Resulting genomic sequences were aligned and compared with all available EnV sequences listed in the National Center for Biotechnology Information databank using the Basic Local Alignment Search Tool

family members that bind and mediate b-catenin activity in the nucleus, are highly associated with the risk of type 2 diabetes. Thus activation of the b-catenin signaling pathway by GLP1 peptide may Lypressin contribute to regulation of pancreatic islet cell proliferation. Given the similarity between GLP1R and GCGR, we speculate that glucagon-mediated activation of bcatenin signaling may play a similar role in regulating liver cell proliferation and regeneration. During development, Wnt/bcatenin signaling often occurs in a temporal and restricted manner, and overactivation of this pathway leads to cancer development. Thus, the relative weak signal may be beneficial to prevent cancer development, while still allowing for self-renewal of specific cellular populations. Overall, our results and others suggested that the function for Lrp5/6 is broader than being a coreceptor for the canonical Wnt signaling pathway and it can serve as a key scaffold molecule to participate in both b-catenin and cAMP/PKA signaling pathways. By engaging with several different GPCRs, it can regulate a range of important physiological functions including embryonic development, bone formation, and metabolism. Our results may help to explain the pleiotropic phenotypes of Lrp5 and 6 mutations. To better understand the physiological role of Lrp5/6 in metabolism, future studies with Lrp5/6 conditional knockout or mutant mice are needed. Materials and Methods Ethics statement All animal work has been carried out in accordance with US National Institutes of Health guidelines. All procedures utilized in this study were evaluating for animal welfare and ethics considerations and were approved by the VARI Institutional Animal Care and Use Committee prior to initiation. Glucagon Induced b-Catenin Signaling Pathway 8 Glucagon Induced b-Catenin Signaling Pathway area represents the background signal of 0.12 determined using Rlu-tagged Lrp5 with soluble YFP as noted. Coexpression of untagged GCGR or Lrp5 competitively reduced the BRET signals obtained between Lrp5-Rlu and GCGR-YFP. Saturation BRET analysis supported the specific interaction of Lrp5 and GCGR. The non-specific bystander type BRET signal was observed when the non-structurally-related CCK1 receptors were co-expressed with Lrp5. Incubation with the natural agonist peptide ligand, glucagon, did not significantly change the Lrp5 and GCGR BRET signal. Data are represented as the means 6 S.E.M. from four to six independent experiments performed in triplicate. Data marked with were significantly different from background signals at the level of p,0.01. doi:10.1371/journal.pone.0033676.g007 Reagents LiCl was purchased from Sigma Aldrich. The antibodies used were mouse anti-b-catenin monoclonal antibody, mouse anti-b-actin antibody, mouse anti-v5 antibody and v5 antibody-conjugated agarose beads, rabbit anti-GCGR antibody , and mouse anti-HA antibody. Forskolin and H89 were obtained from Calbiochem. A Dual Luciferase assay kit was purchased from Promega. GLP-1 peptides, glucagon peptides, and PTH peptides were purchased ” from peptide 2.0. Dickkopf-1 protein was purchased from R&D systems. technology) and exposed to Kodak X-O mat films, which were scanned on an HP ScanJet flat-bed scanner. Alternatively, the blots were exposed by using a Biorad Quantity One ” Gel Box. For reprobing, membranes were stripped using a solution containing 62.5 mM Tris-HCl, 2% SDS, and 100 mM b-mercaptoethanol at 62uC for 45 min. Immunoprecipitation HEK293