ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , LDL-Receptor Related Proteins/immunology , Recombinant Proteins/immunology , Animals , Cell Separation , Epitopes , Flow Cytometry , HeLa Cells , Humans , LDL-Receptor Related Proteins/agonists , LDL-Receptor Related Proteins/biosynthesis , LDL-Receptor Related Proteins/genetics , Low Density Lipoprotein Receptor-Related Protein-6 , Mice , Mice, Inbred BALB C , Microscopy, Fluorescence , Protein Conformation , Protein Structure, Tertiary/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , T Cell Transcription Factor 1/metabolism , Transfection , Wnt Proteins/pharmacology , Wnt3 ProteinABSTRACT
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.