Molecular Characterization of Dopamine D2 Receptor Isoforms Tagged With Green Fluorescent Protein

Abstract

In this study, a cleavable signal peptide fused to the enhanced green fluorescent protein (EGFP) was tagged to the extracellular N-terminus of the human dopamine D2 receptor short and long isoforms (D2S and D2L). Ligand-binding properties of EGFP-tagged receptors were essentially unchanged in comparison to their respective wild-type receptors. The dopamine-mediated activation of both EGFP-D2 isoforms generated a robust inhibition of adenylyl cyclase type 5 in intact cells. In addition, the receptor density of EGFP-D2S and EGFP-D2L in transfected human embryonic kidney 293 (HEK293) cells was not altered when compared to cells transfected with the untagged D2S and D2L. However, the receptor densities of untagged and EGFP-tagged D2L were significantly lower in comparison to those measured with D2S constructs. Moreover, the receptor density of EGFP-D2S and EGFP-D2L was differentially upregulated in cells treated with antipsychotic drugs. As assessed by confocal microscopy, both EGFP-D2 isoforms were present on the cell surface. Notably, in contrast to the predominant plasma membrane localization of EGFP- D2S, EGFP-D2L was visualized both on the plasma membrane and intracellularly before dopamine exposure. Importantly, EGFP-D2S and EGFP-D2L are robustly internalized after dopamine treatment. Overall, our data suggest a differential intracellular sorting of D2S and D2L.

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

“…Because single-receptor detection is not yet operational in or ex vivo, we then used high-resolution single-particle detection in cultured hippocampal neurons to investigate the live surface distribution of D1R. To do so, we designed a receptor tagged on its N terminus with a cyan fluorescent protein (D1R-CFP), which does not affect ligand binding nor dopamine-mediated activation (19). No difference was observed when comparing the overall content of D1R in transfected (D1R-CFP) and nontransfected neurons (Fig.…”

The Physiology, Signaling, and Pharmacology of Dopamine Receptors

“…One widely expressed member of the GRK4 subfamily of GRKs, GRK5 (Premont et al, 1995, can regulate D1 and D2 dopamine receptors in cellular model systems (Tiberi et al, 1996;Iwata et al, 1999;Sedaghat et al, 2006). It has also been reported that long-term treatment with cocaine causes an up-regulation of GRK5 mRNA in the septum (Erdtmann-Vourliotis et al, 2001), and significant alterations in expression have been found in experimental PD animals (Ahmed et al, 2008a), suggesting a possible involvement of this kinase in the regulation of receptor responsiveness to dopaminergic drugs.…”

“…G Protein-Coupled Receptor Kinases. GRK2 is widely expressed in the brain, including primary dopaminergic areas (Arriza et al, 1992) and can phosphorylate and regulate D1, D2, and D3 dopamine receptors in in vitro cellular systems (Tiberi et al, 1996;Iwata et al, 1999;Kim et al, 2001;Kabbani et al, 2002;Sedaghat et al, 2006;Banday et al, 2007). Indirect evidence for the role of this kinase in the regulation of dopamine receptors was provided by the observation of alterations in the GRK2 expression levels in the striatum of animals with experimental parkinsonism (Bezard et al, 2005), rats treated with antipsychotics (Ahmed et al, 2008b), and rats receiving long-term cocaine treatment (Schroeder et al, 2009).…”

“…Similar to GRK2, GRK3 has a ubiquitous pattern of expression in the brain, and in vitro studies using cell culture have indicated a potential role of GRK3 in the regulation of D1, D2, and D3 dopamine receptors (Tiberi et al, 1996;Kim et al, 2001;Kabbani et al, 2002;Sedaghat et al, 2006). Alterations in the GRK3 expression in several brain areas were observed after the longterm treatment of rats with the antipsychotics haloperidol and clozapine or in experimental animal models of PD (Ahmed et al, 2008a,b).…”

Co-expression of the human cannabinoid receptor coding region splice variants (hCB1) affects the function of hCB1 receptor complexes

“…Dopamine D 2Sh receptors are localized to the axonal boutons, while dopamine D 2L receptors are localized to the dendrites (Khan et al, 1998). In addition, dopamine D 2Sh receptor isoforms are predominantly localized at the plasma membrane, whereas dopamine D 2L receptor isoforms are found at the cell membrane and within the cell (Sedaghat et al, 2006). Whether cannabinoid hCB 1 receptor variants also have differential localization in distinct neuronal populations is not yet known.…”