title: Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR).
abstract: The concept of "biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR.
authors.affiliation: From the Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Strachan
authors.affiliation: Key Laboratory of Experimental Teratology of the Ministry of Education, Department of Biochemistry and Molecular Biology, Shandong University, School of Medicine, Jinan, Shandong 250012, China.
authors.forename: Jin-peng
authors.initials: JP
authors.lastname: Sun
authors.affiliation: Trevena Inc., King of Prussia, Pennsylvania 19406.
authors.forename: David H
authors.initials: DH
authors.lastname: Rominger
authors.affiliation: Trevena Inc., King of Prussia, Pennsylvania 19406.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Violin
authors.affiliation: From the Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710.
authors.forename: Seungkirl
authors.initials: S
authors.lastname: Ahn
authors.affiliation: From the Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710.
authors.forename: Alex
authors.initials: A
authors.lastname: Rojas Bie Thomsen
authors.affiliation: From the Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhu
authors.affiliation: From the Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710.
authors.forename: Andrew
authors.initials: A
authors.lastname: Kleist
authors.affiliation: Dipartimento del Farmaco, Istituto Superiore di Sanita, 00161 Rome, Italy, tommaso.costa@iss.it.
authors.forename: Tommaso
authors.initials: T
authors.lastname: Costa
authors.affiliation: From the Department of Medicine, Duke University, Medical Center, Durham, North Carolina 27710, Department of Biochemistry, Duke University, Medical Center, Durham, North Carolina 27710, and Howard Hughes Medical Institute, Duke University, Medical Center, Durham, North Carolina 27710 lefko001@receptor-biol.duke.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000494:Allosteric Regulation; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D044140:Receptor, Angiotensin, Type 1 / Q000819:agonists* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013816:Thermodynamics
chemical_list: D008024:Ligands; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins
keywords: Angiotensin II; Drug Discovery; G Protein-coupled Receptors (GPCR); Molecular Pharmacology; Signal Transduction