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 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.
Highlights
G protein-coupled receptors (GPCRs)4 are a super-gene family of receptor proteins that elicit their effects by activating het
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
As part of our efforts to identify novel dopamine receptorinteracting proteins, we have employed receptor immunoprecipitation (IP) assays along with mass spectrometry. We found that both D1 and D2 dopamine receptors interact with the endoplasmic reticulum (ER) chaperone protein calnexin, a transmembrane protein that resides in the ER where it acts as a chaperone for nascent and newly synthesized glycoproteins
Summary
Materials—HEK293-tsa201 (HEK293T) cells [17] were a gift from Dr Vanitha Ramakrishnan. [3H]SCH23390 (85.0 Ci/mmol) and [3H]methylspiperone (79.5 Ci/mmol) were obtained from PerkinElmer Life Sciences. [3H]cAMP (31.4 Ci/mmol) and the cAMP assay kits were obtained from Diagnostic Products Corp. (Los Angeles, CA). HEK293T cells were transfected via a calcium phosphate precipitation kit (Clontech) according to the manufacturer’s instructions. Immunoprecipitation and Gel Electrophoresis—Cells were removed from culture flasks and collected by centrifugation (300 ϫ g) They were resuspended in 1 ml of solubilization buffer (50 mM HEPES, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 150 mM NaCl, 50 mM NaF, 40 mM sodium pyrophosphate, and Complete-Mini (Roche Applied Science) protease inhibitor mixture) and incubated on ice for 1 h. The lysates were centrifuged at 30,000 ϫ g for 30 min to remove insoluble cell debris and precleared via incubation with protein G-agarose for 3 h. Agarose was removed via centrifugation, and proteins were separated on 4 –12% BisTris NuPAGE gel (Invitrogen) according to the manufacturer’s instructions. Images were collected using a single line excitation (488 nm for D1-GFP and D2-YFP and 563 nm for pDsRed2-ER)
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