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

Neuropharmacology. 2016 Nov;110(Pt A):297-307. doi: 10.1016/j.neuropharm.2016.08.009. Epub 2016 Aug 12.


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 [(35)S] 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.

Keywords: Dopamine D2 receptors; Gαi/o signaling; Internalization; RGS2; β-arrestin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Clathrin / metabolism
  • Cyclic AMP / metabolism
  • Endosomes / drug effects
  • Endosomes / metabolism
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • GTP-Binding Protein alpha Subunit, Gi2 / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Humans
  • Mice
  • Phosphorylation / physiology
  • RGS Proteins / genetics
  • RGS Proteins / metabolism*
  • Receptors, Dopamine D2 / metabolism*
  • beta-Arrestins / metabolism


  • Clathrin
  • DRD2 protein, human
  • RGS Proteins
  • Receptors, Dopamine D2
  • Rgs2 protein, mouse
  • beta-Arrestins
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Cyclic AMP
  • Extracellular Signal-Regulated MAP Kinases
  • GTP-Binding Protein alpha Subunit, Gi2