Elsevier

Neuropharmacology

Volume 110, Part A, November 2016, Pages 297-307
Neuropharmacology

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

https://doi.org/10.1016/j.neuropharm.2016.08.009Get rights and content

Highlights

  • RGS2 negatively modulates dopamine D2 receptor signaling and positively regulates agonist-induced receptor internalization.

  • RGS2 interacts with activated Gαi2 proteins.

  • RGS2 knockdown enhances quinpirole-induced membrane [35S]GTPγS binding and ERK phosphorylation.

  • RGS2 knockdown abolishes agonist-induced D2 receptor internalization.

  • RGS2 knockdown disrupts β-arrestin dissociation from the membrane.

Abstract

Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [35S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

Introduction

Dysfunctional dopamine D2 receptors (D2Rs) are implicated in numerous neurological and psychiatric diseases. Agonists or antagonists of D2Rs have been used for the treatment of Parkinson's disease and schizophrenia [see review in (Beaulieu and Gainetdinov, 2011)]. Thus, it is important to understand the regulation of D2R function. D2Rs are coupled to inhibitory Gαi/o proteins to produce intracellular signaling (Neve et al., 2004). Activation of Gαi/o proteins by D2R agonists promotes the exchange of GDP to GTP on the Gα subunit and subsequent dissociation of G proteins into Gα and Gβγ subunits, which act on various downstream effectors to produce differential cellular and behavioral responses. For example, the inhibitory Gαi/o subunit couples to adenylyl cyclase to inhibit cAMP production whereas the Gβγ subunit stimulates the MAPK signaling cascade. The extent and duration of D2R signaling is critically controlled by the family of regulators of G protein signaling (RGS) proteins that limit G protein activity (Masuho et al., 2013). All RGS proteins contain a RGS domain which binds directly to the activated Gα subunit to facilitate GTP hydrolysis, thus rapidly terminating G protein signaling and receptor responses (Hepler, 1999, Watson et al., 1996). There are more than 20 subtypes of RGS proteins that are distributed in a brain region- and neuron-dependent manner (Gold et al., 1997, Hooks et al., 2008), suggesting that modulation of GPCR signaling by RGS proteins may be receptor-type and brain region-specific.

The majority of D2Rs are localized on postsynaptic non-dopaminergic neurons in the striatum and play an important role in motor function [see review in (Beaulieu and Gainetdinov, 2011)]. Among the members of the RGS family, RGS4, RGS7 and RGS9 are enriched in striatum (Mancuso et al., 2010, McGinty et al., 2008) and have been shown to directly regulate D2R signaling in heterologous expression systems. For example, RGS9 dose-dependently reduces dopamine-stimulated activation of Gαi/o proteins in HEK293 cells stably expressing D2Rs (Masuho et al., 2013). RGS4 overexpression reduces the ability of quinpirole (a D2R/D3R agonist) to inhibit forskolin-stimulated cAMP production in HEK293 cells (Min et al., 2012). Furthermore, there is compelling evidence that RGS9 controls striatal postsynaptic D2R activity and associated motor function (Kovoor et al., 2005, Rahman et al., 2003). In addition to their enriched expression in striatum, D2Rs are also present on the somas and dendrites of midbrain dopamine neurons (Sesack et al., 1994). These receptors serve as autoreceptors to provide negative feedback inhibition of dopamine transmission in the synapse (Bello et al., 2011, Mercuri et al., 1997). Compared to striatal postsynaptic D2Rs, the regulation of midbrain D2R signaling by RGS proteins has yet to be examined. Given the differential distribution patterns of RGS subtypes in the brain, it is likely that the function of midbrain D2Rs (autoreceptors) is regulated by RGS subtypes other than RGS9 proteins because RGS9 is not expressed in dopaminergic neurons (Mancuso et al., 2010). We and others have shown that RGS2 is highly expressed in the cell bodies of midbrain dopaminergic neurons where D2Rs (autoreceptors) are located (Calipari et al., 2014, Labouebe et al., 2007). However, the functional interaction between RGS2 and D2Rs in neuronal-like cell lines has yet to be investigated. Thus, one purpose of the present study was to examine whether RGS2 regulates D2R-mediated G protein signaling in neuroblastoma 2a (N2A) cells. This study will provide the basis for future interrogation of the functional interaction between these two proteins in midbrain dopaminergic neurons.

