Comparison of LRP5 and LRP6 Figure 5. The gap4 region between the most carboxyl terminal PPPSPxS motifs regulates LRP5 activity and PPPSPxS phosphorylation. (A and B) TOPFLASH reporter activity of LRP6, LRP6/5C and its gap1–4 insertional derivatives transfected alone (A) or co- transfected with Wnt1 (B). (C) PPPSPxS phosphorylation at motifs A, C, and E of LRP6, LRP6/5C and its gap1–4 insertional derivatives, as detected via phosphorylation-specific antibodies. Receptor protein levels were detected via the VSVG antibody. The 220 kDa molecular weight marker is visible in the first lane. (D) TOPFLASH reporter activity of *LRP5 and its gap1–4 insertional derivatives that were co-transfected with Wnt1. doi:10.1371/journal.pone.0023537.g005 functional difference between the cytoplasmic domain of these two Wnt receptors. Discussion The LRP5 and LRP6 genes likely arose from duplication of a single ancestral gene approximately 500 million years ago during the emergence of the chordate phylum. Since then LRP5 and LRP6 have retained a relatively high degree structural similarity and paralogous conservation, resulting in partially redundant functions as Wnt receptors in vertebrates. However since this early gene duplication, it is likely that LRP5 and LRP6 have independently evolved to the receptors they are today in the human genome. Genetic studies suggest that LRP6 has a more dominant role in Wnt signal transduction [3]. Consistent with the severity of the developmental phenotypes of Lrp5 and Lrp6 mutant mice [22,23], our comparison of the MEFs deficient for Lrp5 or Lrp6 indicates that a majority of Wnt/b-catenin signaling is mediated through Lrp6 (Figure 1). Moreover we were able to show a dose dependent correlation of Wnt responsiveness in MEFS containing two, one or no functioning Lrp6 alleles. Our data in MEFs in general are consistent with the previous genetic analyses of Lrp5 and Lrp6 compound mutant mice with different allelic combinations [22,23]. Further evidence of the critical importance of LRP6 is found through interspecies protein comparisons. The orthologous conservation of human LRP6 to mouse (98%), chicken (92%) and Xenopus (85%) Lrp6 is greater than human LRP5 to mouse (94%), chicken (88%) and Xenopus (80%) Lrp5 (Percentage numbers represent amino acid sequence identity based on BLAST2 comparisons, data not shown), indicating that LRP6 is more conserved during evolution whereas changes in LRP5 may be more tolerable to the organism. These observations may also in part explain the small number of identified human LRP6 mutations in the literature compared to those of LRP5 [19,38]. In this study we aimed to understand the molecular underpinning that accounts for different LRP5 and LRP6 signaling activities for two considerations. First, both LRP5 and LRP6 are key therapeutic targets for treatment of human diseases including cancer and/or osteoporosis; and secondly, LRP5 and LRP6 share five virtually identical orthologous PPPSPxS motifs, implying that other unknown mechanisms may operate to tune the Wnt receptor activities through these motifs. By generating a series of reciprocal chimeric receptors between LRP5 and LRP6, we found that difference between the cytoplasmic domain of LRP5 and LRP6 underlies, to a significant degree, the different signaling activities of these two receptors (Figure 2). Our previous comprehensive analysis has revealed that the five PPPSPxS motifs, from A to E (Figure S1B), contribute to LRP6 signaling in the rank order of C.D = E.A.B, with the carboxyl C, D, and E cluster making up most of the LRP6 signaling output [28]. Comparing LRP5 and LRP6, we found that motifs A plus B in LRP6 have stronger activity than the corresponding A and B pair in LRP5, while similarly the cluster of motifs C and D and E of LRP6 is more potent than that of LRP5 (Figure 3 and Figure S2). Most strikingly, our analysis identified between motifs D and E a short intervening region, termed gap4, which has significant and reciprocal effects on LRP5 and LRP6 activities. Thus LRP5 with an altered and ‘‘LRP6-like’’ PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23537