RESUMEN
Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. IL-11 has been shown to induce gp130-dependent signaling through the formation of a high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site-directed mutagenesis studies have identified three distinct receptor binding sites of IL-11, which enable it to form this high affinity receptor complex. Here we present data from immunoprecipitation experiments, using differentially tagged forms of ligand and soluble receptor components, which show that multiple copies of IL-11, IL-11R, and gp130 are present in the receptor complex. Furthermore, it is demonstrated that sites II and III of IL-11 are independent gp130 binding epitopes and that both are essential for gp130 dimerization. We also show that a stable high affinity complex of IL-11, IL-11R, and gp130 can be resolved by nondenaturing polyacrylamide gel electrophoresis, and its composition verified by second dimension denaturing polyacrylamide gel electrophoresis. Results indicate that the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all essential for this stable receptor complex to be formed. We therefore propose that IL-11 forms a hexameric receptor complex composed of two molecules each of IL-11, IL-11R, and gp130.
Asunto(s)
Antígenos CD/metabolismo , Interleucina-11/metabolismo , Interleucina-11/farmacología , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Antígenos CD/química , Antígenos CD/genética , Sitios de Unión , Línea Celular , Receptor gp130 de Citocinas , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulinas/química , Interleucina-11/genética , Subunidad alfa del Receptor de Interleucina-11 , Sustancias Macromoleculares , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Mutación/genética , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina-11 , TermodinámicaRESUMEN
Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. A complex of IL-11 and the IL-11 receptor (IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling. In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays. The location of these residues, as predicted from structural studies and a model of IL-11, reveals that mIL-11 has three distinct receptor binding sites. These are structurally and functionally analogous to the previously defined receptor binding sites I, II, and III of interleukin-6 (IL-6). This supports the hypothesis that IL-11 signals via the formation of a hexameric receptor complex and indicates that site III is a generic feature of cytokines that signal via association with gp130.
