Signaling by dopamine regulates D2 receptors trafficking at the membrane

Abstract

sGi2 is a spliced variant of the GTP-binding protein G αi2 . By difference with G αi2 , which is mainly present at the plasma membrane, sGi2 is localized in intracellular compartments. The splicing event generates a novel C-terminal region in sGi2, which is necessary for its intracellular localization. The role of sGi2 is presently unknown, although its intracellular localization might underlie a possible role in the regulation of trafficking of 7TM receptors. Here, we show that sGi2 complexes with dopamine D2 receptors (D2R) in striatal neurons. The sGi2-D2R complex is readily observed in immunoprecipitation studies using specific antibodies for both proteins on mouse striatal extracts, which identify D2-specific bands of >80 KDa suggesting sGi2 interactions with D2R dimers. Importantly, the sGi2-D2R complex in the absence of receptor stimulation is mostly found in intracellular perinuclear areas in primary neuronal cultures. Treatment of neurons with quinpirole, a D2-specific agonist, results into diffusion of D2R and sGi2 staining throughout the cell and into neurites and membranes. This suggests that dopamine could regulate availability of D2 receptors at the cell surface. The formation of sGi2-D2R complex is mediated through the interaction of sGi2 with the third intracellular loop of D2Rs. As functional consequence of the D2R-sGi2 interaction, we observed a reduction of D2 binding sites at the plasma membrane, when the two proteins are co-expressed in transfected cells. Altogether these studies identify sGi2 as a D2R interacting protein involved in the regulation of D2R at the membrane through a dopamine mediated mechanism.

Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes

“…These lines of evidence support a strong link between G protein activation and vesicle formation. To support the importance of Gi protein for vesicular trafficking, recently, an intracellular vesicle-specific splice variant for Gi protein, which regulates vesicular trafficking of dopamine D2 receptors from cellular vesicles to plasma membranes, was identified 58 . Molecular mechanism underlying S1P receptor activation-induced ILV formation in the exosomal MVEs remains to be clarified.…”

Contribution of Kv1.2 Voltage-gated Potassium Channel to D2 Autoreceptor Regulation of Axonal Dopamine Overflow

“…24 h after transfections cells were rinsed with cold PBS and mechanically disrupted using a cell scraper and a glass homogenizer and recovered in membrane extraction buffer (MEB; 125 mM KCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, pH 8.0) and protease and phosphatase inhibitor mixtures. Immunoprecipitation (IP) and Western blotting were performed as described elsewhere (38). Briefly, protein samples (400 g) were pre-cleared via incubation with protein A/G-agarose (Invitrogen) for 2 h. Upon removal of agarose beads, cell lysates were probed using a mouse monoclonal antibody (4H6-7-3) directed against D2R (5 g) overnight at 4°C with agitation.…”

“…For Western blotting, proteins separated by denaturing 10% SDS-PAGE were transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were blocked in 5% nonfat dry milk in PBS, 0.05% Tween 20 and incubated with either anti-Kv1.2 antibody (Alomone Labs) or anti-D2 dopamine receptor antibody (made in-house and previously characterized (38) and anti-actin antibody. For detection, membranes were incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories).…”

Identification of Two Functionally Distinct Endosomal Recycling Pathways for Dopamine D2Receptor

“…For this reason, most antipsychotics currently in use are DRD2 antagonists. The function of DRD2 is determined by its levels on the plasma membrane (PM) (Lin et al, 2001Binda et al, 2002;Free et al, 2007;Kim et al, 2008a,b;Tirotta et al, 2008;Genedani et al, 2010), and DRD2 endocytosis plays an important role in its desensitization following DA stimulation (Kim et al, 2001;Jeanneteau et al, 2004;Kabbani et al, 2004;Macey et al, 2004;Namkung and Sibley, 2004;Sugiura et al, 2004;Bartlett et al, 2005;Genedani et al, 2005;Torvinen et al, 2005;Paspalas et al, 2006;Iizuka et al, 2007;Kim, 2008;Xiao et al, 2009;Celver et al, 2010;Shimokawa et al, 2010). However, the fate of DRD2 following DA-induced endocytosis remains controversial.…”