G protein-coupled receptors (GPCRs) adopt multiple conformational states that can activate or block distinct intracellular signalling pathways, such as those regulated by heterotrimeric G proteins or β-arrestins.
Different agonists for the same receptor can stabilize distinct GPCR conformational states. Agonists that preferentially activate certain intracellular pathways relative to others are referred to as biased agonists.
Structural studies support a model in which GPCRs act as allosteric microprocessors that integrate diverse extracellular and intracellular stimuli to generate distinct conformations that result in varied intracellular responses.
In addition to biased agonists, biased signalling may be encoded by the receptor ('receptor bias') or by the relative expression levels of transducers ('system bias').
Biased signalling is also observed in other receptor families, such as nuclear hormone receptors and receptor tyrosine kinases.
Recent preclinical and clinical work suggests that by more selectively targeting signalling pathways of interest, biased agonists have the potential to increase clinical efficacy while reducing undesirable side effects.
G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and some of the most common drug targets. It is now well established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, GPCR kinases and β-arrestins. While these signalling pathways can be activated or blocked by 'balanced' agonists or antagonists, they can also be selectively activated in a 'biased' response. Biased responses can be induced by biased ligands, biased receptors or system bias, any of which can result in preferential signalling through G proteins or β-arrestins. At many GPCRs, signalling events mediated by G proteins and β-arrestins have been shown to have distinct biochemical and physiological actions from one another, and an accurate evaluation of biased signalling from pharmacology through physiology is crucial for preclinical drug development. Recent structural studies have provided snapshots of GPCR–transducer complexes, which should aid in the structure-based design of novel biased therapies. Our understanding of GPCRs has evolved from that of two-state, on-and-off switches to that of multistate allosteric microprocessors, in which biased ligands transmit distinct structural information that is processed into distinct biological outputs. The development of biased ligands as therapeutics heralds an era of increased drug efficacy with reduced drug side effects.
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Lagerstrom, M. C. & Schioth, H. B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat. Rev. Drug Discov. 7, 339–357 (2008).
Luttrell, L. M., Maudsley, S. & Bohn, L. M. Fulfilling the promise of “biased” G protein-coupled receptor agonism. Mol. Pharmacol. 88, 579–588 (2015).
Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2017).
Smith, J. S. & Rajagopal, S. The β-arrestins: multifunctional regulators of G protein-coupled receptors. J. Biol Chem. 291, 8969–8977 (2016).
Benovic, J. L., Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. β-Adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc. Natl Acad. Sci. USA 83, 2797–2801 (1986).
Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G. & Lefkowitz, R. J. β-Arrestin: a protein that regulates β-adrenergic receptor function. Science 248, 1547–1550 (1990).
Goodman, O. B. Jr et al. β-Arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor. Nature 383, 447–450 (1996).
Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S. & Caron, M. G. Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J. Biol Chem. 274, 32248–32257 (1999).
Laporte, S. A., Oakley, R. H., Holt, J. A., Barak, L. S. & Caron, M. G. The interaction of β-arrestin with the AP-2 adaptor is required for the clustering of β2-adrenergic receptor into clathrin-coated pits. J. Biol Chem. 275, 23120–23126 (2000). References 7–9 are seminal papers describing the role of β-arrestin in GPCR endocytosis.
Hanyaloglu, A. C. & von Zastrow, M. Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu. Rev. Pharmacol. Toxicol. 48, 537–568 (2008).
Gao, H. et al. Identification of β-arrestin2 as a G protein-coupled receptor-stimulated regulator of NF-κB pathways. Mol. Cell 14, 303–317 (2004).
Shenoy, S. K. et al. β-Arrestin-dependent, G protein-independent ERK1/2 activation by the β2 adrenergic receptor. J. Biol Chem. 281, 1261–1273 (2006).
Beaulieu, J. M. et al. An Akt/β-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and behavior. Cell 122, 261–273 (2005).
Luttrell, L. M. et al. β-Arrestin-dependent formation of β2 adrenergic receptor-Src protein kinase complexes. Science 283, 655–661 (1999). This is one of the first studies describing the signalling functions of β-arrestins.
Ahn, S., Kim, J., Hara, M. R., Ren, X. R. & Lefkowitz, R. J. β-Arrestin-2 mediates anti-apoptotic signaling through regulation of BAD phosphorylation. J. Biol Chem. 284, 8855–8865 (2009).
Kendall, R. T. et al. Arrestin-dependent angiotensin AT1 receptor signaling regulates Akt and mTor-mediated protein synthesis. J. Biol Chem. 289, 26155–26166 (2014).
Eichel, K., Jullie, D. & von Zastrow, M. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat. Cell Biol. 18, 303–310 (2016).
Coffa, S., Breitman, M., Spiller, B. W. & Gurevich, V. V. A single mutation in arrestin-2 prevents ERK1/2 activation by reducing c-Raf1 binding. Biochemistry 50, 6951–6958 (2011).
Ferrandon, S. et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat. Chem. Biol. 5, 734–742 (2009).
Calebiro, D. et al. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol. 7, e1000172 (2009).
