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Originally published In Press as doi:10.1074/jbc.M701555200 on March 29, 2007

J. Biol. Chem., Vol. 282, Issue 29, 21285-21300, July 20, 2007
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D1 and D2 Dopamine Receptor Expression Is Regulated by Direct Interaction with the Chaperone Protein Calnexin*

R. Benjamin Free1, Lisa A. Hazelwood2, David M. Cabrera, Heather N. Spalding, Yoon Namkung, Michele L. Rankin, and David R. Sibley3

From the Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-9405

Received for publication, February 21, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 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 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs)4 are a super-gene family of receptor proteins that elicit their effects by activating heterotrimeric G proteins, thereby regulating downstream signaling events. It is estimated that nearly 5% of the human genome consists of GPCRs, and that more than 70% of the current drugs on the market are targeted to these essential receptor proteins (1). Clearly, GPCRs have an established importance in a vast variety of physiological and pathological conditions; but despite the observed significance of GPCR regulation in a variety of diseases, little is known about the specific mechanisms associated with their regulation in terms of cell surface expression. Overall expression of these receptors on the cellular surface likely involves a dynamic balance between surface trafficking, internalization, and degradation. Although significant progress has been made in understanding the internalization mechanisms of GPCRs once they are expressed on the cell surface (2), surprisingly little information is available concerning the mechanisms of GPCR biogenesis and trafficking to the plasma membrane.

Recent studies have suggested that GPCRs are synthesized, assembled, and folded in a conformation-dependent sorting paradigm within the endoplasmic reticulum (ER) (3). This allows for their continued trafficking to the cell surface as functional protein receptors (4). Surface trafficking of newly formed receptors, working in harmony with the more extensively studied endocytotic pathway, determines receptor expression levels and thereby influences the magnitude of the cellular response (for review see Ref. 5). However, the ER processing steps for GPCRs are poorly understood, as is the subsequent regulation of receptor trafficking to the cell surface. It is beginning to emerge that a complex system of regulation exists at the level of the ER that represents critical steps in the trafficking and expression of GPCRs.

Dopamine, a critically important neurotransmitter in the central nervous system and periphery, exerts its biological effects through activating a distinct family of GPCRs. These include five separate receptor proteins (D1–D5), with D1 and D2 representing the most abundant subtypes (6, 7). It has now become clear that dopamine receptors, as well as most GPCRs, exist as components of preformed signaling complexes that can consist of G proteins, enzymes, scaffolding proteins, and other receptors (8–10). Indeed, a number of dopamine receptor-interacting proteins have now been identified, some of which may affect receptor trafficking and expression or may be altered in neuropsychiatric disorders (11, 12).

As part of our efforts to identify novel dopamine receptor-interacting proteins, we have employed receptor immunoprecipitation (IP) assays along with mass spectrometry. By using this approach, we found that both D1 and D2 dopamine receptors interact with the ER chaperone protein calnexin, a transmembrane protein that resides in the ER where it acts as a chaperone for nascent and newly synthesized glycoproteins. Calnexin is involved in protein folding and quality control, which includes retaining misfolded and incomplete proteins in the ER (13–15). If proper folding is achieved, the glycoprotein will be allowed to leave the ER when the association with the lectin is terminated. If not in the proper conformation, the glycoprotein will be retained to ensure folding efficiency and prevent misfolded glycoproteins from exiting the ER (16). We report here that the extent of calnexin-receptor interactions is critical to ensure proper trafficking of D1 and D2 receptors to the cell surface. Our results suggest that calnexin may represent a critically important ER chaperone protein that is involved in regulating the expression of many GPCRs.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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). Cell culture reagents, NuPAGE gels, and gel buffers were purchased from Invitrogen. PNGase F was purchased from New England Biolabs (Boston, MA). All other drugs and buffer components were purchased from Sigma.

Cell Culture and Transfection—HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 50 µg/ml streptomycin, 50 units/ml penicillin, and 10 µg/ml gentamycin. HEK293T cells were transfected via a calcium phosphate precipitation kit (Clontech) according to the manufacturer's instructions. Cath.a-differentiated (CAD) cells were obtained as a gift from Dr. Dona Chikaraishi and grown in DMEM/F-12 supplemented with 8% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. Cells were grown at 37 °C in 5% CO2 and 90% humidity. CAD cells were transferred into serum-free differentiation media consisting of DMEM/F-12 supplemented with 20 µg/ml transferrin (Sigma), 50 ng/ml sodium selenite, 100 µg/ml streptomycin, and 100 units/ml penicillin immediately prior to transfection. CAD cells were transfected using NeuroPORTER reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's instructions.

Several expression constructs were used in this study. An amino-terminal FLAG epitope-tagged construct for the rat D1 receptor (18) was created and named pSFD1, as reported previously by our laboratory (19); pCMV-Tag2B, an expression construct expressing only the FLAG peptide (FLAG tag), was obtained from Stratagene (La Jolla, CA). Rat D1-GFP in a pEGFP-N1 vector was a gift from Dr. Qun-yong Zhou (20). The D2L receptor expression construct containing the FLAG epitope in pSR-D2L was created by our laboratory as described previously (21). Two mutants of the above-mentioned constructs were also made for these studies. The D1-FLAG construct was mutated at positions 4 and 174 (N4Q and N174Q), resulting in D1-GLYT-FLAG, and the D2-FLAG construct was mutated at positions 5, 17, 23, and 175 (N5Q, N17Q, N23Q, and N175Q), resulting in D2-GLYT-FLAG. Mutations were made using QuikChange multisite-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. These mutations were made to eliminate the N-linked glycosylation sites. The human D2S-YFP construct was a gift from Dr. Jonathan Javitch. The calnexin expression construct containing canine calnexin in pcDNA3 was a gift from the laboratory of Dr. David Thomas. pDsRed2-ER vector encodes a fusion protein consisting of red fluorescent protein with the ER targeting sequence of calreticulin fused to the 5' end of DsRed2 and the ER retention sequence KDEL fused to the 3' end of DsRed2. This is used to allow for fluorescent visualization of the endoplasmic reticulum and was purchased from BD Biosciences.

Immunoprecipitation and Gel Electrophoresis—Cells were removed from culture flasks and collected by centrifugation (300 x g). They were then 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 then centrifuged at 30,000 x g for 30 min to remove insoluble cell debris and then precleared via incubation with protein G-agarose for 3 h. The agarose was removed by centrifugation (15,000 x g, 5 min) and discarded. Anti-FLAG M2-agarose (Sigma) was added and incubated with the lysate overnight at 4 °C. The beads were collected via centrifugation and washed three times by resuspension and recentrifugation in solubilization buffer. The agarose was then subjected to a final wash in 1x TE buffer, pH 7.4. Proteins were eluted from the beads using NuPAGE-lithium dodecyl sulfate sample buffer (Invitrogen). Agarose was removed via centrifugation, and proteins were separated on 4–12% BisTris NuPAGE gel (Invitrogen) according to the manufacturer's instructions.

Western Blotting and Gel Staining—Proteins separated by PAGE were transferred onto polyvinylidene difluoride membranes. Membranes were blocked in Superblock (Pierce) prior to incubation with the primary antibody. Primary antibodies used in this study include the following: rat monoclonal anti-D1 dopamine receptor (clone 1-1-F11 S.E6, catalog number D-187, Sigma), rabbit polyclonal anti-calnexin (catalog number SPA-860, StressGen, Victoria, British Columbia, Canada), and rabbit polyclonal anti-D2L/S (catalog number AB5084P, Chemicon, Temecula, CA). After washing, membranes were incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) (anti-rat for the D1 and anti-rabbit for D2 and calnexin). Proteins were visualized via the SuperSignal West Pico chemiluminescent substrate (Pierce) according to the manufacturer's instructions, and images were recorded on film. In some experiments, blots were stripped of all antibodies using Restore Western blot stripping buffer (Pierce) according to the manufacturer's instructions. Removal of all primary antibodies by the buffer was initially checked via incubation with HRP-secondary antibodies followed by ECL and film exposure. In other experiments where total protein was examined, gels were stained using either Coomassie Blue or the SilverQuest staining kit (Invitrogen).

Mass Spectrometry—The identification of PAGE-resolved proteins by mass spectroscopy was carried out by ProtTech, Inc. (Norristown, PA). In brief, proteins in each gel band were digested "in-gel" with modified sequencing grade trypsin (Promega), and the resulting peptide mixture was subjected to tandem mass spectrometry for peptide sequencing. A Finnigan ion trap mass spectrometer LCQ coupled with an HPLC system running a 75 µM ID C18 column was used. Data were acquired in a data-dependent mode. MS/MS spectra were used to search the most recent nonredundant protein data bases, including the Protein Information Resources data base and GenBankTM using ProtQuest software suite from ProtTech. The output of the data base search was manually analyzed and validated to verify proper protein identification.

