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 D1 and D2 dopamine receptors. These protein protein interactions were confirmed using Western blot analysis andco-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 D1-green fluorescent protein and D2-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D1 and D2 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 D1 and D2 receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.