The rate of D2R internalization contributes to the duration and strength of receptor signaling in response to agonist stimulation. D2Rs undergo a complex process of internalization which involves D2R phosphorylation and β-arrestin recruitment to the surface receptors. Upon binding to phosphorylated D2Rs, β-arrestin interacts with a set of endocytic proteins, including clathrin and endocytic adaptor protein 2 (AP2), via a conserved motif in the C terminus tail to facilitate its dissociation from the membrane (Kim et al., 2001, Lan et al., 2009, Laporte et al., 2000, Namkung et al., 2009). Recent evidence indicates that RGS proteins not only act as GTPase-activating proteins, but also play an important role in GPCR internalization. For instance, overexpression of RGS9 inhibits dopamine-induced D2R internalization in HEK293 cells (Celver et al., 2010). Thus, the second purpose of this study was to examine whether RGS2 regulated constitutive and agonist-stimulated D2R internalization. Because D2R internalization is primarily mediated by β-arrestin (Kim et al., 2001), we examined the effect of RGS2 knockdown on β-arrestin-mediated D2R internalization. Here we report that RGS2 negatively modulated D2R-mediated Gαi/o protein signaling. Moreover, RGS2 knockdown prevents agonist-induced D2R internalization by disrupting β-arrestin dissociation from the membrane. The present study provides important new information on the functional interactions between RGS2 and D2Rs which may occur in midbrain dopaminergic neurons.

Section snippets

Chemicals and antibodies

Geneticin and lipofectamine 2000 were purchased from Life Technologies (Carlsbad, CA). [3H] Raclopride and [35S] GTPγS were purchased from Perkin Elmer (Billerica, MA). The Protease and Phosphatase Inhibitor Cocktails and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless stated otherwise. The protein A/G-beads and the HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). All fluorescent secondary antibodies were

Validation of a RGS2 antibody and verification of RGS2 knockdown efficiency by western blotting

We verified the specificity of a commercially available RGS2 antibody. Shown in Fig. 1, 3xHA-RGS2 proteins were detected by both a HA antibody and a RGS2 antibody in N2A cells transfected with 3xHA-RGS2 cDNA. The notable weak expression of the exogenous RGS2 proteins was likely due to the high level of endogenous RGS2 proteins. Using this validated antibody, we examined the knockdown efficiency of RGS2 proteins by RGS2 siRNA. There was a 60.9 ± 1.2% (p < 0.01) reduction in the level of

Discussion

In the current study, we show for the first time that RGS2 negatively modulates D2R-mediated Gαi/o protein signaling in neuroblastoma N2A cells. Moreover, RGS2 critically regulates agonist-stimulated D2R internalization. RGS2 knockdown abolished quinpirole-stimulated D2R internalization and impaired β-arrestin dissociation from the membrane. These data suggest that RGS2 plays an integral role in modulation of both D2R activity and trafficking.

A significant finding of the present study is that

Author contributions

Participated in research design: Luessen, Chen, McCool, Howlett.

Conducted experiments: Luessen, Hinshaw, Sun.

Performed data analysis: Luessen, Hinshaw, Sun, Marrs.

Wrote or contributed to the writing of the manuscript: Luessen, Chen, McCool.

Acknowledgments

This work was supported by NIH grants (DA0006634, DA03690, T32-AA007565, AA023999 and AA014445) and a pilot grant from the Center for Molecular Communication and Signaling at Wake Forest University. The authors would like to thank for the Microscopic Imaging Core Facility in the Department of Biology at Wake Forest University.

References (45)

  • L.J. Leontiadis et al.

    Regulator of G protein signaling 4 confers selectivity to specific G proteins to modulate mu- and delta-opioid receptor signaling

    Cell. Signal.

    (2009)
  • I. Masuho et al.

    Macromolecular composition dictates receptor and G protein selectivity of regulator of G protein signaling (RGS) 7 and 9-2 protein complexes in living cells

    J. Biol. Chem.

    (2013)
  • C. Min et al.

    RGS4 exerts inhibitory activities on the signaling of dopamine D2 receptor and D3 receptor through the N-terminal region

    Pharmacol. Res.