Radioligand Binding Assays—HEK293T (5 x 106 cells) cells were plated in 150-mm tissue culture dishes and transfected as described above. Forty eight hours later, the cells were removed mechanically using calcium-free Earle's balanced salt solution (EBSS). Intact cells were collected by centrifugation and then lysed with 5 mM Tris-HCl and 5 mM MgCl2 at pH 7.4. Homogenates were centrifuged at 20,000 x g for 30 min. The membranes were then resuspended with 50 mM Tris-HCl, pH 7.4. Membrane preparations were incubated for 90 min at room temperature with various concentrations of [3H]SCH23390 (D1 binding) or [3H]methylspiperone (D2 binding) in a reaction volume of 1 ml. Nonspecific binding was determined in the presence of 4 µM (+)-butaclamol. Bound ligand was separated from unbound ligand by filtration through polyethyleneimine-soaked GF/C filters using a Brandel cell harvester. GF/C filter disks were then analyzed via liquid scintillation spectroscopy at a counting efficiency of 60%. Protein concentration was determined using a Bradford protein assay (Bio-Rad).

cAMP Accumulation Assays—Transfected HEK293T cells were seeded into 24-well poly-D-lysine-coated plates (150,000 cells/well for D1; 200,000 cells/well for D2) and cultured for 1 day prior to the experiment. For D1 receptor accumulation assays, cells were washed three times with 200 µl of EBSS per well. Various concentrations of dopamine dissolved in stimulation buffer (DMEM, 20 µM Ro-20-1724, 0.2 mM sodium metabisulfite) were added to each well in a volume of 250 µl for 10 min at 37 °C. For D2 receptor assays, the cells were washed three times with 200 µl of EBSS per well. Various concentrations of dopamine containing 3 µM forskolin and 10 µM propranalol in stimulation buffer were added to each well in a volume of 250 µl for 10 min at 37 °C. The reactions were terminated by adding 200 µl of 3% perchloric acid and incubated on ice for 30 min. 80 µl of 15% KHCO3 was then added to neutralize the acid. The plates remained on ice for an additional 20 min and were then centrifuged at 1,300 x g for 25 min. 50 µl of the supernatant from each well was transferred to a 1.2-ml reaction tube containing 50 µl of cAMP-binding protein, 50 µl of [3H]cAMP, and 150 µl of Tris-EDTA buffer. The reaction was incubated for 90 min at 4 °C. After the incubation, 250 µl of 1% charcoal/dextran mixture was added to each tube and vortexed gently. Tubes were then incubated at 4 °C for 10 min followed by centrifugation (1,300 x g) for 20 min. Radioactivity in the supernatant was then quantified by liquid scintillation spectroscopy at a counting efficiency of 60%. cAMP concentrations were then determined using a standard assay curve according to the manufacturer's instructions.

Confocal Microscopy—1 x 106 HEK293T cells were seeded in 100-mm culture dishes and transfected the next day with either D1-GFP or D2-YFP. For some experiments, cells were also transfected with calnexin. For double-labeling experiments that visualized the ER, the cells were co-transfected with pDsRed2-ER. Cells were cultured for an additional 24 h prior to reseeding at 100,000 cells per poly-D-lysine-coated 35-mm glass bottom culture dishes. Confocal microscopy was performed on a Zeiss laser-scanning confocal microscope (LSM 5 Pascal). Images were collected using a single line excitation (488 nm for D1-GFP and D2-YFP and 563 nm for pDsRed2-ER).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Calnexin as an Interacting Protein for Both D1 and D2 Dopamine Receptors—As part of our efforts to identify novel dopamine receptor-interacting proteins, we employed immunoprecipitation (IP) assays using tagged receptor constructs along with mass spectrometry to identify associated proteins. For these studies, either FLAG-tagged dopamine receptor expression constructs or a vector expressing only the FLAG peptide (FLAG tag) was transfected into HEK293T cells. The cells were then solubilized, and the receptors and associated proteins were immunoprecipitated using anti-FLAG-agarose. The precipitated protein mixtures were then analyzed by SDS-PAGE. Fig. 1A shows an experiment using the D1 dopamine receptor expressed in HEK293T cells. Coomassie Blue staining of the gel reveals the presence of multiple protein species in the immunoprecipitates (Fig. 1A, left lane). A few of these proteins were also found in the negative control cells expressing only the FLAG tag peptide (Fig. 1A, right lane). The proteins precipitated only in the presence of the D1 receptor were tentatively assumed to interact specifically with the receptor. To identify these proteins, specific bands were excised from the gels and digested with trypsin. The eluted peptides were then subjected to LS-MS/MS tandem mass spectrometry as described under "Experimental Procedures." A number of proteins were identified using this approach and are listed in Table 1. The proteins identified in both the D1 receptor and FLAG peptide immunoprecipitates were presumed to be nonspecific in nature. Importantly, we did not observe any proteins that were unique to the FLAG tag peptide samples. Among the putative D1 receptor-interacting proteins was the ER chaperone protein calnexin. We hypothesized from these initial studies that calnexin was involved in dopamine receptor expression, and we therefore chose it for further study.


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  		TABLE 1
Proteins identified by mass spectroscopy that co-immunoprecipitated with the D1 receptor as shown in Fig. 1 NCBI accession numbers are indicated.

 
Fig. 1B shows a representative MS/MS spectrum of a calnexin peptide found in the D1 dopamine receptor sample (Fig. 1A, band 1). This protein was absent in the corresponding gel band (cut in parallel) from the FLAG tag sample lane (Fig. 1A). Three separate peptides corresponding to calnexin sequences were identified (Fig. 1B and Table 2). Mass spectrometry was also performed on Fig. 1A, band 2, and three peptides for the D1 receptor were identified (Table 2). None of these peptides were apparent in the FLAG tag control. Using the D2 receptor, we performed similar experiments (data not shown) as described in Fig. 1, which also identified calnexin in the D2 receptor immunoprecipitates. Table 2 shows that two of the calnexin tryptic fragments identified in the D1 and D2 receptor experiments are identical. These results suggest that calnexin interacts with both D1 and D2 receptors in HEK293T cells.


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  		TABLE 2
Peptides and corresponding parent proteins found in co-IP mass spectrometry experiments

 


Figure 1
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  		FIGURE 1.
Identification of D1 receptor-interacting proteins using immunoprecipitation and mass spectroscopy. A, Coomassie Blue-stained gel of immunoprecipitated proteins. Band number indicates the band cut from the gel and subjected to MS-based sequencing. The D1-FLAG lane shows proteins immunoprecipitated from cells expressing the FLAG-D1 receptor. The FLAG-tag lane shows proteins immunoprecipitated from cells only expressing the FLAG peptide. B, representative MS/MS spectrum obtained from the fragmentation of the precursor ion at m/z 886.6. Liquid chromatography-MS/MS was carried out on the peptide mixture obtained from in-gel digestion of SDS-PAGE-separated protein samples with LCQ ion trap mass spectrometer coupled on line with an HPLC with 75-mm inner diameter C18 column. The precursor ions were selected automatically by the instrument. The peptide sequence was identified by bioinformatics analysis and found to correspond to the parent protein calnexin. Similar spectra were obtained for all reported peptides found in Table 2.

 
Verification of D1 and D2 Receptor Interactions with Calnexin—To verify the interaction of the receptors with calnexin, we performed co-immunoprecipitation and Western blot analyses. Fig. 2A (left panel) shows that the D1 receptor (apparent as a broad diffuse band ~50 kDa in size) is immunoprecipitated only from cells in which it is expressed. Stripping this blot and reprobing it with an antibody to calnexin (Fig. 2A, right panel) reveals that calnexin (apparent as an ~70-kDa band) only immunoprecipitates in the presence of the D1 receptor, and no band was seen in the control FLAG tag lane. To further verify that the D1 receptor and calnexin directly interact, reverse IP experiments were performed using an anti-calnexin antibody. In this case, endogenous calnexin was immunoprecipitated from cells expressing either the D1 receptor or FLAG tag and probed for the presence of the D1 receptor. Fig. 2B (left panel) shows that the D1 receptor (apparent as multiple bands of ~50 kDa) was co-immunoprecipitated with calnexin, but only in cells in which the D1 receptor is expressed. Fig. 2B (right panel) shows the blot stripped of antibodies and re-probed with the anti-calnexin antibody. Dense bands of ~70 kDa are seen in both the D1 receptor-transfected lane and the FLAG tag lane, indicating that calnexin was immunoprecipitated from both cell preparations and was loaded equally on the gel. These data further support a direct protein-protein interaction between the D1 receptor and calnexin.

