ced b-catenin stabilization and b-catenin-mediated transcriptional activity. Second, inhibiting Lrp5/6 function by a dominant negative construct or a functional inhibitor blocked glucagon-mediated b-catenin signaling. One study showed that PTH1R activated b-catenin signaling is Glucagon Induced b-Catenin Signaling Pathway independent of Lrp5/6. However, this conclusion is based on lower expression levels of Lrp5/6 in Chinese Hamster Ovary cells and could reflect cell-type specific differences. An involvement of Lrp6 for PTH 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 peptidemediated cross-talk to b-catenin signaling. Using immunoprecipitation, we found that Lrp5/6 physically interacted with GCGR. Using BRET assay, we further confirmed that ectopic expressed GCGR and Lrp5 do interact specifically on the cell surface. 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. This difference may be due to different receptors, cellular contexts or experimental conditions. Considering that PTH1R and GCGR are different receptors that can interact with Lrp5/6, one model is that these MedChemExpress INK1117 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 22694778 b-catenin pathway. However, pre-association of GCGR with Lrp5/6 on the cell surface can greatly facilitate the signaling communications Glucagon Induced b-Catenin Signaling Pathway 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 but also enhanced glucagon induced cAMP/PKA signaling. It is also consistent with recent studies with PTH1R showing that Lrp6 is not only required for PTH mediated b-catenin signaling 
pathway, but also promotes cAMP/PKA signaling. We found that glucagoninduced b-catenin signaling was dependent on PKA activity, which is consistent with other reports for class B GPCRs such as PTH1R and GLP-1R and suggests that the b-catenin pathway and cAMP/PKA pathway are interconnected. This is different from Wnt proteininduced b-catenin pathway, which does not require PKA activity. Interestingly, treatment of GCGR and 9353797 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. 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 Lr