sequence divergence. This diversity contrasts with the pair of highly homologous coreceptors. The LRP5 and LRP6 extracel- lular domains consist largely of four homologous regions, named E1 to E4 from N- to C-terminal, each containing a YWTD-type b- propeller and EGF-like domain [13]. Each repeat at a similar position in LRP5 and LRP6 is highly conserved, whereas the different repeats within the same protein are considerably more divergent. Interestingly, Bourhis et al. (2010) demonstrated that Wnt9b binds exclusively within the E1-E2 region in vitro, whereas Wnt3a binds only to a fragment containing E3-E4, suggesting that each repeat, or a combination of two adjacent repeats, binds to a different subset of Wnt isoforms [14]. This arrangement may accommodate the diversity of Wnt proteins, and possibly also allow for their differential regulation by LRP5/6 antagonist ligands. By contrast, in Notch and VEGF receptors, whose extracellular regions contain repeats of EGF-like and Ig domains, respectively, binding of multiple ligand isoforms is localized to the same region of one or two repeats, although the presence of other repeats can enhance binding [15–17]. For receptor tyrosine kinases, ligand-induced dimerization stimulates the kinase activity and initiates signaling. While ligand-induced receptor-coreceptor heterodimerization is neces- sary for canonical Wnt signaling, there is no clearly defined role for LRP5/6 or FZD homodimerization. Forced dimerization of different recombinant LRP6 proteins can either activate or inhibit Wnt signaling [18,19]. In this report, we utilize monoclonal antibodies to further explore the functional roles of different regions of LRP6 in signaling by Wnt isoforms and in antagonism by DKK1, as well as the functional consequences of dimerizing endogenous coreceptor. We provide evidence that different subsets of Wnt proteins induce signaling through binding different regions of LRP6, with most Wnts interacting within either E1-E2 or E3- E4, but not both. Individual LRP6 antibodies can inhibit autocrine, or endogenous, Wnt signaling in some tumor cell lines and potentiate autocrine signaling in other tumor cells, likely because the cell lines express different Wnt isoforms. Antibody- mediated dimerization of LRP6 does not induce signaling by itself, but it does potently enhance, or potentiate, signaling in the presence of a Wnt isoform that can simultaneously bind LRP6 and presumably recruit FZD into the complex. With increased understanding of the different Wnt isoforms mediating prolifera- tion, cell fate specification, and stem cell self-renewal in different cancer types and developmental processes, antibodies targeting different regions of LRP6 may prove to be effective agents for selectively modulating these processes. Results Isolation of Wnt Antagonist and Potentiating LRP6 Monoclonal Antibodies To develop candidate therapeutic molecules to manipulate Wnt signaling, we generated antibodies that can either inhibit or enhance signaling induced by Wnt3a protein. We used recombi- nant LRP6 E1-E2-Fc and E3-E4-Fc proteins to select from human synthetic antibody phage libraries and confirmed binding of isolated phage clones to LRP6 by ELISA and FACS [20]. Twenty- four unique antibody heavy chain clones against LRP6 E1-E2 and 22 unique clones against E3-E4 were reformatted and expressed as full-length human IgG1 antibodies. In luciferase reporter gene assays, six of the LRP6 E3-4 (Figure 1A) and none of the E1-E2 antibodies (data not shown) inhibited Wnt3a-induced signaling in HEK293 cells. The YW211.31 antibody recognizing the LRP6 E3-4 domain is the most potent in inhibiting signaling in Wnt3a- stimulated HEK293 cells, with an IC50 of approximately 1 mg/ml Wnts Specify LRP6 mAb Activity (or 6 nM). YW211.31 antibody inhibits Wnt3a-induced LRP6 phosphorylation and b-catenin protein stabilization without affecting levels of LRP6 protein, similar to purified Wnt-binding protein Fzd8CRD-Fc [21] and LRP6-binding protein DKK1 (Figure 1B). YW211.31 antibody can also antagonize mouse LRP6 function since it partially inhibits Wnt3a-induced reporter activity in mouse NIH/3T3 cells and b-catenin protein stabilization in mouse L cells (data not shown). YW211.31 antibody has a binding affinity of about 2 nM by surface plasmon resonance (SPR) and 0.6 nM by Scatchard analysis. To improve affinity and potential potency of YW211.31 antibody, the clone was affinity-matured using His-tagged LRP6 E3-E4 protein and CDR combinatorial libraries in which selected CDR residues were targeted for randomization. Four phage clones showing the most improved affinity by phage competition ELISA were reformatted and expressed as full length human IgGs. The dissociation rate constants of all four affinity-matured IgGs were decreased, leading to improved affinities for the best two antibodies, YW211.31.57 and YW211.31.62, of KD 0.27 and 0.17 nM, respectively. YW211.31.57 and YW211.31.62 also show improved potency in inhibiting signaling in Wnt3a-stimulated HEK293 cells, with IC50 values of approximately 0.1 mg/ml (0.6 nM). None of the antibodies isolated in the screen activated signaling in HEK293 cells in the absence of stimulation with exogenous Wnt3a protein, however five of the LRP6 E1-E2 and two of the E3-E4 antibodies potentiated Wnt3a-induced signaling at least 2-fold. In mouse NIH/3T3 cells, YW210.09, an E1-E2 antibody, also potentiated Wnt3a-induced signaling at least 1.5-fold, indicating that it also recognizes mouse LRP6 (data not shown). In HEK293 cells, the magnitude of enhancement of Wnt3a-induced signaling by YW210.09 antibody is proportional to Wnt3a concentration (Figure 1C). YW210.09 antibody interacts with human LRP6 E1- E2 protein with a KD of 5 nm as measured by SPR analysis. ELISA testing shows that all antagonist and potentiating antibodies specifically bind only the LRP6 protein fragment employed for their isolation, and none recognize both E1-E2 and E3-E4. FACS analysis indicates that soluble LRP6 E1-E4 protein efficiently and completely blocks binding of YW211.31.57 and YW210.09 to HEK293 cells, suggesting that these antibodies do not recognize other cell surface proteins (Figure S1). Effects of LRP6 Monoclonal Antibodies on Autocrine Wnt Signaling We next determined whether the LRP6 antibodies could antagonize or potentiate endogenous, or autocrine, Wnt signaling detected in a variety of tumor cell lines [21–23]. In teratocarci- noma cell lines PA-1 and NTERA-2, YW211.31 antibody inhibits reporter activity induced by autocrine Wnt signaling (Figure 1D) with similar potency to that observed with exogenous Wnt3a (data not shown). In PA1 cells, inhibition of Wnt signaling by YW211.31 antibody is also observed for expression of endogenous Wnt target genes (Figure 1E). The antibody partially inhibits expression of SAX1, GAD1, and APCDD1 that is either induced by exogenous Wnt3a protein or maintained by endogenous, autocrine Wnt signaling. Conversely, repression of LEFTY2 expression by either Wnt3a protein or autocrine Wnt signaling is relieved by YW211.31 antibody. In contrast, YW210.09 antibody potentiates both autocrine Wnt signaling (Figure 1D) and Wnt3a-induced signaling (data not shown) in PA-1 cells by reporter gene assays. Whereas inhibition of Wnt signaling by YW211.31.57 antibody increases progressively with greater antibody concentration, potentiation by YW210.09 and other antibodies decreases at high antibody concentrations in some cell types, such as PA-1 cells. This may suggest that antibody-mediated LRP6 dimerization is PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12682