To ensure that the aforementioned interaction is not unique to HEK293T cells, we performed similar IP experiments using CAD cells, a central nervous system-derived catecholaminergic cell line that can be morphologically differentiated into neuronal phenotypes by the removal of serum (22). Fig. 2C (left panel) shows an immunoblot where CAD cells were transfected with either the D1 receptor or FLAG tag and then immunoprecipitated with anti-FLAG-agarose. Probing the blot with an anti-D1 antibody revealed strong laddered bands that were seen exclusively in the D1 receptor-transfected cells, with the strongest bands centered around ~50 kDa (Fig. 2C, left panel). The lighter bands likely represent other glycosylation variants/intermediates and/or dimers of the D1 receptor. This same blot was then stripped and re-probed with an anti-calnexin antibody revealing a single band of ~70 kDa only in the D1 receptor-transfected cells (Fig. 2C, right panel). These data indicate that the calnexin and D1 receptor interactions are not exclusive to HEK293T cells but also occur in other cells, particularly of a neuronal nature.


Figure 2
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  		FIGURE 2.
Co-immunoprecipitation of D1 receptor and calnexin. A, left panel, HEK293T cells were transfected with D1-FLAG or vector containing only the FLAG peptide (FLAG-tag). Proteins were extracted, immunoprecipitated (IP) using anti-FLAG-agarose, separated by SDS-PAGE, and immunoblotted (IB) as described under "Experimental Procedures." Blots were probed with a monoclonal anti-D1 antibody and visualized using ECL after incubation with an anti-rat HRP-conjugated antibody. Right panel, the blot in the left panel was stripped of all antibodies, re-probed with an anti-calnexin antibody, and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. B, left panel, HEK293T cells were transfected with D1 FLAG or vector containing only the FLAG peptide (FLAG-tag). Proteins were extracted and immunoprecipitated (IP) via incubation with anti-calnexin antibody followed by precipitation with protein-G-agarose. The blot was probed (anti-D1 antibody) and visualized as reported for A. Right panel, the blot in the left panel was stripped of all antibodies, re-probed with an anti-calnexin antibody, and visualized as described for A. Multiple bands are seen in this blot. The top band is calnexin, and the others represent visualization of the antibody used for immunoprecipitation (anti-calnexin antibody); because the IP and the immunoblot antibodies are identical in this assay, they are visible after probing with secondary antibody. C, left panel, CAD cells were transfected with D1-FLAG or vector containing only the FLAG peptide (FLAG-tag). Proteins were extracted, immunoprecipitated (IP) using anti-FLAG-agarose, and immunoblotted (IB) as described under "Experimental Procedures." The blot was probed (anti-D1 antibody) and visualized as described above for A. Right panel, the blot from the left panel was stripped of all antibodies, re-probed with an anti-calnexin antibody, and visualized as described for A. All blots shown are representative of three independent experiments with identical results.

 


Figure 3
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  		FIGURE 3.
Co-immunoprecipitation of the D2 receptor and calnexin. HEK293T cells were transfected with D2-FLAG or vector containing only the FLAG peptide (FLAG-tag). Proteins were extracted, immunoprecipitated (IP) using anti-FLAG-agarose, and immunoblotted (IB) as described under "Experimental Procedures." A, blots were probed with the anti D2 antibody and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. B, the blot from A was stripped of all antibodies, re-probed with an anti-calnexin antibody, and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. Molecular weight markers designate pre-stained molecular weight standards. The blot shown is a representative blot of three independent experiments all showing identical results.

 
We performed similar co-immunoprecipitation analyses to verify the interaction between calnexin and the D2 receptor. Fig. 3 shows an experiment in which HEK293T cells were transfected with either the D2 receptor or FLAG tag, solubilized, and immunoprecipitated with anti-FLAG-agarose followed by SDS-PAGE. The resulting blot was probed with an anti-D2 receptor antibody and visualized using ECL (Fig. 3A). A complex laddering of bands probably corresponding to various glycosylation states of the D2 receptor was seen exclusively in the D2 receptor-transfected lane. In Fig. 3B, the same blot was stripped of antibody and re-probed with an anti-calnexin antibody. A strong band corresponding to calnexin (~70 kDa) was seen in the D2 receptor-transfected lane but not in the FLAG tag lane. These data suggest that calnexin also directly interacts with the D2 receptor.

Characterization of D1 and D2 Receptor Interactions with Calnexin—Calnexin normally binds to N-linked glycans of proteins via its lectin binding domain, and by doing so it assists in the proper folding and maturation of proteins within the ER. However, recent data have suggested that calnexin can also bind to its substrates through glycosylation-independent mechanisms (23, 24). To investigate the mechanism of calnexin-receptor interactions, we made expression constructs of the D1 and D2 receptors that lacked N-linked glycosylation sites. Expression of these constructs followed by Western blot analysis demonstrated that they migrate at lower molecular weights than their wild-type counterparts, consistent with a lack of glycosylation (Fig. 4, A and C). To ensure that the mutant receptors are completely devoid of N-linked glycosylation, control lysates were treated with PNGase F which cleaves at the initial GlcNAc-Asn bond (25). As seen in Fig. 4, A and C, treatment of the receptors with PNGase F resulted in migration patterns identical to those of the mutant receptors, indicating that the mutants lacked N-linked glycosylation. Although the D1 receptor mutant migrated as a single band (~46 kDa), the D2 receptor mutant displayed two prominent bands. Although the exact nature of the higher molecular weight band remains unknown, it may represent a dimer of the nonglycosylated receptor. Clearly, glycosylation is not needed for the formation of this higher molecular weight species as it is present in both the mutant and the PNGase F-treated samples.

We next used these glycosylation-defective receptors to determine the importance of glycosylation for the interactions of calnexin with the receptors. This was assessed by comparing the amount of calnexin that immunoprecipitated with the glycosylation mutants versus the wild-type receptors. As seen in Fig. 4, B and D, the amount of calnexin that immunoprecipitated with the mutant receptors was reduced by about 50% compared with that of the wild-type receptors. Importantly, eliminating the receptor glycosylation only partially blocked the receptor-calnexin interactions, therefore suggesting that a glycosylation-independent mode of interaction with the receptors must also exist.

To ensure that the reduction in receptor-calnexin interactions was because of deglycosylation, and not due to some unknown effect of the mutants, we treated cells expressing the wild-type receptors with drugs known to specifically inhibit protein glycosylation. One drug used in these experiments was tunicamycin, a well characterized specific inhibitor of N-linked protein glycosylation (26–29). Fig. 5A shows that tunicamycin treatment results in the D1 receptor appearing as a single band on SDS-PAGE with a molecular mass of ~49 kDa, similar in size to the predicted nonglycosylated form of the receptor (an identical pattern to that seen with the glycosylation-deficient mutant in Fig. 4A). Stripping of this blot and reprobing with a calnexin antibody show that there is a reduction, but not elimination, of the amount of calnexin that co-immunoprecipitates with the D1 receptor (Fig. 5B). Tunicamycin treatment also results in the D2 receptor appearing primarily as a single species of ~50 kDa, although a minor form of ~70 kDa is still present (Fig. 5C) (an identical pattern to that seen with the glycosylation deficient mutant in Fig. 4C). As with the D1 receptor, tunicamycin treatment reduces, but does not eliminate, the ability of calnexin to co-immunoprecipitate with the D2 receptor (Fig. 5D). These results are completely congruent with those obtained using the glycosylation-defective receptor constructs, and they support the use of tunicamycin as a pharmacological probe of D1 and D2 receptor glycosylation. More importantly, these results further support the notion that calnexin not only interacts with the D1 and D2 receptors through their N-linked glycans, but must also associate with the receptors via direct protein-protein interactions.


Figure 4
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  		FIGURE 4.
D1 and D2 receptor glycosylation mutants. HEK293T cells were transfected with either D1-FLAG or the glycosylation mutant D1-GLYT-FLAG (A and B) or D2-FLAG or the D2 glycosylation mutant D2-GLYT-FLAG (C and D). Proteins were extracted, immunoprecipitated (IP) using anti-FLAG-agarose, either untreated or treated with PNGase F as indicated, separated by SDS-PAGE, and immunoblotted (IB) as described under "Experimental Procedures." Blots were probed with an anti-D1 antibody, an anti-D2 antibody, or an anti-calnexin antibody as indicated and visualized using ECL after incubation with the appropriate HRP-conjugated secondary antibodies. Molecular weight markers designate pre-stained molecular weight standards. The blot shown is representative of three independent experiments all showing identical results.

