Dissecting Molecular Differences between Wnt Coreceptors LRP5 and LRP6 Bryan T. MacDonald, Mikhail V. Semenov¤a, He Huang¤b, Xi He* F. M. Kirby Neurobiology Center, Children’s Hospital, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical b- catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening ‘‘gap4’’ region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity. Citation: MacDonald BT, Semenov MV, Huang H, He X (2011) Dissecting Molecular Differences between Wnt Coreceptors LRP5 and LRP6. PLoS ONE 6(8): e23537. doi:10.1371/journal.pone.0023537 Editor: Wenqing Xu, University of Washington, United States of America Received May 6, 2011; Accepted July 19, 2011; Published August 24, 2011 Copyright: 2011 MacDonald 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 a National Institutes of Health grant (RO1 GM74241) to XH, who was a Leukemia and Lymphoma Society Scholar. BTM and HH were supported by a Ruth L. Kirschstein National Research Service Award and Institutes of Health Research postdoctoral fellowship, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: xi.he@childrens.harvard.edu ¤a Current address: Geriatric Research, Education and Clinical Center (GRECC), Bedford VA Medical Center, Bedford, Massachusetts, United States of America. ¤b Current address: School of Medicine and Biomedical Sciences, University at Buffalo, The State University of New York (SUNY-Buffalo), Buffalo, New York, United States of America. Introduction The Wnt/b-catenin signaling pathway is essential for embry- onic development and adult tissue homeostasis, consequently mutation of many of the components result in human birth defects, cancer and other diseases [1,2]. In the absence of a Wnt ligand, the transcriptional co-activator b-catenin is continuously degraded in the cyptoplasm by a protein complex including the scaffolding protein Axin, tumor suppressor APC (adenomatous polyposis coli), GSK3 (glycogen synthase kinase 3) and CK1a (casein kinase 1a) [3]. The Axin complex mediates CK1a and GSK3 phosphorylation of b-catenin to provide a binding site for the b-Trcp E3 ubiquitin ligase, resulting in b-catenin ubiquitina- tion and subsequent degradation. This process is inhibited when a Wnt ligand brings together two types of receptors: the Frizzled (Fz or FZD) serpentine receptors and the low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or LRP6). The intracellular regions of FZD and LRP5 or LRP6 recruit the cytoplasmic proteins Dishevelled (DVL) and Axin, respectively [3]. Recruit- ment of Axin to the membrane by the Fz- LRP5/6 complex inhibits b-catenin phosphorylation and allows b-catenin levels to accumulate, resulting in b-catenin entering the nucleus and interacting with TCF/LEF (T cell factor/lymphoid enhancer factor) transcription factors to activate Wnt target gene transcrip- tion [4,5]. LRP5 and LRP6 are the two LRP type of Wnt receptors in the human and mouse genome, and are both widely/ubiquitiously expressed [6,7,8,9,10]. Human LRP5 (1615 a.a.) and LRP6 (1613 a.a.) are 70% identical by paralogous conservation, and have a similar domain structure that consists of a large extracellular domain containing four b-propeller plus EGF repeats essential for binding to Wnt and other ligands/antagonists and three LDLR-A repeats [11]. The cytoplasmic region of LRP5/6 contains five highly conserved PPPSPxS motifs that serve as phosphorylation- regulated Axin binding sites [12,13]. LRP5 has a central role in human bone mass regulation. Loss of function mutations in LRP5 result in osteoporosis-pseudoglioma (OPPG) primarily character- ized by low bone mass [14]. On the other hand LRP5 ‘‘gain of function’’ missense mutations, which are clustered in the first b- propeller, cause high bone mass (HBM) disease [15,16], likely as a PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23537