Figure 6. Blocking Lrp5/6 inhibited glucagon-induced b- catenin signaling. A). 293STF cells cultured in 24-well plate were transfected with a combination of GCGR (100 ng), Lrp5 (100 ng), Lrp5ECD (200 ng) and TKRlu (5 ng) plasmids as indicated on day 1, and then were treated with or without 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the TCF-mediated luciferase activity. Triplicate samples were used for each treatment. *p,0.005 compared with the non-treated (NT) group. #p,0.005 compared with the group without the Lrp5ECD transfection. B). DKK1 protein inhibits glucagon- induced b-catenin signaling. 293STF cells cultured in 24-well plate were transfected with pcDNA3.1 and GCGR plasmids (100 ng each) or GCGR and Lrp5 plasmids (100 ng each) along with 5 ng TKRlu plasmid on day 1, and then were treated with 50 nM GCG1-2962 mg/ml DKK1 on day 2. Cells were harvested on day 3 to measure the luciferase activity. Duplicate samples were used for each treatment. *p,0.02 compared with the GCG1-29-treated group. doi:10.1371/journal.pone.0033676.g006 between GCGR and Lrp5/6. So activation of GCGR upon ligand binding can directly cross-talk to Lrp5/6 to transmit downstream b-catenin signaling whereas phosphorylation and activation of Lrp5/6 on the other hand can communicate back to GCGR to boost GCGR mediated cAMP/PKA pathway. This mutual communication is supported by our cell-based reporter data showing that cotransfection of Lrp5 not only enhanced glucagon induced b-catenin signaling (Fig. 4) but also enhanced glucagon induced cAMP/PKA signaling (Fig. S2). It is also consistent with recent studies with PTH1R showing that Lrp6 is not only required for PTH mediated b-catenin signaling pathway [14], but also promotes cAMP/PKA signaling [19]. We found that glucagon- induced b-catenin signaling was dependent on PKA activity (Fig. 5), which is consistent with other reports for class B GPCRs such as PTH1R [10,11,12,14] and GLP-1R [13] and suggests that Glucagon Induced b-Catenin Signaling Pathway the b-catenin pathway and cAMP/PKA pathway are intercon- nected. This is different from Wnt protein–induced b-catenin pathway, which does not require PKA activity [14]. Interestingly, treatment of GCGR and Lrp5 expressing cells with glucagon and Wnt3a conditioned media had a synergistic effect on the b-catenin signaling pathway, suggesting that the cAMP/PKA pathway and the b-catenin pathway reinforce each other (Fig. S4). Glucagon-induced b-catenin signaling is relatively weaker than Wnt protein-induced b-catenin signaling. The relative weak signal was not due to lack of interaction between GCGR and Lrp5/6, but may be intrinsic to GCGR itself. In Frizzled receptors, two residues in the intracellular loops 1 and 3 (Arg263 and Leu443 in human Fz5) and a motif in the C-terminal tail (KTXXXW) play an important role in Dishevelled protein recruitment and Wnt/b- catenin signaling [25,26]. Sequence analysis indicated that GCGR lacks these key residues of Frizzled receptors in its intracellular loops (Fig. S3). The C-terminal motif is not completely conserved in GCGR (RRXXXW in GCGR, Fig. S3). Note that for PTH1R, this motif is better conserved (KSXXXW), which may allow better binding to Dishevelled and more robust b-catenin signaling for PTH1R. What is the physiological consequence of cross-talk to b-catenin signaling from GCGR? Wnt/b-catenin signaling helps to promote stem cell renewal and in many cases favors proliferation over differentiation. Several lines of evidence suggest that Wnt/b- catenin signaling may help pancreatic cells survive and proliferate. First, Wnt/b-catenin signaling is involved in the genesis of pancreatic islets and the proliferation of pancreatic beta cells [27]. Second, polymorphisms in the TCF7L2 gene, one of the LEF/TCF family members that bind and mediate b-catenin activity in the nucleus, are highly associated with the risk of type 2 diabetes [28]. Thus activation of the b-catenin signaling pathway by GLP1 peptide may contribute to regulation of pancreatic islet cell proliferation [13]. Given the similarity between GLP1R and GCGR, we speculate that glucagon-mediated activation of b- catenin signaling may play a similar role in regulating liver cell proliferation and regeneration. During development, Wnt/b- catenin 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 devel- opment, 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 (VARI-IACUC) prior to initiation (VAI-IACUC protocol ID #09-10-025). PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33676