RESUMEN
Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit alpha and recruitment of a common signaling subunit beta (betac). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor alpha and betac subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when alpha subunit alone was anchored. The slow-dissociation effect of betac had a similar impact on GM-CSF receptor stabilization to that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in betac, which together constitute key parts of a cytokine binding epitope. The data argue that betac plays an important role in preventing the ligand-receptor complexes from rapidly dissociating. This slow-dissociation effect of betac explains how, when multiple betac cytokine receptor alpha subunits are present on the same cell surface, selective betac usage can be controlled by sequestration in stabilized cytokine-alpha-betac complexes.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Interleucina-5/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Interleucina-5/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Drosophila , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-5/metabolismoRESUMEN
Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.
Asunto(s)
Complemento C8/química , Complemento C9/química , Fragmentos de Péptidos/química , Subunidades de Proteína/química , Animales , Sitios de Unión/genética , Células COS , Complemento C8/genética , Complemento C8/metabolismo , Complemento C8/fisiología , Complemento C9/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/química , Complejo de Ataque a Membrana del Sistema Complemento/genética , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Vectores Genéticos/síntesis química , Vectores Genéticos/metabolismo , Vectores Genéticos/fisiología , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de LDL/clasificación , Receptores de LDL/genética , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia/genética , Trombospondina 1/química , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMEN
To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.