Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor Sean M. Petersona, Thomas F. Packa, Angela D. Wilkinsb, Nikhil M. Ursa, Daniel J. Urbana, Caroline E. Bassc, Olivier Lichtargeb, and Marc G. Carona,d,e,1 Departments of aCell Biology, dMedicine, and eNeurobiology, Duke University School of Medicine, Durham, NC 27710; bDepartment of Molecular and Human Genetics and Computational and Integrative Biomedical Research Center, Baylor College of Medicine, Houston, TX 77030; and cDepartment of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14214 Edited by Leslie Lars Iversen, University of Oxford, Oxford, United Kingdom, and approved April 20, 2015 (received for review February 9, 2015) The neuromodulator dopamine signals through the dopamine D2 receptors represent the largest targets of drugs on the market. receptor (D2R) to modulate central nervous system functions However, determining the functional contributions of G-protein through diverse signal transduction pathways. D2R is a prominent and β-arrestin signaling pathways to the biological actions of an target for drug treatments in disorders where dopamine function endogenous ligand acting upon its receptor still remains a chal- is aberrant, such as schizophrenia. D2R signals through distinct lenging undertaking. G-protein and β-arrestin pathways, and drugs that are functionally Dopamine (DA) is a neuromodulator that is known to regulate selective for these pathways could have improved therapeutic po- movement, reward, cognition, emotion, and affect. The dopamine tential. How D2R signals through the two pathways is still not well D2 receptor (D2R) is a prominent GPCR that mediates the ac- defined, and efforts to elucidate these pathways have been ham- tions of DA. All typical antipsychotics, such as haloperidol, are pered by the lack of adequate tools for assessing the contribution potent D2R blockers (5), whereas atypical antipsychotics, such as of each pathway independently. To address this, Evolutionary aripiprazole and clozapine, have unique pharmacology, exhibiting Trace was used to produce D2R mutants with strongly biased sig- weak partial agonist activity at D2R or reduced antagonist efficacy, nal transduction for either the G-protein or β-arrestin interactions. respectively (6). Previous studies have demonstrated the ability of These mutants were used to resolve the role of G proteins and D Rs to engage different signal transduction pathways depending β 2 -arrestins in D2R signaling assays. The results show that D2R in- on the cellular complement of G proteins as well as their ability PHARMACOLOGY teractions with the two downstream effectors are dissociable and to regulate different physiological processes (7–9). β-arrestin 2 that G-protein signaling accounts for D2R canonical MAP kinase knockout mice provided robust behavioral and biochemical evi- signaling cascade activation, whereas β-arrestin only activates el- dence for a critical D2R/β-arrestin signaling pathway in the stria- ements of this cascade under certain conditions. Nevertheless, tum (10). Furthermore, neuronal selective deletion of GSK3β,a when expressed in mice in GABAergic medium spiny neurons of putative D2R/β-arrestin 2 effector, could reproduce the pharma- the striatum, the β-arrestin–biased D2R caused a significant poten- cological blockade of D2Rs with antipsychotics (11). Although tiation of amphetamine-induced locomotion, whereas the G protein- thesestudiessuggestthatD2Rs, like many other GPCRs, use biased D2R had minimal effects. The mutant receptors generated pleiotropic signaling pathways to mediate their effects, the brain here provide a molecular tool set that should enable a better DA system is uniquely complex, as diverse responses may also rely β definition of the individual roles of G-protein and -arrestin sig- upon many other determinants. One well-documented variable is naling pathways in D2R pharmacology, neurobiology, and the mode of stimulation of DA receptors, which is a function of associated pathologies. the tonic or phasic release of DA (12). The expression profile of D2R is also complex, being expressed not only in DA synthesizing dopamine | GPCR | functional selectivity | G protein | β-arrestin Significance protein-coupled receptors (GPCRs) are the largest receptor family and transmit the physiological effects of numerous G The dopamine D2 receptor (D R), a G protein-coupled receptor, biologically active molecules. GPCR signal transduction cascades 2 can initiate signaling events through both activation of G account for diverse genomic, biochemical, cellular, and behav- proteins and interactions with β-arrestins. To begin to un- ioral responses including cell fate determination, developmental