 
As an additional test of this hypothesis, we enzymatically removed the carbohydrate residues from the solubilized D1 receptor using PNGase F (see above). Fig. 5E demonstrates that PNGase F treatment eliminates the complex glycosylation of the D1 receptor resulting in a single lower molecular weight band identical to that seen after tunicamycin treatment (cf. Fig. 5A). Fig. 5F shows that PNGase F treatment results in a significant reduction in D1 receptor-calnexin interactions but does not completely eliminate this association. This supports our contention that calnexin interacts with the receptor via glycan-dependent and -independent mechanisms.

To further understand the location and character of the interactions between calnexin and the D1 receptor, we employed IP assays using various D1 mutant receptors previously generated by our laboratory. For these experiments we utilized two different D1 mutants. One mutant, designated D1-T347-FLAG, is a deletion mutant (truncated at position 347) that is missing most of its carboxyl terminus. We have previously shown that the cell surface expression of this mutant is diminished relative to the wild-type receptor (30), but it is fully functional in terms of activating adenylyl cyclase. The second mutant, designated D1–3rd-FLAG, has all of the serine and threonine residues within the third cytoplasmic domain changed to either alanines or valines, thereby eliminating all phosphorylation sites in this region. This mutation has no effect on receptor expression and is fully functional, although it exhibits impaired desensitization (30). These two mutant receptors, along with the wild-type D1 receptor and the FLAG tag construct, were expressed in HEK293T cells, and co-immunoprecipitation assays were performed (Fig. 6A). Importantly, radiolabeled binding assays were conducted to ensure that equal amounts of D1 receptor were loaded onto each lane of the SDS-polyacrylamide gels. As seen in Fig. 6A, 1st lane, a single band of ~70 kDa is detected, indicating that calnexin co-immunoprecipitates with the wild-type D1 receptor as shown previously in Fig. 2A. Interestingly, Fig. 6A, 2nd lane, shows a significant increase in the amount of calnexin that co-immunoprecipitates with the D1-T347-FLAG mutant. In contrast, as shown in Fig. 6A, 3rd lane (Fig. 6A), calnexin co-immunoprecipitates with the D1-3rd-FLAG mutant to a similar extent as with the wild-type D1 receptor. These data indicate that phosphorylation of the third cytoplasmic domain is not necessary for D1 receptor-calnexin interactions. Furthermore, these results indicate that calnexin does not interact with the carboxyl terminus of the D1 receptor. In fact, truncation of the carboxyl terminus actually enhances the interaction of the D1 receptor with calnexin.

To assess if the increased interaction of calnexin with the D1-T347-FLAG mutant was because of increased interactions with the receptor glycans or protein regions, we treated cells expressing the D1-T347-FLAG mutant with tunicamycin. As can be seen in Fig. 6B, there was no effect of tunicamycin treatment on the ability of calnexin to co-immunoprecipitate with the D1-T347-FLAG receptor. These results contrast with those seen with the wild-type receptor (Fig. 5A). These data further indicate that calnexin interacts with the D1 receptor via its protein, as well as its carbohydrate component, and that neither of these interactions occur in the cytoplasmic tail or via phosphorylation of the third cytoplasmic loop.


Figure 5
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  		FIGURE 5.
Characterization of D1 and D2 receptor interactions with calnexin. A, HEK293T cells were transfected with D1-FLAG, and cells were either untreated or treated with 2 µg/ml tunicamycin for 24 h as indicated. Proteins were extracted, immunoprecipitated (IP) using anti-FLAG-agarose, separated by SDS-PAGE, and immunoblotted (IB) as described under "Experimental Procedures." Blots were probed with a monoclonal anti-D1 antibody and visualized using ECL after incubation with an anti-rat HRP-conjugated antibody. B, blot in A was stripped of all antibodies, re-probed with an anti-calnexin antibody, and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. C, HEK293T cells were transfected with D2 FLAG, and cells were either untreated or treated with 2 µg/ml tunicamycin for 24 h as indicated. Proteins were extracted and immunoprecipitated (IP) using anti-FLAG-agarose, separated by SDS-PAGE, and immunoblotted (IB) as described under "Experimental Procedures." The blot was probed (anti-D2 antibody) and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. D, the blot in C was stripped of all antibodies, re-probed with an anti-calnexin antibody, and visualized as described for B. Two bands are seen in each lane of this blot. The top band is calnexin, but the other likely represents visualization of the antibody heavy chain. E and F, effect of enzymatically deglycosylating the solubilized D1 receptor on its ability to associate with calnexin. HEK293T cells were transfected with D1-FLAG, and soluble proteins were extracted as described under "Experimental Procedures." Samples were incubated at 37 °C for 1 h with either 5 units of PNGase F (+) or buffer alone (-). Proteins were then immunoprecipitated (IP) using anti-FLAG-agarose, separated by SDS-PAGE, and immunoblotted (IB) as described under "Experimental Procedures." E, blots were probed with a monoclonal anti-D1 antibody and visualized using ECL after incubation with an anti-rat HRP-conjugated antibody. F, blot in E was stripped of all antibodies, re-probed with an anti-calnexin antibody, and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. Densitometry of two separate blots revealed an ~25% decrease in calnexin immunoreactivity following PNGase F treatment.

 


Figure 6
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  		FIGURE 6.
Characterization of calnexin interactions with D1 receptor mutants. A, HEK293T cells were transfected with either the wild-type D1 receptor (D1-FLAG), the D1 receptor truncated at residue 347 (D1-T347-FLAG), the D1 receptor where all serine/threonine residues in the 3rd cytoplasmic loop of the receptor are mutated (D1-3rd-FLAG), or vector containing only the FLAG peptide (FLAG-tag). Proteins were extracted, and radioligand binding experiments were conducted on the three D1 receptor transfections to ensure equal amounts of D1 receptor in the assays (data not shown). Protein was then immunoprecipitated (IP) using anti-FLAG-agarose and immunoblotted (IB) as described under "Experimental Procedures." Blots were probed with an anti-calnexin antibody and visualized using ECL after incubation with an anti-rabbit HRP-conjugated antibody. The blots shown are representative of three independent experiments. B, HEK293T cells were transfected with the D1-T347-FLAG or FLAG tag constructs and left untreated or treated with tunicamycin as described in Fig. 4. Proteins were extracted, and radioligand binding experiments were conducted on the D1 receptor transfections to ensure equal amounts of D1 receptor in the assays. Blots were probed with an anti-calnexin antibody and visualized as described in A.

 
Effects of Calnexin on D1 and D2 Receptor Expression—To investigate the functional importance of receptor-calnexin interactions, we initially examined the effects of two drugs, tunicamycin and castanospermine, that are known to interfere with the association of calnexin with glycoproteins. As noted above, tunicamycin is a glycosylation inhibitor capable of preventing N-linked glycosylation. Castanospermine is a glucosidase inhibitor that inhibits both glucosidases I and II (31–34), which sequentially trim glycoproteins to mono-glucosylated states that preferentially interact with calnexin (35). Fig. 7, A and B, shows saturation radioligand binding assays using HEK293T cells transfected with the D1 receptor. Cells were treated with either castanospermine for 48 h (Fig. 7A), with tunicamycin for 24 h (Fig. 7B), or left untreated followed by membrane binding assays using the D1-selective antagonist [3H]SCH23390. No significant change is seen in the maximum binding capacity (Bmax) or the affinity (Kd) of [3H]SCH23390 for the D1 receptor (Fig. 7, A and B, and Table 3).


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  		TABLE 3
Effects of drug treatments and mutations on D1 and D2 receptor binding

 
Similar experiments were conducted with HEK293T cells expressing the D2 receptor (Fig. 7, C and D). In this case, saturation binding assays using the D2-selective radioligand [3H]methylspiperone revealed that both castanospermine and tunicamycin treatments reduced the total number of D2 receptors without any effect on their affinity for the radioligand (Fig. 7, C and D, and Table 3). Thus, total D2 receptor expression appears to be more sensitive to the glycan-dependent calnexin interactions than that of the D1 receptor.

The assays shown in Fig. 7, A–D, are designed to measure the total complement of membrane-associated receptors, which include those on the cell surface as well as any receptors residing in internal membranous structures or vesicles. As it was of interest to assess the effects of calnexin on the trafficking of receptors to the cell surface, we employed an intact cell binding assay that utilizes a hydrophilic radioligand, [3H]sulpiride, that only labels D2 receptors on the cell surface (21). Fig. 7, E and F, shows intact cell saturation binding assays with [3H]sulpiride after treatment with tunicamycin or castanospermine. As can be seen, both of these treatments significantly reduced the expression of the D2 receptor at the cell surface, with tunicamycin having a greater effect than castanospermine (Table 3). Interfering with D2 receptor-calnexin interactions not only reduces cell surface receptor expression but also decreases the total amount of D2 receptors expressed within the cell.

