A B C LRP6 E1 E2 E3 E4 Wnt FZD 3a YW211.31 mAb LRP6 YW211 .31 Wnt 3a One-armed YW211.31 mAb LRP6 OA-YW211 .31 Wnt 3a Active Wnt3a signaling Active Wnt1 signaling Inactive Wnt3a signaling Potentiated Wnt1 signaling LRP6 Wnt 1 E1 E2 E3 E4 FZD LRP6 Wnt YW211 1 .31 Wnt 1 FZD FZD LRP6 OA-YW211 .31 Wnt 1 FZD Wnt 1 Wnt 3a LRP6 YW210.09 YW211 .31 Inactive Wnt1 and Wnt3a signaling Wnts Specify LRP6 mAb Activity No mAbs YW211.31 + YW210.09 mAbs Inactive Wnt3a signaling Active Wnt1 signaling Figure 8. Model of LRP6 interactions with antibodies and Wnt isoforms. (A–B) Wnt3a and Wnt3 induce signaling by binding the E3-E4 region of LRP6 and recruiting FZD into the complex, whereas Wnt1, Wnt9b, and other Wnt isoforms in this class (Figure 3C) bind the E1-E2 region. YW211.31.57 and YW210.09 antibodies recognize LRP6 regions E3-E4 and E1-E2, respectively, to inhibit signaling by only the Wnt isoforms that bind these regions. Both antibodies can bind an LRP6 molecule simultaneously to antagonize signaling in the presence of Wnt isoforms from both classes. Each antibody can potentiate signaling by the class of Wnt isoforms that bind regions outside its epitope, through crosslinking coreceptors and allowing Wnt to recruit FZD. (C) A one-armed antibody can inhibit signaling by blocking Wnt binding, but cannot potentiate since it does not dimerize LPR6. doi:10.1371/journal.pone.0012682.g008 levels are already maximal. We did detect a significant increase in bone mineral density in the calvaria explant model with YW210.09 antibody, and it will be important to confirm that this is mediated by potentiation of Wnt signaling in osteoblasts. While b-catenin dependent Wnt signaling is known to be required for intramembranous ossification of skull bones during embryogenesis, PLoS ONE | www.plosone.org 13 September 2010 | Volume 5 | Issue 9 | e12682