derstand the contribution of these events in the physiology of reprogramming, olfactory, taste and light sensation, as well as the dopamine system, D2R was mutated to be functionally complex behaviors mediated by neuromodulators (1). The di- selective for each signaling pathway. The engineered receptors versity of responses to a particular hormone or neuromodulator is are functional in vitro and in vivo. Furthermore, both functions dictated not only by its cognate receptor but also by the ability of are essentially dissociable and mediate different physiological that receptor to engage distinct signaling pathways. For a number and pharmacological responses. These tools provide an addi- of GPCRs, their propensity to activate distinct G proteins can tional but previously unavailable approach to elucidate the role elicit diverse responses depending on the cellular environment (2). of biased signaling in the multiple physiological actions of the However, an even more subtle but intriguing mode of signaling dopamine system. has been attributed to the ability of a receptor to activate signaling pathways independent of G-protein activation, through the scaf- Author contributions: S.M.P., T.F.P., A.D.W., N.M.U., D.J.U., O.L., and M.G.C. designed folding of signaling complexes by β-arrestin, a component of the research; S.M.P. and T.F.P. performed research; C.E.B. contributed new reagents/analytic GPCR desensitization and internalization machinery (3). These tools; S.M.P. and M.G.C. analyzed data; and S.M.P., A.D.W., O.L., and M.G.C. wrote the paper. two signaling modes harbor distinct functional properties, and in Conflict of interest statement: M.G.C. has received compensation from Lundbeck as a instances the same ligand can act as an agonist for one pathway member of their Psychopharmacology Advisory Board and is a consultant for Omeros but antagonist at the other. The selective or biased activation of Corp. M.G.C. also owns equity in Acadia Pharmaceuticals. a given pathway is commonly referred to as “functional selectivity” This article is a PNAS Direct Submission. and can be easily demonstrated in heterologous systems especially 1To whom correspondence should be addressed. Email: [email protected]. when biased small molecule ligands are available (4). Biased This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. GPCR ligands may have high therapeutic potential as these 1073/pnas.1502742112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1502742112 PNAS | June 2, 2015 | vol. 112 | no. 22 | 7097–7102 Downloaded by guest on September 30, 2021 neurons of the substantia nigra and ventral tegmental area where was used to predict residue changes. EA models the evolutionary they function as presynaptic autoreceptors but also in GABAergic relationship between genotype and phenotype as a smooth pro- medium spiny neurons (MSNs), cholinergic interneurons of the cess upon which a mutation causes a small perturbation. Ex- striatum, and cortical neurons (13), where they function as post- plicitly, if γ is the genotype sequence and φ is the fitness phenotype, synaptic receptors. Thus, understanding the contributions of EA postulates an evolutionary function f between them exists, functional selectivity at D2R in intact biological systems is a such that challenge that cannot be elucidated in heterologous systems alone. To develop tools where this challenge can begin to be fðγÞ = φ, [1] addressed, the Evolutionary Trace (ET) (14) approach was used to engineer D2R mutants that selectively interact with either and f is differentiable so that the Evolutionary Action point [Gprot] [βarr] Gproteinsorβ-arrestins, designated D2Rand D2R, mutation Δγ on fitness is respectively. These mutants show separation of G-protein and β-arrestin interactions, and expression of these mutants in vivo in f′ðγÞ · Δγ = Δφ. [2] the mouse striatum provides proof-of-concept for their biological activity and discrete functions. In practice, f remains unknown, but its derivative (or gradient) f′ is given by ET, and Δγ is given by substitution odds. The Evolu- Results tionary Action Eq. 2 is thus generally solvable for coding muta- Evolutionary Trace-Guided Mutagenesis of D2R. The ET method tion of proteins and quantifies the effect of mutations over identifies amino acids that determine the function of a protein multiple scales, spanning molecular, clinical, and population ge- and map its functional sites when a structure is available (15, 16). netics effects (21–24). Here, for each residue identified in Fig. 1 ET algorithms exploit protein orthologs and paralogs to corre- A and B, mutations were predicted and scored by EA according late sequence variations with phylogenetic divergences and