Irannejad, R. et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013). References 19–21 are impactful studies demonstrating sustained G protein signalling from endosomes following endocytosis regulated by β-arrestins.
Roth, B. L. & Chuang, D. M. Multiple mechanisms of serotonergic signal transduction. Life Sci. 41, 1051–1064 (1987).
Luttrell, L. M. Minireview: More than just a hammer: ligand “bias” and pharmaceutical discovery. Mol. Endocrinol. 28, 281–294 (2014).
Jarpe, M. B. et al. [D-Arg1,D-Phe5,D-Trp7,9,Leu11]Substance P acts as a biased agonist toward neuropeptide and chemokine receptors. J. Biol Chem. 273, 3097–3104 (1998).
Urban, J. D. et al. Functional selectivity and classical concepts of quantitative pharmacology. J. Pharmacol. Exp. Ther. 320, 1–13 (2007).
Violin, J. D. & Lefkowitz, R. J. β-Arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol Sci. 28, 416–422 (2007).
Spengler, D. et al. Differential signal transduction by five splice variants of the PACAP receptor. Nature 365, 170–175 (1993).
Kenakin, T. Agonist-receptor efficacy. II. Agonist trafficking of receptor signals. Trends Pharmacol Sci. 16, 232–238 (1995).
Gurwitz, D. et al. Discrete activation of transduction pathways associated with acetylcholine m1 receptor by several muscarinic ligands. Eur. J. Pharmacol. 267, 21–31 (1994).
Wei, H. et al. Independent β-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc. Natl Acad. Sci. USA 100, 10782–10787 (2003).
Whalen, E. J., Rajagopal, S. & Lefkowitz, R. J. Therapeutic potential of β-arrestin- and G protein-biased agonists. Trends Mol. Med. 17, 126–139 (2011).
Violin, J. D., Crombie, A. L., Soergel, D. G. & Lark, M. W. Biased ligands at G-protein-coupled receptors: promise and progress. Trends Pharmacol Sci. 35, 308–316 (2014).
Wisler, J. W., Xiao, K., Thomsen, A. R. & Lefkowitz, R. J. Recent developments in biased agonism. Curr. Opin. Cell Biol. 27, 18–24 (2014).
Rajagopal, S., Rajagopal, K. & Lefkowitz, R. J. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. Drug Discov. 9, 373–386 (2010).
Samama, P., Cotecchia, S., Costa, T. & Lefkowitz, R. J. A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model. J. Biol Chem. 268, 4625–4636 (1993).
Changeux, J. P. & Edelstein, S. J. Allosteric mechanisms of signal transduction. Science 308, 1424–1428 (2005).
Kenakin, T. P. Biased signalling and allosteric machines: new vistas and challenges for drug discovery. Br. J. Pharmacol. 165, 1659–1669 (2012).
De Lean, A., Stadel, J. M. & Lefkowitz, R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor. J. Biol Chem. 255, 7108–7117 (1980).
Gentry, P. R., Sexton, P. M. & Christopoulos, A. Novel allosteric modulators of G protein-coupled receptors. J. Biol Chem. 290, 19478–19488 (2015).
Onaran, H. O., Rajagopal, S. & Costa, T. What is biased efficacy? Defining the relationship between intrinsic efficacy and free energy coupling. Trends Pharmacol Sci. 35, 639–647 (2014).
Kenakin, T. Gaddum Memorial Lecture 2014: receptors as an evolving concept: from switches to biased microprocessors. Br. J. Pharmacol. 172, 4238–4253 (2015).
Galandrin, S., Oligny-Longpre, G. & Bouvier, M. The evasive nature of drug efficacy: implications for drug discovery. Trends Pharmacol Sci. 28, 423–430 (2007).
Wardell, S. E., Marks, J. R. & McDonnell, D. P. The turnover of estrogen receptor α by the selective estrogen receptor degrader (SERD) fulvestrant is a saturable process that is not required for antagonist efficacy. Biochem. Pharmacol. 82, 122–130 (2011).
Norris, J. D. et al. Peptide antagonists of the human estrogen receptor. Science 285, 744–746 (1999).
Paige, L. A. et al. Estrogen receptor (ER) modulators each induce distinct conformational changes in ERα and ERβ. Proc. Natl Acad. Sci. USA 96, 3999–4004 (1999).
Heldring, N. et al. Estrogen receptors: how do they signal and what are their targets. Physiol Rev. 87, 905–931 (2007).
Kovalenko, M. et al. Phosphorylation site-specific inhibition of platelet-derived growth factor β-receptor autophosphorylation by the receptor blocking tyrphostin AG1296. Biochemistry 36, 6260–6269 (1997).
Girnita, L. et al. β-Arrestin and Mdm2 mediate IGF-1 receptor-stimulated ERK activation and cell cycle progression. J. Biol Chem. 282, 11329–11338 (2007).
Arey, B. J. et al. Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms. Mol. Endocrinol. 11, 517–526 (1997).
Arey, B. J. et al. Differing pharmacological activities of thiazolidinone analogs at the FSH receptor. Biochem. Biophys. Res. Commun. 368, 723–728 (2008).