Surface expression was also investigated despite no significant change in the total number of cellular D1 receptors after glycosylation interference. Unfortunately, a cell surface-restricted radioligand is not available for the D1 receptor, thus negating the ability to assess cell surface expression in this fashion. As an alternative approach, we performed cAMP accumulation assays that reflected the functional D1 receptors expressed at the cell surface. After treating cells with tunicamycin or castanospermine, dopamine dose-response curves were generated, resulting in internal cAMP accumulation (Fig. 8). Castanospermine treatment resulted in a significant (~30%) decrease in the maximum accumulation of cAMP in response to dopamine, with no significant change in the potency of dopamine (Fig. 8A). Similarly, treatment with tunicamycin resulted in an ~30% reduction of Emax, whereas there was no change in the EC50 for dopamine (Fig. 8B). Taken together, these data show that inhibition of calnexin-receptor interactions results in a decrease in D1 receptor-mediated signaling, which is consistent with a decrease in receptor expression at the cell surface.


Figure 7
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  		FIGURE 7.
Effects of tunicamycin and castanospermine on D1 and D2 receptor expression. HEK293T cells were transfected with either D1 receptor (A and B) or D2 receptor (C–F) expression constructs and then treated with either 1 mM castanospermine for 48 h or 2 µg/ml tunicamycin for 24 h. Independent untreated controls were run for each drug treatment. Cells were then harvested, membranes prepared, and radioligand binding assays performed for D1 receptors ([3H]SCH23390) (A and B) or D2 receptors ([3H]methylspiperone) (C and D) as described under "Experimental Procedures." In addition, intact cell radioligand binding assays were performed for D2 receptors using [3H]sulpiride (E and F). A and B, data are normalized to Bmax values of untreated control cells for each individual experiment and are expressed as representative curves. Bmax values for untreated controls ranged from 1 to 5 pmol/mg protein. No statistically significant differences were seen between control and either drug treatment group. C and D, data are normalized to Bmax values of untreated control cells for each individual experiment and are expressed as representative curves. Bmax values for untreated controls ranged from 4 to 12 pmol/mg protein. E, representative saturation binding curve for [3H]sulpiride binding to intact HEK293T cells transfected with D2. Data show a representative curve from three individual experiments normalized to Bmax values of untreated control cells. Bmax value for the untreated control on this curve was 3.1 pmol/mg protein. F, histogram of single point [3H]sulpiride binding assays using 6 nM [3H]sulpiride, performed on HEK293T cells treated as indicated. Data are normalized to untreated control binding values for each individual experiment and are expressed as means ± S.E., n = 5. Values for untreated control ranged from 2 to 3 pmol/mg protein. Average binding parameters from experiments shown in A–E (n = 5) are shown in Table 3.

 
To ensure that the results observed with tunicamycin and castanospermine are specific to blockade of receptor glycosylation, we performed similar experiments using the glycosylation-defective constructs described above. When radiolabeled binding assays were performed on the D1-GLYT mutant, an increase in total cellular binding was seen (Table 3). However, when functional cAMP accumulation assays were performed with the D1-GLYT mutant, an ~27% decrease was seen in the Emax value with no change in EC50 (percent control Emax, 100% versus 64.2% ± 9.7, n = 3, for D1-FLAG and D1-GLYT, respectively). These data indicate a reduction in the surface expression of the mutant receptor despite an increase in overall expression levels. Membrane binding assays for the D2-GLYT mutant demonstrated a significant (~85%) decrease in overall expression, paralleling results seen with glycosylation inhibitor treatment. Overall, the results with the glycosylationdefective mutants are in excellent agreement with those obtained using tunicamycin, which inhibits N-linked glycosylation of proteins.

To further investigate the importance of calnexin on D1 and D2 receptor expression, we used chimeric proteins in which the D1 receptor was fused with GFP, and the D2 receptor was fused with YFP, thus enabling their subcellular visualization using confocal fluorescence microscopy. This approach has the added advantage of allowing visualization of receptor proteins that are not capable of binding ligands in intact cells, such as receptors in prefolded or misfolded states. Cells were transfected with D1-GFP or D2-YFP and then left untreated or treated with either castanospermine or tunicamycin and visualized. Fig. 9, A and D, shows untreated (control) D1 and D2 receptors. Nearly all receptor protein is located at the surface of the cell plasma membrane with very little intracellular fluorescence. Fig. 9, B (D1) and E (D2), shows representative cells after treatment for 24 h with tunicamycin. These cells show both punctate and diffuse intracellular staining in addition to surface staining. Fig. 9, C (D1) and F (D2), are representative cells after treatment with castanospermine for 24 h. These cells also show discrete pockets of bright staining and intracellular clusters. Taken together, these data show that attenuating the ability of calnexin to interact with either the D1 or D2 receptors results in decreased trafficking of the receptors to the cell surface. In the case of the D2 receptor, there is also a reduction in the total amount of receptor capable of interacting with ligands (Fig. 7, C and D).

Another approach for determining the biological importance of the receptor-calnexin interactions is to study the effect of calnexin overexpression on receptor expression and function. In Fig. 10, we overexpressed calnexin by transfecting it along with the D1 receptor in HEK293T cells. Co-immunoprecipitation experiments indicated that this was associated with a >2-fold increase in calnexin association with the receptor (data not shown). Surprisingly, we found that overexpression of calnexin decreases the D1 receptor expression level to about one-third of control with no change in the affinity of the receptor for the radioligand (Fig. 10A). Similarly, when D1 receptor function was examined (Fig. 10B), dopamine-stimulated cAMP accumulation was decreased by ~50% when calnexin was overexpressed without a change in agonist potency. These data demonstrate that overexpression of calnexin decreases the total cellular complement of D1 receptors and causes a concomitant decrease in D1 receptor function.


Figure 8
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  		FIGURE 8.
Effect of castanospermine or tunicamycin treatment on D1 receptor-stimulated cAMP accumulation. HEK293T cells were transfected with D1 receptor expression plasmid as described under "Experimental Procedures." 24 h later, cells were plated in 24-well plates at a density of 150,000 cells per well and were either untreated, treated with 1 mM castanospermine for 48 h (A), or treated with 2 µg/ml tunicamycin for 24 h (B). Cells were stimulated with the indicated concentrations of dopamine followed by cAMP determinations as described under "Experimental Procedures." Separate untreated controls were run for each drug treatment. Control Emax values ranged from 11 to 52 pmol/150,000 cells. Data were normalized for each individual experiment as a percentage of the Emax value of the untreated control and are expressed as mean ± S.E., n = 5. Average EC50 values expressed as logM ± S.E. are as follows: A, -7.6 ± 0.1 (untreated) and -7.4 ± 0.2 (castanospermine-treated); B, -7.5 ± 0.1 (untreated), and -7.0 ± 0.3 (tunicamycin-treated).

 


Figure 9
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  		FIGURE 9.
Confocal fluorescence microscopy of D1-GFP and D2-YFP after treatment with tunicamycin or castanospermine. A–C, HEK293T cells were transfected with D1-GFP, and 24 h later were transferred onto 35-mm glass bottom culture dishes and either not treated (A), treated with 2 µg/ml tunicamycin (B), or treated with 1 mM castanospermine (C) for 24 h. Cells were then visualized using a laser-scanning confocal microscope. Images are taken at x1000 using single line excitation (488 nm) and are representative images from blinded experiments, done three separate times with similar results. D–F, HEK293T cells were transfected with D2-YFP, and 24 h later transferred onto 35-mm glass bottom culture dishes and either not treated (D), treated with 2 µg/ml tunicamycin (E), or treated with 1 mM castanospermine (F) for 24 h. Cells were then visualized using a laser-scanning confocal microscope. Images are taken at x1000 using single line excitation (488 nm) and are representative images from blinded experiments, done three separate times with similar results.

 
To determine whether calnexin overexpression also affects D2 receptor expression and function, radioligand binding and functional assays were performed after calnexin co-transfection. Fig. 11A shows that the maximum binding capacity for [3H]methylspiperone is decreased by >50% with a slight change in receptor affinity for the radioligand (control Kd = 0.40 nM ± 0.04 versus treated Kd = 0.25 nM ± 0.01, n = 4). To assess the function of the D2 receptor, we examined its ability to inhibit forskolin-stimulated cAMP accumulation. As shown in Fig. 11B, under control conditions, dopamine dose-dependently inhibits cAMP accumulation with a maximum response of nearly 50%. Calnexin overexpression decreases the maximum D2 receptor-mediated cAMP inhibition by about half without a change in the EC50 value for dopamine (Fig. 11B). These data indicate that overexpression of calnexin also affects D2 receptor expression and function in a negative manner consistent with a decrease of receptor surface expression.


