RESUMEN
The leptin receptor (LR), a member of the class I cytokine receptor family, is composed of a single subunit. Its extracellular domain consists of two so-called cytokine receptor homology domains, separated by an Ig-like domain, and two additional fibronectin type III modules. Requirements for LR activation were examined using a complementation strategy. Two LR mutants, LR-FFY-Deltabox 1 and LR-F3, deficient in Janus kinase or signal transducer and activator of transcription (STAT) activation, respectively, were only able to generate a STAT3-dependent signal when coexpressed. Based on the requirements for Janus kinase/STAT signaling, and on the lack of complementation with similar receptor constructs, but containing the extracellular domain of the homodimeric erythropoietin receptor, this observation can be explained only by higher order LR clustering. Using a panel of deletion mutants we were able to define a role for the cytokine receptor homology 1 and Ig-like domains in leptin signaling. Moreover, we demonstrate a nonredundant function for the individual receptor chains within the homomeric LR complex. Based on these data, we propose a possible model for LR clustering.
Asunto(s)
Regulación de la Expresión Génica/genética , Receptores de Superficie Celular/genética , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Eritropoyetina/farmacología , Humanos , Leptina/farmacología , Ratones , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos , Receptores de Superficie Celular/metabolismo , Receptores de Leptina , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.
Asunto(s)
Cisteína/química , Fibronectinas/química , Receptores de Superficie Celular/metabolismo , Animales , Western Blotting , Células COS , Línea Celular , Membrana Celular/metabolismo , Análisis por Conglomerados , Citocinas/química , Proteínas de Unión al ADN/química , Dimerización , Disulfuros/química , Relación Dosis-Respuesta a Droga , Fibronectinas/metabolismo , Eliminación de Gen , Genes Reporteros , Vectores Genéticos , Humanos , Inmunoprecipitación , Leptina/química , Ligandos , Ratones , Mutación , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Leptina , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/química , TransfecciónRESUMEN
Despite the impact of the leptin system on body weight and other physiologic processes, little is known about the binding of leptin to its receptor. The extracellular domain of the leptin receptor consists of two cytokine receptor homology (CRH) domains separated by an immunoglobulin-like domain, and followed by two juxtamembrane fibronectin type III modules. The CRH2 domain functions as a high-affinity binding site for leptin, and we previously demonstrated interaction with helices A and C of leptin. In this work, we constructed a homology model for the leptin/CRH2 complex and performed a detailed mutation analysis of the CRH2/leptin interface. Using both cell-based and in vitro binding assays using the isolated CRH2 domain, we show the critical role of hydrophobic interactions between Leu 13 and Leu 86 of leptin and Leu 504 in CRH2 in leptin binding and signalling. This binding pattern closely resembles the interaction of other four-helix bundle long chain cytokines with the CRH domain of their cognate receptors.
Asunto(s)
Leptina/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Sitios de Unión/genética , Sitios de Unión/fisiología , Peso Corporal/genética , Peso Corporal/fisiología , Células COS , Chlorocebus aethiops , Humanos , Leptina/genética , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Receptores de Superficie Celular/genética , Receptores de LeptinaRESUMEN
The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.