Yanofsky, S. D. et al. Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists. J. Biol Chem. 281, 13226–13233 (2006).
Kjelsberg, M. A., Cotecchia, S., Ostrowski, J., Caron, M. G. & Lefkowitz, R. J. Constitutive activation of the α1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J. Biol Chem. 267, 1430–1433 (1992).
Chen, X., Bai, B., Tian, Y., Du, H. & Chen, J. Identification of serine 348 on the apelin receptor as a novel regulatory phosphorylation site in apelin-13-induced G protein-independent biased signaling. J. Biol Chem. 289, 31173–31187 (2014).
Wanka, L. et al. C-Terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment. Cell. Signal. 29, 233–239 (2017).
Rajagopal, S. et al. β-Arrestin-but not G protein-mediated signaling by the “decoy” receptor CXCR7. Proc. Natl Acad. Sci. USA 107, 628–632 (2010).
Levoye, A., Balabanian, K., Baleux, F., Bachelerie, F. & Lagane, B. CXCR7 heterodimerizes with CXCR4 and regulates CXCL12-mediated G protein signaling. Blood 113, 6085–6093 (2009).
Decaillot, F. M. et al. CXCR7/CXCR4 heterodimer constitutively recruits β-arrestin to enhance cell migration. J. Biol Chem. 286, 32188–32197 (2011).
Wang, Y. et al. CXCR4 and CXCR7 have distinct functions in regulating interneuron migration. Neuron 69, 61–76 (2011).
Smith, J. S. et al. C-X-C motif chemokine receptor 3 splice variants differentially activate beta-arrestins to regulate downstream signaling pathways. Mol. Pharmacol. 92, 136–150 (2017).
Berchiche, Y. A. & Sakmar, T. P. CXC chemokine receptor 3 alternative splice variants selectively activate different signaling pathways. Mol. Pharmacol. 90, 483–495 (2016).
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).
Nakajima, K. & Wess, J. Design and functional characterization of a novel, arrestin-biased designer G protein-coupled receptor. Mol. Pharmacol. 82, 575–582 (2012).
Hu, J. et al. A G protein-biased designer G protein-coupled receptor useful for studying the physiological relevance of Gq/11-dependent signaling pathways. J. Biol Chem. 291, 7809–7820 (2016).
Siuda, E. R. et al. Optodynamic simulation of beta-adrenergic receptor signalling. Nat. Commun. 6, 8480 (2015).
Spangler, S. M. & Bruchas, M. R. Optogenetic approaches for dissecting neuromodulation and GPCR signaling in neural circuits. Curr. Opin. Pharmacol. 32, 56–70 (2017).
Reiter, E. & Lefkowitz, R. J. GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol. Metab. 17, 159–165 (2006).
Zidar, D. A., Violin, J. D., Whalen, E. J. & Lefkowitz, R. J. Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands. Proc. Natl Acad. Sci. USA 106, 9649–9654 (2009).
Riggs, B. L. & Hartmann, L. C. Selective estrogen-receptor modulators — mechanisms of action and application to clinical practice. N. Engl. J. Med. 348, 618–629 (2003).
Urs, N. M. et al. Distinct cortical and striatal actions of a β-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties. Proc. Natl Acad. Sci. USA 113, E8178–E8186 (2016). This paper presents a physiological example of how system bias can influence β-arrestin signalling.
Schattauer, S. S., Kuhar, J. R., Song, A. & Chavkin, C. Nalfurafine is a G-protein biased agonist having significantly greater bias at the human than rodent form of the kappa opioid receptor. Cell. Signal. 32, 59–65 (2017).
Shenoy, S. K. & Lefkowitz, R. J. β-Arrestin-mediated receptor trafficking and signal transduction. Trends Pharmacol Sci. 32, 521–533 (2011).
Kim, J. et al. Functional antagonism of different G protein-coupled receptor kinases for β-arrestin-mediated angiotensin II receptor signaling. Proc. Natl Acad. Sci. USA 102, 1442–1447 (2005).
Busillo, J. M. et al. Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling. J. Biol Chem. 285, 7805–7817 (2010).
Nobles, K. N. et al. Distinct phosphorylation sites on the β2-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin. Sci. Signal. 4, ra51 (2011).
Butcher, A. J. et al. Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code. J. Biol Chem. 286, 11506–11518 (2011). References 72–75 are some of the key papers demonstrating 'barcode' patterns, indicating how site-specific phosphorylation regulates GPCR signalling.
Poulin, B. et al. The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner. Proc. Natl Acad. Sci. USA 107, 9440–9445 (2010).
Luo, J., Busillo, J. M., Stumm, R. & Benovic, J. L. G. Protein-coupled receptor kinase 3 and protein kinase C phosphorylate the distal C-terminal tail of the chemokine receptor CXCR4 and mediate recruitment of β-arrestin. Mol. Pharmacol. 91, 554–566 (2017).
Shukla, A. K. et al. Distinct conformational changes in β-arrestin report biased agonism at seven-transmembrane receptors. Proc. Natl Acad. Sci. USA 105, 9988–9993 (2008).