Figure 10
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  		FIGURE 10.
Effect of overexpressing calnexin on D1 receptor expression and function. HEK293T cells were transfected with either 15 µg of D1 receptor and 15 µg of empty pcDNA vector or 15 µg of D1 receptor and 15 µg of calnexin expression vector. 24 h later cells were either washed and remained in the plate (A) or were plated in 24-well plates at a density of 150,000 cells per well (B). Cells were assayed 48 h after transfection. A, radioligand binding saturation assays using [3H]SCH23390 at the indicated concentrations. Bmax values for D1 receptor + pcDNA ranged from 2.0 to 11.9 pmol/mg. Kd values expressed as nM ± S.E. are 0.15 ± 0.05 (D1 receptor + pcDNA) and 0.18 ± 0.06 (D1 receptor + calnexin). Data are expressed as mean ± S.E., n = 3. B, cells were stimulated with the indicated concentration of dopamine, and cAMP accumulation was assessed as described under "Experimental Procedures." Emax values for D1 receptor + pcDNA ranged from 54.8 to 10.7 pmol/150,000 cells. Data were normalized for each individual experiment as a percentage of the Emax value of the untreated control and is expressed as mean ± S.E., n = 3. Average EC50 values expressed as logM ± S.E. are -6.9 ± 0.1 (D1 receptor + pcDNA) and -6.6 ± 0.1 (D1 receptor + calnexin).

 
Taking the D1 and D2 data together, it appears that there may be a level of calnexin expression (endogenous) that is optimal for receptor maturation and biosynthesis, but if this level is exceeded, calnexin may actually retard receptor trafficking to the cell surface. To explore this further, we examined the subcellular distribution of the D1 and D2 receptors after calnexin overexpression using confocal fluorescence microscopy. As demonstrated in Fig. 12, when HEK293T cells were transfected with either D1-GFP (Fig. 12A) or D2-YFP (Fig. 12C), most receptor protein is located at the cell surface. However, when calnexin is overexpressed in these cells (Fig. 12, B and D), the receptors are mostly found in discrete punctate oval-like clusters within the cells. This is consistent with the receptors not traveling to the cell surface in the presence of a high level of calnexin protein, suggesting a retardation of trafficking.


Figure 11
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  		FIGURE 11.
Effect of overexpressing calnexin on D2 receptor expression and function. HEK293T cells were transfected with either 15 µg of D2 receptor and 15 µg of empty pcDNA vector or 15 µg of D2 receptor and 15 µg of calnexin expression vector. 24 h later, cells were washed and either remained in the plate (A) or were plated in 24-well plates at a density of 200,000 cells per well (B). Cells were assayed 48 h after transfection. A, radioligand binding assays of these cells using [3H]methylspiperone at the indicated concentrations. Bmax values for D2 receptor + pcDNA ranged from 2.4 to 3.0 pmol/mg. Data were calculated as pmol/mg protein and then normalized for each individual experiment as a percentage of the Bmax value of D2 receptor + pcDNA controls. Kd values expressed as nM ± S.E. are 0.03 ± 0.01(D2 receptor + pcDNA) and 0.03 ± 0.01 (D2 receptor + calnexin). Data are expressed as mean ± S.E., n = 4. B, cells were stimulated with 1 µM forskolin in the presence of the indicated concentrations of dopamine, and cAMP accumulation was assessed as described under "Experimental Procedures." Data were normalized for each individual experiment as a percentage of the Emax value of the untreated control and is expressed as mean ± S.E., n = 3. Average EC50 values expressed as logM ± S.E. are -8.4 ± 0.2 (D2 receptor + pcDNA) and -7.5 ± 0.2 (D2 receptor + calnexin).

 
We have hypothesized that calnexin is acting as a chaperone protein for the D1 and D2 receptors via ER retention properties similar to its effects on other proteins. If this is the case, the receptor proteins that are internally retained after overexpression of calnexin should be localized to the ER. To examine this, we conducted double-labeling experiments using pDsRed2-ER, a vector designed for fluorescent labeling of the ER in living cells. The pDsRed2-ER is an expression vector that encodes a fusion protein consisting of red fluorescent protein, the ER targeting sequence of calreticulin, and the ER retention sequence KDEL. Cells were transfected with D1-GFP, pDsRed2-ER, and calnexin, followed by confocal fluorescence microscopy at either 488 nm (GFP) or 543 nM (DsRed2). As observed previously, when calnexin is overexpressed in these cells, the D1 receptor does not traffic to the cell surface but is retained in intracellular punctate oval clusters (Fig. 13A). These clusters are also labeled by the ER marker protein (Fig. 13B), indicating that they represent endoplasmic reticula. When the images are merged, it is clear that the punctate intracellular clusters of receptors are located in the ER (Fig. 13C). These data are consistent with calnexin acting as an ER retention protein that, if overexpressed, results in newly synthesized D1 receptors being retained in the ER.


Figure 12
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  		FIGURE 12.
Confocal fluorescence microscopy of D1-GFP and D2-YFP after overexpression of calnexin. HEK293T cells were transfected with either D1-GFP and pcDNA vector (A), D1-GFP and calnexin (B), D2-YFP and pcDNA vector (C), or D2-YFP and calnexin (D). Twenty four hours later cells were transferred into 35-mm glass bottom culture dishes. 48 h after transfection, cells were visualized using a laser-scanning confocal microscope. Images are taken at x1000 using single line excitation (488 nm) and are representative images from blinded experiments performed three times with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major finding of this study is our identification of calnexin as a novel interacting protein for both D1 and D2 dopamine receptors. Calnexin functions as both an ER retention protein and chaperone, thus enabling the proper folding and assembly of glycoproteins prior to their export to the Golgi. We determined that interaction with calnexin in the ER is critical for the optimum expression of both D1 and D2 receptor subtypes. Interestingly, the interaction of calnexin with the receptors appears to be tightly regulated as either decreasing (via receptor mutagenesis or pharmacological inhibitors) or increasing (via overexpression) calnexin-receptor interactions diminished receptor trafficking and expression. Clearly, there is an optimal degree or duration of calnexin interaction that results in the maximum amount of receptor being expressed at the cell surface. Calnexin thus joins a growing list of proteins being identified that directly interact with dopamine and other GPCRs to regulate their expression, signaling, subcellular location, and desensitization (8, 9).

The interaction of calnexin with D1 and D2 receptors appears to be complex and dependent upon two different mechanisms. Previously, most studies have suggested that calnexin specifically associates with glycoproteins based on their N-linked oligosaccharide side chains, preferring monoglucosylated oligosaccharides (13). This has been extensively demonstrated for many calnexin-protein interactions where the interaction is completely inhibited in the absence of glycosylation. However, newer data suggest that calnexin may also target and bind non-glycosylated membrane proteins (14, 23, 24). In one case, calnexin has been shown to bind to transmembrane regions of the proteolipid protein (14). These data suggest that calnexin can interact with proteins via glycan-dependent as well as glycan-independent mechanisms. Our current results also support a dual mechanism of interaction of D1 and D2 receptors with calnexin. Receptor mutants lacking N-linked glycosylation sites, as well as inhibiting receptor glycosylation with tunicamycin, diminished but did not completely block the interaction of calnexin with the receptors. Furthermore, the increased association of calnexin with a trafficking-impaired D1 receptor mutant was not affected by inhibiting receptor glycosylation (Fig. 6). Taken together, these results suggest that calnexin associates with D1 and D2 receptors via their protein components, as well as their carbohydrate side chains.


Figure 13
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  		FIGURE 13.
Confocal fluorescence microscopy of D1-GFP and the ER marker pDsRed2-ER after overexpression of calnexin. HEK293T cells were transfected with D1-GFP, pDsRed2, and calnexin as described under "Experimental Procedures." Twenty four hours later, cells were transferred into 35-mm glass bottom culture dishes and then visualized 48 h after transfection. A, visualization of cells at a single line excitation of 488 nm, detecting D1-GFP. Arrows indicate punctate clusters similar to those seen in Fig. 10 with calnexin overexpression. Cells marked with an asterisk indicate cells transfected with D1-GFP but did not co-transfect with either calnexin or DsRed2-ER. B, visualization of cells at x1000 using a single line excitation of 543 nm, detecting DsRed2-ER. The endoplasmic reticulum appears red. C, overlay of A and B, clearly indicating overlap of the punctate clusters found with calnexin overexpression demonstrating that the receptor is localized to the ER. Images are representative of three separate experiments.

