Detection of endogenous LRP6 expressed on human cells by monoclonal antibodies specific for the native conformation.

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.

[1]  A. Myers,et al.  Common genetic variation within the Low-Density Lipoprotein Receptor-Related Protein 6 and late-onset Alzheimer's disease , 2007, Proceedings of the National Academy of Sciences.

[2]  Jayaram Radhakrishnan,et al.  LRP6 Mutation in a Family with Early Coronary Disease and Metabolic Risk Factors , 2007, Science.

[3]  Hideki Yamamoto,et al.  Multiplicity of the interactions of Wnt proteins and their receptors. , 2007, Cellular signalling.

[4]  A. Einstein,et al.  Inside out , 1991, Nature.

[5]  Fumiaki Ito,et al.  Cytoskeletal reorganization by soluble Wnt‐3a protein signalling , 1998, Genes to cells : devoted to molecular & cellular mechanisms.

[6]  S. Blacklow,et al.  Requirement for natively unstructured regions of mesoderm development candidate 2 in promoting low-density lipoprotein receptor-related protein 6 maturation. , 2007, Biochemistry.

[7]  Christof Niehrs,et al.  Casein kinase 1 gamma couples Wnt receptor activation to cytoplasmic signal transduction. , 2005, Nature.

[8]  Nam-Chul Ha,et al.  Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling , 2008, PloS one.

[9]  R. Mann,et al.  Boca‐dependent maturation of β‐propeller/EGF modules in low‐density lipoprotein receptor proteins , 2004, The EMBO journal.

[10]  Harold P. Erickson,et al.  C-terminal opening mimics 'inside-out' activation of integrin α5β1 , 2001, Nature Structural Biology.

[11]  J. Takagi,et al.  A murine monoclonal antibody that binds N-terminal extracellular segment of human protease-activated receptor-4. , 2008, Hybridoma.

[12]  Akira Kikuchi,et al.  Caveolin Is Necessary for Wnt-3a-Dependent Internalization of LRP6 and Accumulation of β-Catenin , 2006 .

[13]  Yan Li,et al.  LDL-receptor-related protein 6 is a receptor for Dickkopf proteins , 2001, Nature.

[14]  J. Takagi,et al.  Classification of ‘activation’ antibodies against integrin β1 chain , 1997, FEBS letters.

[15]  Christof Niehrs,et al.  Wnt Induces LRP6 Signalosomes and Promotes Dishevelled-Dependent LRP6 Phosphorylation , 2007, Science.

[16]  D. Strickland,et al.  Diverse roles for the LDL receptor family , 2002, Trends in Endocrinology & Metabolism.

[17]  J. Hsieh,et al.  Mesd Encodes an LRP5/6 Chaperone Essential for Specification of Mouse Embryonic Polarity , 2003, Cell.

[18]  R. Mann,et al.  Boca, an Endoplasmic Reticulum Protein Required for Wingless Signaling and Trafficking of LDL Receptor Family Members in Drosophila , 2003, Cell.

[19]  A. Bafico,et al.  Analysis of Endogenous LRP6 Function Reveals a Novel Feedback Mechanism by Which Wnt Negatively Regulates Its Receptor , 2007, Molecular and Cellular Biology.

[20]  W. Schneider,et al.  LDL receptor relatives at the crossroad of endocytosis and signaling , 2003, Cellular and Molecular Life Sciences CMLS.

[21]  J. Herz The LDL Receptor Gene Family (Un)Expected Signal Transducers in the Brain , 2001, Neuron.

[22]  J. Abreu,et al.  Inhibition of GSK3 Phosphorylation of β-Catenin via Phosphorylated PPPSPXS Motifs of Wnt Coreceptor LRP6 , 2009, PloS one.

[23]  Stuart A. Aaronson,et al.  A Novel Mechanism for Wnt Activation of Canonical Signaling through the LRP6 Receptor , 2003, Molecular and Cellular Biology.

[24]  Chika Yokota,et al.  Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions , 2007, Development.