Kinetic analysis of the interleukin-13 receptor complex

Interleukin (IL)-13 is a key cytokine associated with the asthmatic phenotype. It signals via its cognate receptor, a complex of IL-13 receptor ?1 chain (IL-13R?1) with IL-4R?; however, a second protein, IL-13R?2, also binds IL-13. To determine the binding contributions of the individual components of the IL-13 receptor to IL-13, we have employed surface plasmon resonance and equilibrium binding assays to investigate the ligand binding characteristics of shIL-13R?1, shIL-13R?2, and IL-4R?. shIL-13R?1 bound IL-13 with moderate affinity (KD = 37.8 ± 1.8 nM, n = 10), whereas no binding was observed for hIL-4R?. In contrast, shIL-13R?2 produced a high affinity interaction with IL-13 (KD = 2.49 ± 0.94 nM n = 10). IL-13R?2 exhibited the binding characteristics of a negative regulator with a fast association rate and an exceptional slow dissociation rate. Although IL-13 interacted weakly with IL-4R? on its own (KD > 50 µM), the presence of hIL-4R? significantly increased the affinity of shIL-13R?1 for IL-13 but had no effect on the binding affinity of IL-13R?2. Detailed kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in which IL-13 first binds to IL-13R?1 and this then recruits IL-4? to stabilize a high affinity interaction.Abbreviations: IL, interleukin; shIL-13R?1, soluble human IL-13 receptor ? chain 1; shIL-13R?2, soluble human IL-13 receptor ? chain 2; sIL-4R?, soluble human IL-4 receptor; SPR, surface plasmon resonance, RU resonance units; STAT, signal transducers and activators of transcription; SA chip, streptavidin-coated sensor chip