D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin.
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 protein-protein 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.
Free
R Benjamin
RB
Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-9405, USA.
Hazelwood
Lisa A
LA
Cabrera
David M
DM
Spalding
Heather N
HN
Namkung
Yoon
Y
Rankin
Michele L
ML
Sibley
David R
DR
D037281:Calnexin / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D047468:Immunoprecipitation; D007700:Kinetics; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D017447:Receptors, Dopamine D1 / Q000737:chemistry*; D017448:Receptors, Dopamine D2 / Q000737:chemistry*
D010455:Peptides; D011134:Polysaccharides; D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2; D037281:Calnexin; D000242:Cyclic AMP