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 “20008854 target=’resource_window’> 22948146 signaling communications Glucagon Induced b-Catenin Signaling Pathway LY341495 chemical information 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 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 Lrp5/6, but may be intrinsic to GCGR itself. In Frizzled receptors, two residues in the intracellular loops 1 and 3 and a motif in the C-terminal tail play an important role in Dishevelled protein recruitment and Wnt/bcatenin signaling. Sequence analysis indicated that GCGR lacks these key residues of Frizzled receptors in its intracellular loops. The C-terminal motif is not completely conserved in GCGR. Note that for PTH1R, this motif is better conserved, 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/bcatenin 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. 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. Thus activation of the b-catenin signaling pathway by GLP1 peptide may 
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 devel