Charest, P. G., Terrillon, S. & Bouvier, M. Monitoring agonist-promoted conformational changes of β-arrestin in living cells by intramolecular BRET. EMBO Rep. 6, 334–340 (2005).
Namkung, Y. et al. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET. Nat. Commun. 7, 12178 (2016).
Lee, M. H. et al. The conformational signature of β-arrestin2 predicts its trafficking and signalling functions. Nature 531, 665–668 (2016).
Nuber, S. et al. β-Arrestin biosensors reveal a rapid, receptor-dependent activation/deactivation cycle. Nature 531, 661–664 (2016). References 81 and 82 are studies that link β-arrestin conformation with function.
Hernandez, P. A. et al. Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat. Genet. 34, 70–74 (2003).
Balabanian, K. et al. WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12. Blood 105, 2449–2457 (2005).
Barak, L. S., Oakley, R. H., Laporte, S. A. & Caron, M. G. Constitutive arrestin-mediated desensitization of a human vasopressin receptor mutant associated with nephrogenic diabetes insipidus. Proc. Natl Acad. Sci. USA 98, 93–98 (2001).
Wootten, D. et al. The extracellular surface of the GLP-1 receptor is a molecular trigger for biased agonism. Cell 165, 1632–1643 (2016). This paper presents important mechanistic work on how receptor mutants can bias signalling responses.
Wootten, D. et al. Key interactions by conserved polar amino acids located at the transmembrane helical boundaries in Class B GPCRs modulate activation, effector specificity and biased signalling in the glucagon-like peptide-1 receptor. Biochem. Pharmacol. 118, 68–87 (2016).
Wootten, D. et al. A hydrogen-bonded polar network in the core of the glucagon-like peptide-1 receptor is a fulcrum for biased agonism: lessons from class B crystal structures. Mol. Pharmacol. 89, 335–347 (2016).
Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).
Venkatakrishnan, A. J. et al. Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region. Nature 536, 484–487 (2016).
Katritch, V., Cherezov, V. & Stevens, R. C. Diversity and modularity of G protein-coupled receptor structures. Trends Pharmacol Sci. 33, 17–27 (2012).
Zhang, H. et al. Structure of the Angiotensin receptor revealed by serial femtosecond crystallography. Cell 161, 833–844 (2015).
Zhang, H. et al. Structural basis for ligand recognition and functional selectivity at angiotensin receptor. J. Biol Chem. 290, 29127–29139 (2015).
Choe, H. W. et al. Crystal structure of metarhodopsin II. Nature 471, 651–655 (2011).
Standfuss, J. et al. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471, 656–660 (2011).
Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474, 521–525 (2011).
Jaakola, V. P. et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008).
Xu, F. et al. Structure of an agonist-bound human A2A adenosine receptor. Science 332, 322–327 (2011).
Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).
Manglik, A. et al. Crystal structure of the micro-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012).
Huang, W. et al. Structural insights into micro-opioid receptor activation. Nature 524, 315–321 (2015).
Zheng, Y. et al. Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540, 458–461 (2016).
Oswald, C. et al. Intracellular allosteric antagonism of the CCR9 receptor. Nature 540, 462–465 (2016).
Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013).
Jazayeri, A. et al. Extra-helical binding site of a glucagon receptor antagonist. Nature 533, 274–277 (2016).
Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).
Liang, Y. L. et al. Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118–123 (2017).
Zhang, Y. et al. Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248–253 (2017). References 107 and 108 are structural studies utilizing cryo-EM to solve GPCR structures in complex with G proteins. Cryo-EM technology will likely play an important role in providing critical structural insights into biased signalling.
Zhang, H. et al. Structural basis for selectivity and diversity in angiotensin II receptors. Nature 544, 327–332 (2017).
Burg, J. S. et al. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor. Science 347, 1113–1117 (2015).
Carpenter, B., Nehme, R., Warne, T., Leslie, A. G. & Tate, C. G. Structure of the adenosine A2A receptor bound to an engineered G protein. Nature 536, 104–107 (2016).
Staus, D. P. et al. Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature 535, 448–452 (2016).
Kim, Y. J. et al. Crystal structure of pre-activated arrestin p44. Nature 497, 142–146 (2013).
Kang, Y. et al. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523, 561–567 (2015).
Szczepek, M. et al. Crystal structure of a common GPCR-binding interface for G protein and arrestin. Nat. Commun. 5, 4801 (2014).
Renaud, J. P. et al. Biophysics in drug discovery: impact, challenges and opportunities. Nat. Rev. Drug Discov. 15, 679–698 (2016).
Westfield, G. H. et al. Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex. Proc. Natl Acad. Sci. USA 108, 16086–16091 (2011).
Shukla, A. K. et al. Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature 512, 218–222 (2014).
Kumari, P. et al. Functional competence of a partially engaged GPCR-β-arrestin complex. Nat. Commun. 7, 13416 (2016).
Cahill, T. J. et al. Distinct conformations of GPCR-β-arrestin complexes mediate desensitization, signaling, and endocytosis. Proc. Natl Acad. Sci. USA 114, 2562–2567 (2017).
