RESUMEN
LRP6 is a cell surface molecule that plays a critical role in the Wnt signaling pathway, and is implicated in numerous human diseases. Studies of cellular signaling mediated by LRP6 have relied on overexpression experiments, due to the lack of good monoclonal antibodies (mAbs) reactive with native LRP6 ectodomain. By using native recombinant LRP6 ectodomain fragment produced in mammalian expression system, we succeeded in developing a panel of anti-human LRP6 mAbs. Selected mAbs were capable of staining endogenous LRP6 on cell surface by using flow cytometry and immunofluorescence microscopy, and enriching detergent-solubilized LRP6 from cell lysate by immunoprecipitation.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Relacionadas con Receptor de LDL/inmunología , Proteínas Recombinantes/inmunología , Animales , Separación Celular , Epítopos , Citometría de Flujo , Células HeLa , Humanos , Proteínas Relacionadas con Receptor de LDL/agonistas , Proteínas Relacionadas con Receptor de LDL/biosíntesis , Proteínas Relacionadas con Receptor de LDL/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Conformación Proteica , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor 1 de Transcripción de Linfocitos T/metabolismo , Transfección , Proteínas Wnt/farmacología , Proteína Wnt3RESUMEN
R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.