    (2012)
  • Y. Namkung et al.

    G protein-coupled receptor kinase-mediated phosphorylation regulates post-endocytic trafficking of the D2 dopamine receptor

    J. Biol. Chem.

    (2009)
  • M.P. Papakonstantinou et al.

    RGS2 and RGS4 proteins: new modulators of the kappa-opioid receptor signaling

    Cell. Signal.

    (2015)
  • Z. Rahman et al.

    RGS9 modulates dopamine signaling in the basal ganglia

    Neuron

    (2003)
  • M. Rodriguez-Munoz et al.

    RGS14 prevents morphine from internalizing Mu-opioid receptors in periaqueductal gray neurons

    Cell. Signal.

    (2007)
  • J.J. Tesmer et al.

    Structure of RGS4 bound to AlF4–activated G(i alpha1): stabilization of the transition state for GTP hydrolysis

    Cell

    (1997)
  • G. Tulipano et al.

    Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes

    J. Biol. Chem.

    (2004)
  • S.E. Bartlett et al.

    Dopamine responsiveness is regulated by targeted sorting of D2 receptors

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • J.M. Beaulieu et al.

    The physiology, signaling, and pharmacology of dopamine receptors

    Pharmacol. Rev.

    (2011)
  • E.P. Bello et al.

    Cocaine supersensitivity and enhanced motivation for reward in mice lacking dopamine D2 autoreceptors

    Nat. Neurosci.

    (2011)
  • Cited by (14)

    • Attenuation of nicotine-induced rewarding and antidepressant-like effects in male and female mice lacking regulator of G-protein signaling 2

      2022, Pharmacology Biochemistry and Behavior
      Citation Excerpt :

      For example, recent studies have shown that activation of D2/D3 dopamine receptors mediate the rapid antidepressant-like effects of ketamine (Li et al., 2015). Moreover, downregulation of RGS2 has shown to result in internalization of D2 receptors in neuroblastoma cells (Luessen et al., 2016). Thus, decreased D2/D3 receptors signaling in mice lacking RGS2 could also be responsible for the decreased nicotine-induced antidepressant-like effects in these mice.

    • Dopamine D2 autoreceptor interactome: Targeting the receptor complex as a strategy for treatment of substance use disorder

      2020, Pharmacology and Therapeutics
      Citation Excerpt :

      Moreover, RGS2 suppresses β2 adrenergic receptor-mediated Gαi/o signaling in HEK293 cells and cardiomyocytes (Chakir et al., 2011). Lastly, RGS2 negatively modulates D2R-mediated G-protein signaling (Luessen et al., 2016). Knockdown of RGS2 via siRNAs in N2A cells stably expressing D2SRs increases quinpirole-stimulated [35S]GTPγS binding and enhances inhibition of forskolin-stimulated cAMP production confirming the ability of RGS2 to antagonize D2R-mediated Gαi/o signaling in a biological relevant system.

    • Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

      2019, Journal of Biological Chemistry
      Citation Excerpt :

      Because β-arrestin regulates internalization of many GPCRs, including 5-HT1AR (32, 33), the functional consequence of ethanol-induced β-arrestin2 reduction on GPCR internalization was examined using 5-HT1AR as a receptor model system. The internalization of 5-HT1AR was measured by immunocytochemistry using an antibody-feeding protocol described previously (34). We first assessed the kinetics of 8-OH-DPAT–stimulated 5-HT1AR internalization.

    • Biased signaling agonist of dopamine D3 receptor induces receptor internalization independent of β-arrestin recruitment

      2019, Pharmacological Research
      Citation Excerpt :

      CHO-D2R or CHO-D3R cells were treated with 10 μM DA or vehicle at 37 °C for 60 min and internalized receptors were determined by [3H]methylspiperone binding. Consistent with previous studies [12,22,24,28–30], DA (10 μM, 60 min) promoted significant internalization of CHO-D2R, with about 35% of surface receptors being translocated to the cell interior (Fig. 1A). However, DA did not induce CHO-D3R internalization, a finding in agreement with previous reports on HEK293-D3R cells [12,22,24].

    View all citing articles on Scopus
    View full text