 
The protein-protein interaction domains that mediate calnexin association with the D1 and D2 receptors are at present unclear and require further study. Calnexin clearly does not bind to the long carboxyl terminus of the D1 receptor, as an increased association was observed with the Thr-347 mutant where the carboxyl region is almost completely eliminated. Calnexin contains a single transmembrane spanning domain (34), and it is conceivable that calnexin could interact with D1/D2 receptors, or other GPCRs, via their transmembrane domains as has been demonstrated for the proteolipid protein (14). Alternatively, calnexin may interact with other nonmembranous regions of the receptors. These possibilities are not mutually exclusive and are currently under investigation.

The fact that calnexin can interact with glycoproteins via two distinct mechanisms raises the possibility that these different interactions may serve different functions. Although calnexin functions as a chaperone protein to assist in protein folding and assembly, it also serves as an ER retention protein that prevents misfolded proteins from exiting the ER and going to the Golgi. Prolonged retention in the ER subjects the glycoprotein to a quality control process involving further modification by slow acting {alpha}-mannosidase and shuttling to an ER-associated degradation (ERAD) pathway (35, 36). Interestingly, we found that the increased interaction of calnexin with a trafficking-impaired mutant of the D1 receptor was insensitive to glycosylation inhibitors (Fig. 6), suggesting that the increased association was because of protein-protein interactions. Conversely, inhibiting glycosylation of the wild-type D1 receptor decreased calnexin interactions and cell surface trafficking. It is thus tempting to speculate that the glycan-dependent receptor-calnexin interactions may mediate the chaperone functions of calnexin, whereas the glycan-independent protein-protein interactions may be more relevant to ER retention and quality control.

Further evidence for an ER retention function of calnexin was obtained using calnexin overexpression assays, which resulted in a very distinct pattern of receptor localization as determined by confocal microscopy. Similar staining patterns have been documented previously for cystic fibrosis transmembrane conductance regulator protein subsequent to overexpression of calnexin, where it was determined that the cystic fibrosis transmembrane regulator protein remained in concentric membranous bodies of the ER (37). Our double labeling experiments indeed determined that the D1 receptor is localized in the ER following overexpression of calnexin. This confirms that calnexin acts as an ER retention protein for receptors when it is overexpressed. Interestingly, we see no such patterns when the subcellular location of the receptors is visualized after treatment of the cells with glycosylation inhibitors. Instead, the receptors appear to be in diffuse intracellular clusters located throughout the cell. These data indicate that the mechanism of receptor down-regulation via calnexin overexpression is distinct from that of receptor down-regulation via inhibition of glycosylation. Interestingly, similar results using overexpression of calnexin and other ER retention proteins such as BiP/GRP78 and DRiP78 have been observed (10, 38, 39). Overexpression of these ER chaperone/retention proteins resulted in diminished transport of their substrates from the ER to the Golgi, possibly by shifting the equilibrium away from, or blockade of, a cargo-selection process.

With respect to glycan-dependent mechanisms of the calnexin interaction, it was interesting to note that there were some differences with respect to how this regulated the expression of the D1 and D2 receptors. Inhibiting glycosylation of the D1 receptor diminished its cell surface trafficking. It should be noted that these results contrast with those of Karpa et al. (28) who found that N-linked glycosylation was not required for D1 receptor localization to the plasma membrane. The reason(s) for this discrepancy is not clear, although Karpa et al. (28) utilized the human D1 receptor, while we performed experiments with the rat receptor protein. In contrast to the D1 receptor results, inhibiting glycosylation decreased both cell surface trafficking and the overall cellular expression of the D2 receptor. The reason for this is unknown, but it may involve the differences in glycosylation levels of the two proteins. Although the D1 receptor has only two identified N-linked glycosylation sites, the D2 receptor has four such consensus sequences for N-linked glycosylation. Potentially, the D2 receptor is more heavily glycosylated than the D1 receptor and may be more likely to degrade in the absence of glycosylation.

Another role that calnexin may play in D1 and D2 receptor expression is that of oligomeric assembly in the ER. GPCR dimers are now known to regulate a number of functional properties of these receptors (for reviews see Refs. 40 and 41). One important aspect of dimerization is its potential to play a critical role in the surface trafficking of at least some GPCRs (42–44). The mechanisms underlying GPCR dimerization within the ER remain elusive but may involve folding states of the receptors through interactions with chaperone proteins. Indeed, ER chaperone proteins have been implicated in folding of several receptors for peptide hormones, including V2 vasopressin (45, 46), luteinizing hormone, follicle-stimulating hormone, and thyrotropin receptors (47, 48). Interaction of GPCRs with calnexin may facilitate the formation of dimers prior to trafficking to the cell surface. It is known that D1 and D2 receptors exist as dimers (41) on the cell surface. In our Western blot analyses, both D1 (Fig. 2, A and C) and D2 (Fig. 3) receptors exhibited higher molecular weight species indicative of oligomeric complexes. Interestingly, after co-precipitating the D1 receptor with an anti-calnexin antibody (Fig. 2B), only a lower molecular weight species corresponding to a receptor monomer is observed. This suggests that calnexin may bind exclusively to incompletely assembled monomeric receptors. These data lead to an hypothesis of calnexin acting as a retention protein for individual subunits, assisting in the formation of completely assembled dimeric/oligomeric receptors.

In summary, calnexin is a D1 and D2 dopamine receptor-interacting protein with multifaceted effects on the trafficking and expression of both receptors. Our findings suggest that optimal levels of calnexin interaction are essential for proper expression of fully assembled and functional dopamine receptors on the cell surface. This GPCR expression/trafficking pathway is likely to be under precise cellular control, as alterations have a profound influence on receptor expression and subsequent signaling. These findings further lead to the possibility of using GPCR-chaperone complexes as targets for the manipulation of receptor expression.

Note Added in Proof—A paper by Rosenbaum et al. (Rosenbaum, E. E., Hardie, R. C., and Colley, N. J. (2006) Neuron 49, 229–241) showing that Calnexin is essential for rhodopsin maturation, Ca2+ regulation, and photoreceptor cell survival was published while this manuscript was under review.


   FOOTNOTES
 
* This work was supported in part by the Intramural Research Program of NINDS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of an NINDS Competitive Fellowship from the National Institutes of Health. Back

2 Recipient of an NIGMS Pharmacology Research Associate Program Fellowship. Back

3 To whom correspondence should be addressed: Molecular Neuropharmacology Section, NINDS, National Institutes of Health, 5625 Fishers Ln., Rm. 4S-04, MSC 9405, Bethesda, MD 20892-9405. Tel.: 301-496-9316; Fax: 301-480-3726; E-mail: sibley{at}helix.nih.gov.

4 The abbreviations used are: GPCR, G protein-coupled receptor; ER, endoplasmic reticulum; CAD, Cath.a differentiated cells; GFP, green fluorescent protein; YFP, yellow fluorescent protein; DMEM, Dulbecco's modified essential medium; EBSS, Earle's balanced salt solution; TBST, Tris-buffered saline containing 0.05% Tween 20; IP, immunoprecipitation; MS/MS, tandem mass spectrometry; HRP, horseradish peroxidase; PNGase F, peptide N-glycosidase F; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl) propane-1,3-diol; HPLC, high pressure liquid chromatography. Back