Thomsen, A. R. et al. GPCR-G protein-β-arrestin super-complex mediates sustained G protein signaling. Cell 166, 907–919 (2016). This is a negative stain EM study that provides a potential structural basis for how β-arrestin and G proteins can orchestrate prolonged intracellular GPCR signalling.
Kahsai, A. W. et al. Multiple ligand-specific conformations of the β2-adrenergic receptor. Nat. Chem. Biol. 7, 692–700 (2011).
Isogai, S. et al. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature 530, 237–241 (2016).
Perez-Aguilar, J. M., Shan, J., LeVine, M. V., Khelashvili, G. & Weinstein, H. A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2. J. Am. Chem. Soc. 136, 16044–16054 (2014).
Kim, I. M. et al. β-Blockers alprenolol and carvedilol stimulate β-arrestin-mediated EGFR transactivation. Proc. Natl Acad. Sci. USA 105, 14555–14560 (2008).
Kim, I. M. et al. β-Arrestin1-biased β1-adrenergic receptor signaling regulates microRNA processing. Circ. Res. 114, 833–844 (2014).
Wisler, J. W. et al. A unique mechanism of β-blocker action: carvedilol stimulates β-arrestin signaling. Proc. Natl Acad. Sci. USA 104, 16657–16662 (2007).
Liu, J. J., Horst, R., Katritch, V., Stevens, R. C. & Wuthrich, K. Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335, 1106–1110 (2012).
Kahsai, A. W. et al. Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor. Nat. Chem. Biol. 12, 709–716 (2016).
Nygaard, R. et al. The dynamic process of β2-adrenergic receptor activation. Cell 152, 532–542 (2013).
Manglik, A. et al. Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell 161, 1101–1111 (2015).
Mary, S. et al. Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor. Proc. Natl Acad. Sci. USA 109, 8304–8309 (2012).
Wacker, D. et al. Structural features for functional selectivity at serotonin receptors. Science 340, 615–619 (2013).
Yang, F. et al. Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR. Nat. Commun. 6, 8202 (2015).
Ariens, E. J. Affinity and intrinsic activity in the theory of competitive inhibition. I. Problems and theory. Arch. Intern. Pharmacodynamie Therapie 99, 32–49 (1954).
Stephenson, R. P. A modification of receptor theory. Br. J. Pharmacol. Chemother. 11, 379–393 (1956).
Furchgott, R. F. in in Advances in Drug Research Vol. 3 (eds Harper, N. J. & Simmonds, A. B.) 21–55 (Academic Press, 1966).
Black, J. W. & Leff, P. Operational models of pharmacological agonism. Proc. R. Soc. Lond. B Biol Sci. 220, 141–162 (1983).
Rajagopal, S. et al. Quantifying ligand bias at seven-transmembrane receptors. Mol. Pharmacol. 80, 367–377 (2011).
Kenakin, T., Watson, C., Muniz-Medina, V., Christopoulos, A. & Novick, S. A simple method for quantifying functional selectivity and agonist bias. ACS Chem. Neurosci. 3, 193–203 (2012).
Griffin, M. T., Figueroa, K. W., Liller, S. & Ehlert, F. J. Estimation of agonist activity at G protein-coupled receptors: analysis of M2 muscarinic receptor signaling through Gi/o, Gs, and G15 . J. Pharmacol. Exp. Ther. 321, 1193–1207 (2007).
Snyder, J. C., Rochelle, L. K., Lyerly, H. K., Caron, M. G. & Barak, L. S. Constitutive internalization of the leucine-rich G protein-coupled receptor-5 (LGR5) to the trans-Golgi network. J. Biol Chem. 288, 10286–10297 (2013).
Onaran, H. O. et al. Systematic errors in detecting biased agonism: analysis of current methods and development of a new model-free approach. Sci. Rep. 7, 44247 (2017). This is a detailed comparison of different methods to quantify biased signalling.
Gregory, K. J., Hall, N. E., Tobin, A. B., Sexton, P. M. & Christopoulos, A. Identification of orthosteric and allosteric site mutations in M2 muscarinic acetylcholine receptors that contribute to ligand-selective signaling bias. J. Biol Chem. 285, 7459–7474 (2010).
Strachan, R. T. et al. Divergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR). J. Biol. Chem. 289, 14211–14224 (2014).
Moffat, J. G., Vincent, F., Lee, J. A., Eder, J. & Prunotto, M. Opportunities and challenges in phenotypic drug discovery: an industry perspective. Nat. Rev. Drug Discov. 16, 531–543 (2017).
Jones, L. H. & Bunnage, M. E. Applications of chemogenomic library screening in drug discovery. Nat. Rev. Drug Discov. 16, 285–296 (2017).
Horvath, P. et al. Screening out irrelevant cell-based models of disease. Nat. Rev. Drug Discov. 15, 751–769 (2016).
Swinney, D. C. & Anthony, J. How were new medicines discovered? Nat. Rev. Drug Discov. 10, 507–519 (2011).
Whistler, J. L., Chuang, H. H., Chu, P., Jan, L. Y. & von Zastrow, M. Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction. Neuron 23, 737–746 (1999).
Zhang, J. et al. Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness. Proc. Natl Acad. Sci. USA 95, 7157–7162 (1998).
