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Elucidation of G-protein and ?-arrestin functional selectivity at the dopamine D2 receptor.

ABSTRACT: The neuromodulator dopamine signals through the dopamine D2 receptor (D2R) to modulate central nervous system functions through diverse signal transduction pathways. D2R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D2R signals through distinct G-protein and ?-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D2R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D2R mutants with strongly biased signal transduction for either the G-protein or ?-arrestin interactions. These mutants were used to resolve the role of G proteins and ?-arrestins in D2R signaling assays. The results show that D2R interactions with the two downstream effectors are dissociable and that G-protein signaling accounts for D2R canonical MAP kinase signaling cascade activation, whereas ?-arrestin only activates elements of this cascade under certain conditions. Nevertheless, when expressed in mice in GABAergic medium spiny neurons of the striatum, the ?-arrestin-biased D2R caused a significant potentiation of amphetamine-induced locomotion, whereas the G protein-biased D2R had minimal effects. The mutant receptors generated here provide a molecular tool set that should enable a better definition of the individual roles of G-protein and ?-arrestin signaling pathways in D2R pharmacology, neurobiology, and associated pathologies.

SUBMITTER: Peterson SM

PROVIDER: S-EPMC4460444 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor.

Peterson Sean M SM Pack Thomas F TF Wilkins Angela D AD Urs Nikhil M NM Urban Daniel J DJ Bass Caroline E CE Lichtarge Olivier O Caron Marc G MG

Proceedings of the National Academy of Sciences of the United States of America 20150511 22

The neuromodulator dopamine signals through the dopamine D2 receptor (D2R) to modulate central nervous system functions through diverse signal transduction pathways. D2R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D2R signals through distinct G-protein and β-arrestin pathways, and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D2R signals through the two pathways is sti ...[more]

PMID: 25964346

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Project description:Functional selectivity (or biased agonism) is a property exhibited by some G protein-coupled receptor (GPCR) ligands, which results in the modulation of a subset of a receptor's signaling capabilities and more precise control over complex biological processes. The dopamine D2 receptor (D2R) exhibits pleiotropic responses to the biogenic amine dopamine (DA) to mediate complex central nervous system functions through activation of G proteins and β-arrestins. D2R is a prominent therapeutic target for psychological and neurological disorders in which DA biology is dysregulated and targeting D2R with functionally selective drugs could provide a means by which pharmacotherapies could be developed. However, factors that determine GPCR functional selectivity in vivo may be multiple with receptors, ligands and transducers contributing to the process. We have recently described a mutagenesis approach to engineer biased D2R mutants in which G protein-dependent ([Gprot]D2R) and β-arrestin-dependent signaling ([βarr]D2R) were successfully separated (Peterson, et al. PNAS, 2015). Here, permutations of these mutants were used to identify critical determinants of the D2R signaling complex that impart signaling bias in response to the natural or synthetic ligands. Critical residues identified in generating [Gprot]D2R and [βarr]D2R conferred control of partial agonism at G protein and/or β-arrestin activity. Another set of mutations that result in G protein bias was identified that demonstrated that full agonists can impart unique activation patterns, and provided further credence to the concept of ligand texture. Finally, the contributions and interplay between different transducers indicated that G proteins are not aberrantly activated, and that receptor kinase and β-arrestin activities are inextricably linked. These data provide a thorough elucidation of the feasibility and malleability of D2R functional selectivity and point to means by which novel in vivo therapies could be modeled.

Project description:PurposeThe G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors.MethodsTo test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4.ResultsImmunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation.ConclusionsThe results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.

Dataset Information

Elucidation of G-protein and ?-arrestin functional selectivity at the dopamine D2 receptor.

Publications

Elucidation of G-protein and β-arrestin functional selectivity at the dopamine D2 receptor.

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