Modulation of b-Catenin Signaling by Glucagon Receptor Activation Jiyuan Ke1*., Chenghai Zhang1., Kaleeckal G. Harikumar4, Cassandra R. Zylstra-Diegel2, Liren Wang 1 , Laura E. Mowry2, Laurence J. Miller4, Bart O. Williams 2 , H. Eric Xu1,3* 1 Laboratory of Structural Sciences, Van Andel Research Institute, Grand Rapids, Michigan, United States of America, 2 Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, Michigan, United States of America, 3 VARI/SIMM Center, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People’s Republic of China, 4 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona, United States of America Abstract The glucagon receptor (GCGR) is a member of the class B G protein–coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced b-catenin stabilization and activated b-catenin–mediated transcription. Activation of b-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor–related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated b-catenin signaling, we examined the role of Lrp5 in glucagon-induced b-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced b-catenin stabilization and TCF promoter–mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced b-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and b-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations. Citation: Ke J, Zhang C, Harikumar KG, Zylstra-Diegel CR, Wang L, et al. (2012) Modulation of b-Catenin Signaling by Glucagon Receptor Activation. PLoS ONE 7(3): e33676. doi:10.1371/journal.pone.0033676 Editor: Cara Gottardi, Northwestern University Feinberg School of Medicine, United States of America Received October 10, 2011; Accepted February 14, 2012; Published March 16, 2012 Copyright: 2012 Ke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants R21RR024887 to BOW, R01DK46577 to LJM, and 5R01GM087413 to HEX. This work was also supported in part by the Jay and Betty Van Andel Foundation. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jiyuan.ke@vai.org (JK); eric.xu@vai.org (HEX) . These authors contributed equally to this work. Introduction G protein–coupled receptors (GPCRs) with seven-transmem- brane domains form a large family that respond to extracellular signals by activating heterotrimeric G proteins. The glucagon receptor (GCGR) is a class B GPCR. Its ligand, glucagon, is a 29- amino acid peptide secreted by the islet A cells of the endocrine pancreas. The binding of glucagon to its receptor activates the cAMP/protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) pathways [1,2]. The major action of glucagon is to increase glucose production from the liver by stimulating glycogenolysis and gluconeogenesis. Together with insulin, glucagon is an important regulator of glucose homeostasis. Frizzled (Fz) receptors, which are known as atypical GPCRs, are receptors for the Wnt family of secreted glycoproteins [3]. The binding of Wnt ligands to Fz receptors activates either canonical or noncanonical Wnt pathways depending on the cellular context [4]; these are distinct from classical GPCR signaling pathways. The Wnt/b-catenin pathway is initiated by Wnt protein binding simultaneously to a Fz receptor and its co-receptor low-density- lipoprotein receptor–related protein 5/6 (Lrp5/6), causing dis- ruption of the destruction complex which normally targets b- catenin for ubiquitin-dependent proteasomal degradation. This ultimately results in accumulation of b-catenin in the cytosol, which can then translocate into the nucleus to activate Wnt target gene expression with TCF transcription factors [5]. The canonical Wnt pathway plays an essential role in many stages of development, in stem cell renewal, and in tissue homeostasis [3,6,7]. Lrp5 and Lrp6 belong to a subfamily of low-density- lipoprotein receptor–related proteins that are indispensable components of the canonical Wnt signaling pathway [5]. A number of classical GPCRs have been shown to cross-talk or activate the b-catenin pathway in a Wnt-independent manner by various mechanisms. The stimulation of a-adrenergic receptors on cardiomyocytes (acting through the heterotrimeric G alpha subunit, Gq), or prostaglandin E2 receptors on colon cancer cells (acting through the G alpha subunit, Gs) results in the stabilization of b-catenin and the activation of b-catenin signaling [8,9]. Activation of parathyroid hormone receptor type 1 (PTH1R) increases b-catenin levels and b-catenin-mediated transcription through cAMP/PKA-dependent inactivation of glycogen synthase kinase 3b (GSK-3b) in UMR106 [10] and Saos-2 mouse osteoblastic cells [11], and through both PKA and PKC PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33676