, as well as the recently discovered cyt okine, IL-15, We have also studied the extracellular domains of the mo use receptor subunits for the first time and have used these as well a s mouse-human hybrid receptors to probe the mechanism of assembly of t hese complexes, We show that no additional proteins are required to re produce the properties of these complexes in vitro. In addition, kinet ic studies with site-specific analogs of IL-2 and the mouse-human rece ptor hybrids clearly indicate that the extracellular domains of alpha and beta can together readily bind ligand with kinetic properties dist inct from those of the constituent subunits, In contrast, a complex co ntaining ligand and the extracellular domains of beta and gamma was co mparatively difficult to assemble and required prolonged exposure to I L-2, Our method enabled us to calculate the stoichiometry of these com plexes and to determine that anchoring these subunits is necessary to efficiently drive complex formation. The kinetic and equilibrium diffe rences between the mouse and human receptor complexes, and between IL- 2 and IL-15 binding to these receptors clarify the roles of the alpha and gamma subunits in the differential response of cells to different cytokines that may be present simultaneously in the environment.