Glucagon Induced b-Catenin Signaling Pathway Figure 5. Glucagon-induced b-catenin signaling is dependent on PKA activity. A). 293STF cells cultured in 24-well plate were transfected with 100 ng GCGR and 5 ng TKRlu plasmids on day 1 and then treated with GCG1-29 (50 nM), GCG9-29 (50 nM), or GCG1-29 (50 nM) and PKA inhibitor H89 (10 mM) 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 group. #p,0.005 compared with GCG1-29-treated group. B). 293STF cells cultured in 24-well plate were transfected with 100 ng GCGR, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with GCG1-29, H89, or both on day 2. Cells were harvested on day 3 to measure the TCF-mediated luciferase activity. Duplicate samples were used for each treatment. *p,0.02 compared with the non-treated (NT) group. #p,0.005 compared with GCG1-29-treated group. doi:10.1371/journal.pone.0033676.g005 independent of Lrp5/6 [15]. However, this conclusion is based on lower expression levels of Lrp5/6 in Chinese Hamster Ovary (CHO) cells and could reflect cell-type specific differences. An involvement of Lrp6 for PTH [14] and of Lrp5/6 for glucagon mediated b-catenin signaling may indicate that there is a common mechanism of signaling for some class B GPCRs including PTH1R, GLP-1R, and GCGR. Indeed, cotransfection of Lrp5 can also enhance GLP-1 peptide–mediated cross-talk to b-catenin signaling (Fig. 4C). Using immunoprecipitation, we found that Lrp5/6 physically interacted with GCGR (Fig. 7A–B, S1). Using BRET assay, we further confirmed that ectopic expressed GCGR and Lrp5 do interact specifically on the cell surface (Fig. 7 C–E). We found that this interaction is ligand-independent in both experiments, which is somewhat different from previous report which showed that PTH1R interacts with Lrp5 in a ligand-independent manner, but with Lrp6 in a ligand-dependent manner [14]. This difference may be due to different receptors, cellular contexts or experimen- tal conditions. Considering that PTH1R and GCGR are different receptors that can interact with Lrp5/6, one model is that these interactions may occur via a common adaptor protein to which GCGR, PTH1R, and Lrp5/6 all can bind, e.g. a G-protein complex. Our data suggested that association of GCGR and Lrp5 alone is not sufficient for activation of the downstream b-catenin pathway. In addition, ligand binding is required, presumably through inducing conformational changes of GCGR and phosphorylation of Lrp5/6 to activate the downstream b-catenin pathway. However, pre-association of GCGR with Lrp5/6 on the cell surface can greatly facilitate the signaling communications PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33676