D1 and D2 Dopamine Receptor Expression Is Regulated by Direct Interaction with the Chaperone Protein Calnexin

Abstract

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D 1 and D 2 dopamine receptors. These proteinprotein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D 1 -green fluorescent protein and D 2 -yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D 1 and D 2 receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D 1 and D 2 receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

Kinin B1 receptor homo-oligomerization is required for receptor trafficking to the cell surface

“…B1R was found to interact with calnexin, a chaperone that promotes both of the above functions, which suggests that this chaperone participates in B1R folding control. This is consistent with work on the D1 and D2 dopamine receptors, which were also found to interact with calnexin in HEK293 cells [27]. The same study showed that overexpression of calnexin resulted in decreased cell surface receptor expression, and the receptors mostly appeared in discreet punctate oval-like clusters within the cell [27].…”

Brain region-specific N-glycosylation and lipid rafts association of the rat mu opioid receptor

“…N-glycosylation has been found to regulate some GPCRs with regard to their export trafficking [6] (Li et al, in press), ligand-induce internalization [7] (Li et al, in press) and degradation [8] (Li et al, in press). Recently, specific sugar binding receptors (lectins) have been shown to be important for the surface expression of membrane proteins including GPCRs [9][10][11].…”

Intranasal Delivery of a Peptide with Antidepressant-Like Effect

“…In addition to existing as unique receptors, dopamine receptors can also couple with other proteins and receptors to form functional heterodimers that activate signaling cascades, independent from those activated by each component receptor (Maggio et al, 2009;Missale et al, 2010). Dopamine receptors also form heterodimers through direct protein-protein interactions between D1 and D2 receptors (Free et al, 2007;Pei et al, 2010;Perreault et al, 2010;So et al, 2009), D1 and D3 receptors in the striatum (Ridray et al, 1998;Schwartz et al, 1998), D2 and D5 receptors (O'Dowd et al, 2013;So et al, 2009), D1 receptors and NMDA receptors (NMDARs) Pei et al, 2004), and D5 and GABA-A receptors (Liu et al, 2000), among other transmembrane and cytoplasmic proteins. Dopamine D1 and D2 receptors couple in this manner and the functional heterodimer may play a role in the pathogenesis of major depressive disorder (MDD) (Pei et al, 2010;Wong and Liu, 2012).…”