   ACKNOWLEDGMENTS
 
We acknowledge the DNA sequencing facility, NINDS (National Institutes of Health), for generating all sequence data. We also thank Dr. Vanitha Ramakrishnan for the HEK293tsa201 cells, Dr. Qun-Yong Zhou for the rat D1-GFP construct, Dr. Jonathan Javitch for the D2-YFP construct, Dr. David Thomas for the calnexin construct, Dr. Dona Chikaraishi for the CAD cells, and Paul Marinec for confocal microscopy assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lundstrom, K. (2005) IDrugs 8, 909-913[Medline] [Order article via Infotrieve]
  2. Marchese, A., Chen, C., Kim, Y. M., and Benovic, J. L. (2003) Trends Biochem. Sci. 28, 369-376[CrossRef][Medline] [Order article via Infotrieve]
  3. Van Craenenbroeck, K., Clark, S. D., Cox, M. J., Oak, J. N., Liu, F., and Van Tol, H. H. (2005) J. Biol. Chem. 280, 19350-19357[Abstract/Free Full Text]
  4. Duvernay, M. T., Filipeanu, C. M., and Wu, G. (2005) Cell. Signal. 17, 1457-1465[CrossRef][Medline] [Order article via Infotrieve]
  5. Tan, C. M., Brady, A. E., Nickols, H. H., Wang, Q., and Limbird, L. E. (2004) Annu. Rev. Pharmacol. Toxicol. 44, 559-609[CrossRef][Medline] [Order article via Infotrieve]
  6. Sibley, D. R., and Monsma, F. J., Jr. (1992) Trends Pharmacol. Sci. 13, 61-69[CrossRef][Medline] [Order article via Infotrieve]
  7. Missale, C., Nash, S. R., Robinson, S. W., Jaber, M., and Caron, M. G. (1998) Physiol. Rev. 78, 189-225[Abstract/Free Full Text]
  8. Bockaert, J., Fagni, L., Dumuis, A., and Marin, P. (2004) Pharmacol. Ther. 103, 203-221[CrossRef][Medline] [Order article via Infotrieve]
  9. Bergson, C., Levenson, R., Goldman-Rakic, P. S., and Lidow, M. S. (2003) Trends Pharmacol. Sci. 24, 486-492[CrossRef][Medline] [Order article via Infotrieve]
  10. Bermak, J. C., and Zhou, Q. Y. (2001) Mol. Interv. 1, 282-287[Abstract/Free Full Text]
  11. Bai, J., He, F., Novikova, S. I., Undie, A. S., Dracheva, S., Haroutunian, V., and Lidow, M. S. (2004) Biol. Psychiatry 56, 427-440[CrossRef][Medline] [Order article via Infotrieve]
  12. Laurin, N., Misener, V. L., Crosbie, J., Ickowicz, A., Pathare, T., Roberts, W., Malone, M., Tannock, R., Schachar, R., Kennedy, J. L., and Barr, C. L. (2005) Mol. Psychiatry 10, 1117-1125[CrossRef][Medline] [Order article via Infotrieve]
  13. Hammond, C., Braakman, I., and Helenius, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917[Abstract/Free Full Text]
  14. Swanton, E., High, S., and Woodman, P. (2003) EMBO J. 22, 2948-2958[CrossRef][Medline] [Order article via Infotrieve]
  15. Molinari, M., Eriksson, K. K., Calanca, V., Galli, C., Cresswell, P., Michalak, M., and Helenius, A. (2004) Mol. Cell 13, 125-135[CrossRef][Medline] [Order article via Infotrieve]
  16. Labriola, C., Cazzulo, J. J., and Parodi, A. J. (1995) J. Cell Biol. 130, 771-779[Abstract/Free Full Text]
  17. Heinzel, S. S., Krysan, P. J., Calos, M. P., and DuBridge, R. B. (1988) J. Virol. 62, 3738-3746[Abstract/Free Full Text]
  18. Monsma, F. J., Jr., Mahan, L. C., McVittie, L. D., Gerfen, C. R., and Sibley, D. R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6723-6727[Abstract/Free Full Text]
  19. Gardner, B., Liu, Z. F., Jiang, D., and Sibley, D. R. (2001) Mol. Pharmacol. 59, 310-321[Abstract/Free Full Text]
  20. Bermak, J. C., Li, M., Bullock, C., Weingarten, P., and Zhou, Q. Y. (2002) Eur. J. Cell Biol. 81, 77-85[CrossRef][Medline] [Order article via Infotrieve]
  21. Namkung, Y., and Sibley, D. R. (2004) J. Biol. Chem. 279, 49533-49541[Abstract/Free Full Text]
  22. Qi, Y., Wang, J. K., McMillian, M., and Chikaraishi, D. M. (1997) J. Neurosci. 17, 1217-1225[Abstract/Free Full Text]
  23. Danilczyk, U. G., and Williams, D. B. (2001) J. Biol. Chem. 276, 25532-25540[Abstract/Free Full Text]
  24. Wanamaker, C. P., and Green, W. N. (2005) J. Biol. Chem. 280, 33800-33810[Abstract/Free Full Text]
  25. Maley, F., Trimble, R. B., Tarentino, A. L., and Plummer, T. H., Jr. (1989) Anal. Biochem. 180, 195-204[CrossRef][Medline] [Order article via Infotrieve]
  26. McDowell, W., and Schwarz, R. T. (1988) Biochimie (Paris) 70, 1535-1549
  27. Zhang, R., Cai, H., Fatima, N., Buczko, E., and Dufau, M. L. (1995) J. Biol. Chem. 270, 21722-21728[Abstract/Free Full Text]
  28. Karpa, K. D., Lidow, M. S., Pickering, M. T., Levenson, R., and Bergson, C. (1999) Mol. Pharmacol. 56, 1071-1078[Abstract/Free Full Text]
  29. Meissner, J. D., Naumann, A., Mueller, W. H., and Scheibe, R. J. (1999) Biochem. J. 338, 561-568[CrossRef][Medline] [Order article via Infotrieve]
  30. Kim, O. J., Gardner, B. R., Williams, D. B., Marinec, P. S., Cabrera, D. M., Peters, J. D., Mak, C. C., Kim, K. M., and Sibley, D. R. (2004) J. Biol. Chem. 279, 7999-8010[Abstract/Free Full Text]
  31. Elbein, A. D. (1991) FASEB J. 5, 3055-3063[Abstract]
  32. Keller, S. H., Lindstrom, J., and Taylor, P. (1996) J. Biol. Chem. 271, 22871-22877[Abstract/Free Full Text]
  33. Ermonval, M., Duvet, S., Zonneveld, D., Cacan, R., Buttin, G., and Braakman, I. (2000) Glycobiology 10, 77-87[Abstract/Free Full Text]
  34. Helenius, A., and Aebi, M. (2004) Annu. Rev. Biochem. 73, 1019-1049[CrossRef][Medline] [Order article via Infotrieve]
  35. Hebert, D. N., Garman, S. C., and Molinari, M. (2005) Trends Cell Biol. 15, 364-370[CrossRef][Medline] [Order article via Infotrieve]
  36. Sitia, R., and Braakman, I. (2003) Nature 426, 891-894[CrossRef][Medline] [Order article via Infotrieve]
  37. Okiyoneda, T., Harada, K., Takeya, M., Yamahira, K., Wada, I., Shuto, T., Suico, M. A., Hashimoto, Y., and Kai, H. (2004) Mol. Biol. Cell 15, 563-574[Abstract/Free Full Text]
  38. Jackson, M. R., Cohen-Doyle, M. F., Peterson, P. A., and Williams, D. B. (1994) Science 263, 384-387[Abstract/Free Full Text]
  39. Reddy, P. S., and Corley, R. B. (1998) BioEssays 20, 546-554[CrossRef][Medline] [Order article via Infotrieve]
  40. Bouvier, M. (2001) Nat. Rev. Neurosci. 2, 274-286[CrossRef][Medline] [Order article via Infotrieve]
  41. George, S. R., O'Dowd, B. F., and Lee, S. P. (2002) Nat. Rev. Drug Discov. 1, 808-820[CrossRef][Medline] [Order article via Infotrieve]
  42. Lee, S. P., O'Dowd, B. F., Ng, G. Y., Varghese, G., Akil, H., Mansour, A., Nguyen, T., and George, S. R. (2000) Mol. Pharmacol. 58, 120-128[Abstract/Free Full Text]
  43. Salahpour, A., Angers, S., Mercier, J. F., Lagace, M., Marullo, S., and Bouvier, M. (2004) J. Biol. Chem. 279, 33390-33397[Abstract/Free Full Text]
  44. Bulenger, S., Marullo, S., and Bouvier, M. (2005) Trends Pharmacol. Sci. 26, 131-137[CrossRef][Medline] [Order article via Infotrieve]
  45. Morello, J. P., Salahpour, A., Laperriere, A., Bernier, V., Arthus, M. F., Lonergan, M., Petaja-Repo, U., Angers, S., Morin, D., Bichet, D. G., and Bouvier, M. (2000) J. Clin. Investig. 105, 887-895[Medline] [Order article via Infotrieve]
  46. Morello, J. P., Salahpour, A., Petaja-Repo, U. E., Laperriere, A., Lonergan, M., Arthus, M. F., Nabi, I. R., Bichet, D. G., and Bouvier, M. (2001) Biochemistry 40, 6766-6775[CrossRef][Medline] [Order article via Infotrieve]
  47. Siffroi-Fernandez, S., Giraud, A., Lanet, J., and Franc, J. L. (2002) Eur. J. Biochem. 269, 4930-4937[Medline] [Order article via Infotrieve]
  48. Rozell, T. G., Davis, D. P., Chai, Y., and Segaloff, D. L. (1998) Endocrinology 139, 1588-1593[Abstract/Free Full Text]

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