The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

• Published in: The Journal of biological chemistry 2013-08, Vol.288 (32), p.22942-22960
• Main Authors: Jensen, Dane D., Godfrey, Cody B., Niklas, Christian, Canals, Meritxell, Kocan, Martina, Poole, Daniel P., Murphy, Jane E., Alemi, Farzad, Cottrell, Graeme S., Korbmacher, Christoph, Lambert, Nevin A., Bunnett, Nigel W., Corvera, Carlos U.
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - pharmacology; Arrestin; Arrestins - antagonists & inhibitors; Arrestins - genetics; Arrestins - metabolism; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins; beta-Cyclodextrins - pharmacology; Bile Acid; Cholagogues and Choleretics - pharmacology; Cyclic AMP - genetics; Cyclic AMP - metabolism; Deoxycholic Acid - pharmacology; Endocytosis; Endocytosis - drug effects; Endocytosis - physiology; Endosomes - genetics; Endosomes - metabolism; Enzyme Inhibitors - pharmacology; ErbB Receptors - antagonists & inhibitors; ErbB Receptors - genetics; ErbB Receptors - metabolism; G Protein-coupled Receptors (GPCR); G-Protein-Coupled Receptor Kinase 2 - genetics; G-Protein-Coupled Receptor Kinase 2 - metabolism; G-Protein-Coupled Receptor Kinase 5 - genetics; G-Protein-Coupled Receptor Kinase 5 - metabolism; HEK293 Cells; Humans; Lipid Raft; Membrane Microdomains - genetics; Membrane Microdomains - metabolism; Mitogen-Activated Protein Kinase 1 - genetics; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - genetics; Mitogen-Activated Protein Kinase 3 - metabolism; Oleanolic Acid - pharmacology; Phenylalanine - analogs & derivatives; Phenylalanine - pharmacology; Protein Transport - drug effects; Protein Transport - physiology; Quinazolines - pharmacology; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Signal Transduction; Thiophenes - pharmacology; Tyrphostins - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.455774

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists. Background: The TGR5 bile acid receptor controls energy balance, inflammation, and digestion, but TGR5 signaling is poorly understood. Results: TGR5 does not interact with β-arrestins, internalize, or desensitize, but signals from plasma membrane rafts. Conclusion: TGR5 transmits sustained signals close to the cell surface. Significance: Understanding TGR5 signaling will facilitate design of TGR5 agonists for metabolic, inflammatory, and digestive disorders.