Melief, E. J., Miyatake, M., Bruchas, M. R. & Chavkin, C. Ligand-directed c-Jun N-terminal kinase activation disrupts opioid receptor signaling. Proc. Natl Acad. Sci. USA 107, 11608–11613 (2010).
Bohn, L. M., Gainetdinov, R. R., Lin, F. T., Lefkowitz, R. J. & Caron, M. G. μ-Opioid receptor desensitization by β-arrestin-2 determines morphine tolerance but not dependence. Nature 408, 720–723 (2000).
Bohn, L. M. et al. Enhanced morphine analgesia in mice lacking β-arrestin 2. Science 286, 2495–2498 (1999).
Groer, C. E. et al. An opioid agonist that does not induce μ-opioid receptor-arrestin interactions or receptor internalization. Mol. Pharmacol. 71, 549–557 (2007).
Soergel, D. G. et al. Biased agonism of the μ-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers. Pain 155, 1829–1835 (2014).
Viscusi, E. R. et al. A randomized, phase 2 study investigating TRV130, a biased ligand of the μ-opioid receptor, for the intravenous treatment of acute pain. Pain 157, 264–272 (2016).
Manglik, A. et al. Structure-based discovery of opioid analgesics with reduced side effects. Nature 537, 185–190 (2016). This is a manuscript describing the design of a biased agonist, from a structure-based approach to physiological effects.
Land, B. B. et al. The dysphoric component of stress is encoded by activation of the dynorphin κ-opioid system. J. Neurosci. 28, 407–414 (2008).
White, K. L. & Roth, B. L. Psychotomimetic effects of kappa opioid receptor agonists. Biol Psychiatry 72, 797–798 (2012).
Bruchas, M. R. et al. Selective p38α MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. Neuron 71, 498–511 (2011).
Land, B. B. et al. Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking. Proc. Natl Acad. Sci. USA 106, 19168–19173 (2009).
Ehrich, J. M. et al. Kappa opioid receptor-induced aversion requires p38 MAPK activation in VTA dopamine neurons. J. Neurosci. 35, 12917–12931 (2015).
Brust, T. F. et al. Biased agonists of the kappa opioid receptor suppress pain and itch without causing sedation or dysphoria. Sci. Signal. 9, ra117 (2016).
White, K. L. et al. The G protein-biased κ-opioid receptor agonist RB-64 is analgesic with a unique spectrum of activities in vivo. J. Pharmacol. Exp. Ther. 352, 98–109 (2015).
Maillet, E. L. et al. Noribogaine is a G-protein biased κ-opioid receptor agonist. Neuropharmacology 99, 675–688 (2015).
White, K. L. et al. Identification of novel functionally selective κ-opioid receptor scaffolds. Mol. Pharmacol. 85, 83–90 (2014).
Rives, M. L., Rossillo, M., Liu-Chen, L. Y. & Javitch, J. A. 6′-Guanidinonaltrindole (6′-GNTI) is a G protein-biased κ-opioid receptor agonist that inhibits arrestin recruitment. J. Biol Chem. 287, 27050–27054 (2012).
Lovell, K. M. et al. Structure-activity relationship studies of functionally selective kappa opioid receptor agonists that modulate ERK 1/2 phosphorylation while preserving G protein over βarrestin2 signaling bias. ACS Chem. Neurosci. 6, 1411–1419 (2015).
Zhou, L. et al. Development of functionally selective, small molecule agonists at kappa opioid receptors. J. Biol Chem. 288, 36703–36716 (2013).
Melief, E. J. et al. Duration of action of a broad range of selective κ-opioid receptor antagonists is positively correlated with c-Jun N-terminal kinase-1 activation. Mol. Pharmacol. 80, 920–929 (2011).
Munro, T. A. et al. Long-acting kappa opioid antagonists nor-BNI, GNTI and JDTic: pharmacokinetics in mice and lipophilicity. BMC Pharmacol. 12, 5 (2012).
Carroll, F. I. & Carlezon, W. A. Jr. Development of kappa opioid receptor antagonists. J. Med. Chem. 56, 2178–2195 (2013).
Chavkin, C. & Koob, G. F. Dynorphin, dysphoria, and dependence: the stress of addiction. Neuropsychopharmacology 41, 373–374 (2016).
Van't Veer, A. & Carlezon, W. A. Jr. Role of κ-opioid receptors in stress and anxiety-related behavior. Psychopharmacology 229, 435–452 (2013).
Howes, O. D. & Kapur, S. The dopamine hypothesis of schizophrenia: version III — the final common pathway. Schizophr. Bull. 35, 549–562 (2009).
Free, R. B. et al. Discovery and characterization of a G protein-biased agonist that inhibits β-arrestin recruitment to the D2 dopamine receptor. Mol. Pharmacol. 86, 96–105 (2014).
Shonberg, J. et al. A structure-activity analysis of biased agonism at the dopamine D2 receptor. J. Med. Chem. 56, 9199–9221 (2013).
Chen, X. et al. Structure-functional selectivity relationship studies of β-arrestin-biased dopamine D2 receptor agonists. J. Med. Chem. 55, 7141–7153 (2012).
