Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies Yan Gong1, Eric Bourhis2, Cecilia Chiu3, Scott Stawicki3, Venita I. DeAlmeida1, Bob Y. Liu1, Khanhky Phamluong4, Tim C. Cao5, Richard A. D. Carano5, James A. Ernst2,6, Mark Solloway4, Bonnee Rubinfeld1, Rami N. Hannoush2, Yan Wu 3 , Paul Polakis1, Mike Costa1* 1 Department of Cancer Targets, Genentech Research & Early Development, South San Francisco, California, United States of America, 2 Department of Protein Engineering, Genentech Research & Early Development, South San Francisco, California, United States of America, 3 Department of Antibody Engineering, Genentech Research & Early Development, South San Francisco, California, United States of America, 4 Department of Molecular Biology, Genentech Research & Early Development, South San Francisco, California, United States of America, 5 Department of Tumor Biology and Angiogenesis, Genentech Research & Early Development, South San Francisco, California, United States of America, 6 Department of Protein Chemistry, Genentech Research & Early Development, South San Francisco, California, United States of America Abstract b-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states. Citation: Gong Y, Bourhis E, Chiu C, Stawicki S, DeAlmeida VI, et al. (2010) Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies. PLoS ONE 5(9): e12682. doi:10.1371/journal.pone.0012682 Editor: Carl-Philipp Heisenberg, Max Planck Institute of Molecular Cell Biology and Genetics, Germany Received April 20, 2010; Accepted August 19, 2010; Published September 13, 2010 Copyright: 2010 Gong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Genentech Research & Early Development. The funder did have roles in study design, data collection and analysis, decision to publish, and preparation of the manuscript. Competing Interests: All authors are employed by Genentech Research & Early Development. This does not alter their adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: mcosta@gene.com Introduction Similar to most other morphogen and growth factor signaling pathways, mammalian Wnt signaling is deployed multiple times during development and tissue homeostasis [1]. These events differentially utilize 19 ligands, 10 receptors, and multiple coreceptors, including LRP5/6, Ror1/2, and Ryk [2]. In addition, different secreted antagonists that bind either Wnts, such as SFRP1/2/3/4/5 and WIF1, or bind LRP5/6, including DKK1/ 2/4 and SOST, modulate interactions between ligands and receptors. These membrane and extracellular proteins and their multiple isoforms provide for differential regulation at the level of expression and combinatorial protein interactions. Most Wnt isoforms appear to be capable of binding coreceptor LRP5/6, and this engagement specifies canonical, or b-catenin dependent, Wnt signaling. Wnt-mediated heterodimerization of LRP5/6 and FZD triggers phosphorylation of the LRP5/6 intracellular domain and Axin binding [3–5]. DVL is brought into the complex by directly binding both Axin and FZD, and DVL oligomerization likely enlarges these protein complexes on the cytoplasmic face of the membrane to sequester GSK3 and inhibit its phosphorylation and destabilization of b-catenin [6–12]. The unusually large number of ligand isoforms that mediate mammalian canonical Wnt signaling display considerable primary PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12682