result of disruption of binding and inhibition of LRP5 by its antagonists DKK1 and Sclerostin/SOST [17,18]. Only one or two disease-causing LRP6 missense mutations have been found so far, including the one associated with coronary artery disease and osteoporosis [19], reflecting the likelihood that a severe or complete loss of function of LRP6 is incompatible with embryogenesis. Mouse models of the LRP5 [8,20] and LRP6 [21] mutations recapitulate the human disorders. Lrp52/2 mice and heterozygous Lrp6+/2 mice are viable and exhibit OPPG/ osteoporosis phenotypes demonstrating their overlapping func- tions in at least some aspects of bone development/homeostasis [22]. However Lrp62/2 phenotypes are much more severe, as embryos resemble a composite phenotype of several canonical Wnt loss of function mutations and typically die during late stages of embryogenesis [7]. The severity of the Lrp62/2 phenotype is made worse by removing one or both alleles of Lrp5 and the resulting embryos die very early in development [23]. Indeed Lrp5/6 double knockout mutants fail to undergo gastrulation and closely resemble the phenotype of Wnt3 knockout mice [23,24]. Collectively these data demonstrate that LRP6 has a more prominent role than LRP5 during development and that LRP5 and LRP6 together are essential for transducing Wnt/b-catenin signaling. Other studies have peripherally compared the relative activities of LRP5 and LRP6. Expression of LRP6 alone, but not LRP5 alone, is able to induce a secondary axis in Xenopus embryos [25]. Similarly in mammalian cell culture, LRP5 only weakly activates the Wnt pathway in the absence of an exogenous Wnt, in contrast to the highly active LRP6 [25,26]. On the other hand, coexpression of LRP5 and Wnt synergistically activates b-catenin signaling in both Xenopus embryos and mammalian cells [27]. These results are consistent with genetic studies that LRP6 appears to be the stronger of the two receptors in the Wnt pathway. Given the importance of LRP5 and LRP6 in development and human diseases, we chose to dissect the molecular basis that distinguishes and regulates LRP5 and LRP6 functions by generating a series of LRP5/6 chimeric proteins. Our results suggest that despite highly conserved PPPSPxS motifs shared by both receptors, LRP6 cytoplasmic domain (LRP6C) harbors stronger signaling activity than that of LRP5 (LRP5C), likely owing to the fact that PPPSPxS motifs are more readily phosphorylated in LRP6 than in LRP5. We further identified between the two most carboxyl PPPSPxS motifs an intervening region that appears to account for most of the difference in phosphorylation and signaling capacity between LRP5C and LRP6C. Finally we show that LRP5/LRP6-binding to Axin is likely direct without GSK3 acting as an intermediary physical bridge. Results A predominant role of Lrp6 in Wnt signaling in MEFs To compare the activity of the endogenous Lrp5 and Lrp6 we derived mouse embryonic fibroblasts (MEFs) from the wild-type (WT) +/+, Lrp52/2, and Lrp62/2 embryonic day 14 (E14) embryos. We analyzed the expression of the endogenous receptors and confirmed the absence of protein in our knockout cells (Figure 1A). Furthermore we detected roughly half as much Lrp6 protein in MEFS derived from Lrp6+/2 heterozygotes. We found that WT MEFs treated with Wnt3a conditioned media (CM) displayed a strong increase in cytosolic b-catenin levels compared to cells treated with the control CM (Figure 1B). Lrp52/2 MEFS, which only express the endogenous Lrp6 protein, displayed a slightly diminished response to Wnt3a compared to WT MEFs Comparison of LRP5 and LRP6 (Figure 1B). However Lrp62/2 MEFs, which only express the endogenous Lrp5 protein, exhibited a much weaker response to Wnt3a CM. We found similar results when using Wnt1 CM in the Lrp52/2 and Lrp62/2 knockout MEFS (data not shown). Additionally the Lrp52/2; Lrp6+/2 MEFS, which contained only one allele of Lrp6, displayed a higher Wnt response compared to Lrp62/2 cells containing two alleles of Lrp5 (Figure 1B). With the caveat that we do not know the abundance of Lrp5 or Lrp6 proteins on the cell surface, these results suggest that a majority of Wnt/b-catenin signaling in MEFs is mediated through Lrp6. LRP6C exhibits stronger signaling activity than LRP5C We set out to generate chimeric receptors between LRP5 and LRP6 to dissect their functional differences (Figure 2A and Figure S1A). In the process we found that the signal peptide of LRP5 is less active than that of LRP6, as *LRP5, in which the signal peptide of LRP6 replaced that of LRP5, exhibited a modest increase in the total protein level compared to the WT LRP5 (Figure 2B and 2C). Expression of *LRP5, but not of LRP5, showed a modest Wnt pathway activation in the Wnt responsive TOPFLASH luciferase assay, although this activation remained much weaker than that by expression of LRP6 alone (Figure 2D). In the presence of Wnt1 co-expression, however, *LRP5 and LRP5 exhibited virtually identical activity (Figure 2E). We speculate that under our experimental conditions of LRP5 or LRP5* over-expression, only a portion, but not all, of LRP5 or *LRP5 is activated by Wnt1, possibly due to availability of specific Fz proteins (and/or other factors such as LRP chaperones). For comparison of the mature LRP5 and LRP6 proteins (i.e., after the removal of the signal peptide) we made various LRP5/LRP6 chimeric proteins using *LRP5 and LRP6. We first generated *LRP5/6C and LRP6/5C by swapping the cytoplasmic domain between *LRP5 and LRP6 (Figure 2F). Compared to *LRP5, *LRP5/6C displayed higher activity when expressed alone or in the presence of Wnt1 (Figures 2G and 2H). On the contrary LRP6/5C was much weaker and in fact was mostly inactive alone or in synergy with Wnt1 (Figure 2G and 2H). In repeating experiments LRP6/5C sometimes behaved as a dominant negative receptor such that the co-expression of LRP6/ 5C suppressed Wnt1-stimulated reporter activity. We noticed that the difference of signaling activity by over-expressed *LRP5 versus LRP6 in the absence of Wnt1 co-expression was drastic (about 8 folds, Figure 2G); in the presence of Wnt1 co-expression both *LRP5 and LRP6 were significantly activated but the difference between *LRP5 and LRP6 was quite smaller (about 2 folds, Figure 2H). One possibility is that under over-expression condition only a portion, but not all, of *LRP5 or LRP6 is activated by Wnt1 due to the availability of specific (and endogenous) Fz proteins. We also note the caveat that signaling by over-expressed *LRP5 or LRP6 alone may or may not be Fz-dependent. To further map the activity difference between LRP5C and LRP6C, we utilized a conserved PstI site located after PPPSPxS motif B in both LRP5 and LRP6 to divide the cytoplasmic domain into two parts, which contain motifs A plus B, and motifs C plus D plus E, respectively (Figure 3A and Figure S1B). These series of *LRP5 and LRP6 chimeric receptors were expressed at compa- rable levels, respectively (Figure 3B, data not shown). *LRP5/6AB and *LRP5/6CDE, like *LRP5/6C, displayed higher activity compared to *LRP5 alone (Figure 3C). In the presence of Wnt1, *LRP5/6CDE was similar to the *LRP5/6C, and both showed stronger activity than *LRP5/6AB, which in turn was more active than *LRP5 (Figure 3D). Reciprocally, we observed signaling activities in the following rank order: LRP6.LRP6/5AB (which PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23537