title: The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity.
abstract: Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production by modulating cAMP accumulation and genes that control gluconeogenesis. These interactors may be useful targets to control glucose homeostasis in T2D.
authors.affiliation: Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada; Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
authors.forename: Junfeng
authors.initials: J
authors.lastname: Han
authors.affiliation: Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Sean
authors.initials: S
authors.lastname: Froese
authors.affiliation: Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Feihan F
authors.initials: FF
authors.lastname: Dai
authors.affiliation: Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, M5S1A8, Canada.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Robitaille
authors.affiliation: Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Alpana
authors.initials: A
authors.lastname: Bhattacharjee
authors.affiliation: Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
authors.forename: Weiping
authors.initials: W
authors.lastname: Jia
authors.affiliation: Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, M5S1A8, Canada.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Angers
authors.affiliation: Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Wheeler
authors.affiliation: Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
authors.forename: Li
authors.initials: L
authors.lastname: Wei
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins; D002460:Cell Line; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D005786:Gene Expression Regulation; D005943:Gluconeogenesis / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods; D043562:Receptors, G-Protein-Coupled; D018027:Receptors, Glucagon / Q000819:agonists* / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D002352:Carrier Proteins; D043562:Receptors, G-Protein-Coupled; D018027:Receptors, Glucagon; D000242:Cyclic AMP; D005947:Glucose