Allen, J. A. et al. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc. Natl Acad. Sci. USA 108, 18488–18493 (2011).
Park, S. M. et al. Effects of β-arrestin-biased dopamine D2 receptor ligands on schizophrenia-like behavior in hypoglutamatergic mice. Neuropsychopharmacology 41, 704–715 (2016).
Urs, N. M., Peterson, S. M. & Caron, M. G. New concepts in dopamine D2 receptor biased signaling and implications for schizophrenia therapy. Biol Psychiatry 81, 78–85 (2016).
Sexton, P. M., Findlay, D. M. & Martin, T. J. Calcitonin. Curr. Med. Chem. 6, 1067–1093 (1999).
Andreassen, K. V. et al. Prolonged calcitonin receptor signaling by salmon, but not human calcitonin, reveals ligand bias. PLoS ONE 9, e92042 (2014).
Furness, S. G. et al. Ligand-dependent modulation of G protein conformation alters drug efficacy. Cell 167, 739–749.e11 (2016).
Bachelerie, F. et al. International Union of Basic and Clinical Pharmacology. LXXXIX. Update on the extended family of chemokine receptors and introducing a new nomenclature for atypical chemokine receptors. Pharmacol Rev. 66, 1–79 (2014).
Steen, A., Larsen, O., Thiele, S. & Rosenkilde, M. M. Biased and G protein-independent signaling of chemokine receptors. Front. Immunol. 5, 277 (2014).
Muller, M., Carter, S., Hofer, M. J. & Campbell, I. L. Review: The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 in neuroimmunity — a tale of conflict and conundrum. Neuropathol. Appl. Neurobiol. 36, 368–387 (2010).
Groom, J. R. & Luster, A. D. CXCR3 ligands: redundant, collaborative and antagonistic functions. Immunol. Cell Biol. 89, 207–215 (2011).
Rashighi, M. et al. CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo. Sci. Transl Med. 6, 223ra23 (2014).
Rajagopal, S. et al. Biased agonism as a mechanism for differential signaling by chemokine receptors. J. Biol Chem. 288, 35039–35048 (2013).
Zohar, Y. et al. CXCL11-dependent induction of FOXP3-negative regulatory T cells suppresses autoimmune encephalomyelitis. J. Clin. Invest. 124, 2009–2022 (2014).
Kohout, T. A. et al. Differential desensitization, receptor phosphorylation, β-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7. J. Biol Chem. 279, 23214–23222 (2004).
Drury, L. J. et al. Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways. Proc. Natl Acad. Sci. USA 108, 17655–17660 (2011).
Corbisier, J., Gales, C., Huszagh, A., Parmentier, M. & Springael, J. Y. Biased signaling at chemokine receptors. J. Biol Chem. 290, 9542–9554 (2015).
Corbisier, J., Huszagh, A., Gales, C., Parmentier, M. & Springael, J. Y. Partial agonist and biased signaling properties of the synthetic enantiomers J113863/UCB35625 at chemokine receptors CCR2 and CCR5. J. Biol Chem. 292, 575–584 (2016).
Felker, G. M. et al. Heart failure therapeutics on the basis of a biased ligand of the angiotensin-2 type 1 receptor. Rationale and design of the BLAST-AHF study (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure). JACC Heart Fail. 3, 193–201 (2015).
Abraham, D. M. et al. β-Arrestin mediates the Frank-Starling mechanism of cardiac contractility. Proc. Natl Acad. Sci. USA 113, 14426–14431 (2016).
Liu, C. H. et al. Arrestin-biased AT1R agonism induces acute catecholamine secretion through TRPC3 coupling. Nat. Commun. 8, 14335 (2017).
Donato, M. & Gelpi, R. J. Adenosine and cardioprotection during reperfusion — an overview. Mol. Cell. Biochem. 251, 153–159 (2003).
Welihinda, A. A., Kaur, M., Greene, K., Zhai, Y. & Amento, E. P. The adenosine metabolite inosine is a functional agonist of the adenosine A2A receptor with a unique signaling bias. Cell. Signal. 28, 552–560 (2016).
Vecchio, E. A. et al. The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling. Biochem. Pharmacol. 117, 46–56 (2016).
Aurelio, L. et al. Allosteric modulators of the adenosine A1 receptor: synthesis and pharmacological evaluation of 4-substituted 2-amino-3-benzoylthiophenes. J. Med. Chem. 52, 4543–4547 (2009).
Valant, C. et al. Separation of on-target efficacy from adverse effects through rational design of a bitopic adenosine receptor agonist. Proc. Natl Acad. Sci. USA 111, 4614–4619 (2014).
Chuo, C. H. et al. VCP746, a novel A1 adenosine receptor biased agonist, reduces hypertrophy in a rat neonatal cardiac myocyte model. Clin. Exp. Pharmacol. Physiol. 43, 976–982 (2016).
Baltos, J. A. et al. Structure-activity analysis of biased agonism at the human adenosine A3 receptor. Mol. Pharmacol. 90, 12–22 (2016).
