Aaron Novikoff1, Shannon L O'Brien2, Miriam Bernecker3, Gerald Grandl3, Maximilian Kleinert3, Patrick J Knerr4, Kerstin Stemmer3, Martin Klingenspor5, Anja Zeigerer6, Richard DiMarchi7, Matthias H Tschöp8, Brian Finan4, Davide Calebiro9, Timo D Müller10. 1. Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany; Division of Metabolic Diseases, Department of Medicine, Technische Universität München, D-80333 Munich, Germany. 2. Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, B15 2TT, UK; Center of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, Birmingham, B15 2TT, UK. 3. Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany. 4. Novo Nordisk Research Center Indianapolis, Indianapolis, IN 46241, USA. 5. Chair for Molecular Nutritional Medicine, School of Life Sciences, Technical University of Munich, 85354 Freising, Germany. 6. German Center for Diabetes Research (DZD), Neuherberg, Germany; Institute for Diabetes and Cancer, Helmholtz Center Munich, 85764 Neuherberg, Germany; Joint Heidelberg-IDC Translational Diabetes Program, Inner Medicine 1, Heidelberg University Hospital, Heidelberg, Germany. 7. Department of Chemistry, Indiana University, Bloomington, IN 47405, USA. 8. German Center for Diabetes Research (DZD), Neuherberg, Germany; Division of Metabolic Diseases, Department of Medicine, Technische Universität München, D-80333 Munich, Germany; Helmholtz Zentrum München, Neuherberg, Germany; Technische Universität München, München, Germany. 9. Institute of Metabolism and Systems Research, University of Birmingham, Birmingham, B15 2TT, UK; Center of Membrane Proteins and Receptors (COMPARE), Universities of Nottingham and Birmingham, Birmingham, B15 2TT, UK. Electronic address: D.Calebiro@bham.ac.uk. 10. Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany; Department of Pharmacology and Experimental Therapy, Institute of Experimental and Clinical Pharmacology and Toxicology, Eberhard Karls University Hospitals and Clinics, 72076 Tübingen, Germany. Electronic address: timo.mueller@helmholtz-muenchen.de.
Abstract
OBJECTIVE: We assessed the spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide. METHODS: Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live-cell HILO microscopy. RESULTS: Relative to native and acylated GLP-1 agonists, MAR709 and tirzepatide showed preserved maximal cAMP production despite partial Gαs recruitment paralleled by diminished ligand-induced receptor internalization at both target receptors. Despite MAR709's lower internalization rate, GLP-1R co-localization with Rab11-associated recycling endosomes was not different between MAR709 and GLP-1R specific mono-agonists. CONCLUSIONS: Our data indicated that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics relative to native peptides, semaglutide, and matched mono-agonist controls. These findings support the hypothesis that the structure of GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking.
OBJECTIVE: We assessed the spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide. METHODS: Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live-cell HILO microscopy. RESULTS: Relative to native and acylated GLP-1 agonists, MAR709 and tirzepatide showed preserved maximal cAMP production despite partial Gαs recruitment paralleled by diminished ligand-induced receptor internalization at both target receptors. Despite MAR709's lower internalization rate, GLP-1R co-localization with Rab11-associated recycling endosomes was not different between MAR709 and GLP-1R specific mono-agonists. CONCLUSIONS: Our data indicated that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics relative to native peptides, semaglutide, and matched mono-agonist controls. These findings support the hypothesis that the structure of GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking.
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