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

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.

Nucleus Accumbens Microcircuit Underlying D2-MSN-Driven Increase in Motivation

“…Finally, all sections were stained with 4’,6-diamidino-2-phenylindole (DAPI; 1 mg ml −1 ). Anti-D1R and anti-D2R antibodies were previously validated ( Luedtke et al, 1999 ; Basu et al, 2004 ; Luessen et al, 2016 ; Extended Data Fig. 5-1).…”

Biomarker discovery in attention deficit hyperactivity disorder: RNA sequencing of whole blood in discordant twin and case-controlled cohorts

“…RGS2 knockout mice exhibit increased fear learning, spatial learning, and neophobia [ 59 ]. Further, RGS2 modulates the activity and internalization of the dopamine D2 receptor in neuroblastoma cells [ 60 ], and has been implicated in dopamine receptor signaling during amphetamine self-administration [ 61 ].…”

Chronic intermittent ethanol exposure selectively alters the expression of Gα subunit isoforms and RGS subtypes in rat prefrontal cortex

“…We used four validated RGS antibodies against RGS2 (Luessen et al , 2016), RGS4 (Schwendt et al , 2006), RGS7 (Orlandi et al, 2015) and RGS19 (Ji et al, 2010) to assess their protein levels (Fig. 4).…”

“…CIE increased the mRNA and protein levels of RGS2, RGS4, RGS7 and RGS19. RGS2 is known to modulate the signaling of D2 receptor-mediated Gαi/o protein (Luessen et al , 2016) and the coupling between GABA receptors and GIRK channels (Labouebe et al , 2007). Thus, increased RGS2 protein level may contribute to ethanol-induced dysregulation of GPCR and GIRK channel activity by rapidly uncoupling of activated Gα subunits and GPCR/channel.…”