Barak, L. S. et al. ML314: a biased neurotensin receptor ligand for methamphetamine abuse. ACS Chem. Biol. 11, 1880–1890 (2016).
Peddibhotla, S. et al. Discovery of ML314, a brain penetrant non-peptidic β-arrestin biased agonist of the neurotensin NTR1 receptor. ACS Med. Chem. Lett. 4, 846–851 (2013).
Quoyer, J. et al. Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein. Proc. Natl Acad. Sci. USA 110, E5088–E5097 (2013).
Wacker, D. et al. Crystal structure of an LSD-bound human serotonin receptor. Cell 168, 377–389.e12 (2017).
Mallipeddi, S., Janero, D. R., Zvonok, N. & Makriyannis, A. Functional selectivity at G-protein coupled receptors: advancing cannabinoid receptors as drug targets. Biochem. Pharmacol. 128, 1–11 (2016).
Ahn, K. H., Mahmoud, M. M., Shim, J. Y. & Kendall, D. A. Distinct roles of β-arrestin 1 and β-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1). J. Biol Chem. 288, 9790–9800 (2013).
Fay, J. F. & Farrens, D. L. Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1. Proc. Natl Acad. Sci. USA 112, 8469–8474 (2015).
Mancini, A. D. et al. β-Arrestin recruitment and biased agonism at free fatty acid receptor 1. J. Biol Chem. 290, 21131–21140 (2015).
Christopoulos, A. Advances in G protein-coupled receptor allostery: from function to structure. Mol. Pharmacol. 86, 463–478 (2014).
Kenakin, T. & Christopoulos, A. Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat. Rev. Drug Discov. 12, 205–216 (2013).
Rajagopal, S. Quantifying biased agonism: understanding the links between affinity and efficacy. Nat. Rev. Drug Discov. 12, 483 (2013).
Kenakin, T. & Christopoulos, A. Measurements of ligand bias and functional affinity. Nat. Rev. Drug Discov. 12, 483 (2013).
Breton, B. et al. Multiplexing of multicolor bioluminescence resonance energy transfer. Biophys. J. 99, 4037–4046 (2010).
Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat. Struct. Mol. Biol. 22, 362–369 (2015).
Devost, D. et al. Conformational profiling of the AT1 angiotensin II receptor reflects biased agonism, G protein coupling, and cellular context. J. Biol Chem. 292, 5443–5456 (2017).
Barak, L. S., Ferguson, S. S., Zhang, J. & Caron, M. G. A β-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. J. Biol Chem. 272, 27497–27500 (1997).
Inoue, A. et al. TGFα shedding assay: an accurate and versatile method for detecting GPCR activation. Nat. Methods 9, 1021–1029 (2012).
The authors thank J. Silverman for helpful discussion on error propagation and for design and implementation of the online biased calculator resource. We thank T. Pack, M. Caron and C. Chavkin for comments on sections of the manuscript. This work is supported by NIH Grants T32GM7171 (J.S.S.), HL16037 (R.J.L.), HL114643 (S.R.) and GM122798 (S.R.); the Duke Medical Scientist Training Program (J.S.S.); a Burroughs Wellcome Career Award for Medical Scientists (S.R.). R.J.L. is an HHMI Investigator.
R.J.L. is a co-founder and shareholder of Trevena.
- G protein-coupled receptor kinases
(GRKs). A family of serine/threonine kinases that phosphorylate the intracellular residues of a G protein-coupled receptor following agonist binding, which is often required for β-arrestin recruitment and signalling.
Multifunctional adaptor proteins that regulate G protein-coupled receptor (GPCR) signalling through desensitization and internalization and by promoting signalling through a wide variety of pathways. They also regulate non-GPCR targets, such as receptor tyrosine kinases.
The ability of a ligand to generate a quantifiable response after binding to a receptor.
A measurement of how well a ligand binds to a receptor, commonly expressed in terms of a dissociation constant (Kd). Affinity depends on cellular context, and therefore affinity for a G protein-coupled receptor is influenced by transducers, such as G proteins and β-arrestins.
- Ligand bias
Biased signalling encoded in the ligand that generates a distinct ligand–receptor conformation relative to a reference ligand.
- Allosteric modulators
Ligands that bind to an allosteric site of the receptor and affect receptor responses to orthosteric ligands. Some allosteric modulators are capable of generating biased responses.
- Biased agonists
Ligands that selectively enhance or attenuate some, but not all, of the signalling pathways available to a receptor compared with a reference ligand (usually an endogenous agonist).
- Receptor bias
Biased signalling encoded by differences in receptor structure or conformation compared with the 'wild-type' receptor.
- System bias
Biased signalling directed by the relative expression of receptor transducers, such as increased expression of G proteins, G protein-coupled receptor kinases and/or β-arrestins.
- Orthosteric site
The site on a receptor to which the endogenous ligand binds.
- Allosteric site
A binding site on a receptor that is different from the orthosteric site.
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Smith, J., Lefkowitz, R. & Rajagopal, S. Biased signalling: from simple switches to allosteric microprocessors. Nat Rev Drug Discov 17, 243–260 (2018). https://doi.org/10.1038/nrd.2017.229
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