Luessen Deborah J, Hinshaw Tyler P, Sun Haiguo, Howlett Allyn C, Marrs Glen, McCool Brian A, Chen Rong
Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC, 27157, USA.
Department of Biology, Wake Forest University, Winston-Salem, NC, 27106, USA.
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.
多巴胺 D2 受体（D2Rs）的表达失调和功能异常与药物成瘾、帕金森病和精神分裂症有关。在本研究中，我们检测了 D2Rs 是否受 G 蛋白信号调节因子 2（RGS2）调控，RGS2 是 RGS 家族的成员之一，通过加速 GTP 酶活性来调节 G 蛋白信号。利用神经母细胞瘤 2a（N2A）细胞，我们发现 RGS2 可被氟化铝激活的 Gαi2 蛋白免疫沉淀。与乱序 siRNA 处理相比，RGS2 siRNA 敲低增强了膜结合的 [(35)S] GTPγS 与活化的 Gαi/o 蛋白的结合，增强了对 cAMP 积累的抑制作用，并在存在 D2/D3R 激动剂喹吡罗的情况下增加了 ERK 磷酸化。这些数据表明 RGS2 是 D2R 介导的 Gαi/o 信号的负调节因子。此外，通过免疫细胞化学评估，RGS2 敲低略微增加了组成型 D2R 内化，并显著消除了喹吡罗诱导的 D2R 内化。RGS2 敲低并不影响激动剂诱导的β-抑制蛋白膜募集；然而，在长时间喹吡罗处理后，它阻止了β-抑制蛋白从膜上解离，在此期间β-抑制蛋白在对照细胞中从膜上移开。此外，共聚焦显微镜对β-抑制蛋白内吞后命运的分析表明，喹吡罗处理导致β-抑制蛋白在对照细胞中以时间依赖性方式转运至早期和再循环内体，而在 RGS2 敲低细胞中β-抑制蛋白未发生向这些内体的转运。β-抑制蛋白转运受损可能导致了 RGS2 敲低细胞中喹吡罗刺激的 D2R 内化的消除。因此，RGS2 对于β-抑制蛋白介导的 D2R 内化是不可或缺的。本研究揭示了 RGS2 蛋白对 D2R 信号和内化的一种新的调控作用。
