Reciprocal Modulation of Function between the D1 and D2 Dopamine Receptors and the Na+,K+-ATPase*

It is well documented that dopamine can increase or decrease the activity of the Na+,K+-ATPase (NKA, sodium pump) in an organ-specific fashion. This regulation can occur, at least partially, via receptor-mediated second messenger activation and can promote NKA insertion or removal from the plasma membrane. Using co-immunoprecipitation and mass spectrometry, we now show that, in both brain and HEK293T cells, D1 and D2 dopamine receptors (DARs) can exist in a complex with the sodium pump. To determine the impact of NKA on DAR function, biological assays were conducted with NKA and DARs co-expressed in HEK293T cells. In this system, expression of NKA dramatically decreased D1 and D2 DAR densities with a concomitant functional decrease in DAR-mediated regulation of cAMP levels. Interestingly, pharmacological inhibition of endogenous or overexpressed NKA enhanced DAR function without altering receptor number or localization. Similarly, DAR function was also augmented by small interfering RNA reduction of the endogenous NKA. These data suggest that, under basal conditions, NKA negatively regulates DAR function via protein-protein interactions. In reciprocal fashion, expression of DARs decreases endogenous NKA function in the absence of dopamine, implicating DAR proteins as regulators of NKA activity. Notably, dopamine stimulation or pertussis toxin inhibition of D2 receptor signaling did not alter NKA activity, indicating that the D2-mediated decrease in NKA function is dependent upon protein-protein interactions rather than signaling molecules. This evidence for reciprocal regulation between DARs and NKA provides a novel control mechanism for both DAR signaling and cellular ion balance.

array of dopaminergic processes throughout the body. DARs are subdivided into two families based on their pharmacological and genetic profiles. The D1 family of receptors, containing the D 1 and D 5 subtypes, couple to the heterotrimeric G-protein G s and positively regulate adenylyl cyclase activity. The D2 family of DARs consists of the D 2 , D 3 , and D 4 receptor subtypes. These receptors couple to inhibitory G i/o proteins and reduce adenylyl cyclase activity. Within the central nervous system, dopamine and its receptors regulate locomotion, addiction, cognition, and learning and memory formation (1)(2)(3)(4)(5); nonneuronal DARs modulate blood pressure and digestive function (6 -8). Dysregulation of dopaminergic pathways and receptors has been implicated in several diseases, including schizophrenia, Parkinson disease, and hypertension. Despite the breadth of dopaminergic functions and disorders, the molecular control and coordination of dopamine signaling are still being elucidated.
It is now appreciated that DARs and other G-protein-coupled receptors can couple to a variety of proteins to form a large protein complex termed the "signalplex" (9 -11). Identification of the numerous protein partners in the signalplex is critical to understanding the molecular mechanisms responsible for tissue-specific dopamine functions and various dopaminergic disorders. Several dopamine receptor-interacting proteins (DRIPs) have recently been discovered (10,12). These identified DRIPs regulate a variety of functions, including cell signaling and receptor trafficking and retention. Using co-immunoprecipitation coupled with mass spectroscopy analysis, we have now identified the ␣1 subunit of the Na ϩ ,K ϩ -ATPase (sodium pump, NKA) as a DRIP in both D 1 and D 2 DAR signalplexes.
The NKA is a ubiquitous membrane protein that actively hydrolyzes ATP to maintain the Na ϩ /K ϩ gradient across the plasma membrane. The NKA consists of a large, catalytic ␣ subunit and a smaller ␤ subunit. The ␣1 subunit is expressed in virtually all tissues, including neurons and glia in the central nervous system (13). Neuronally, the NKA is responsible for maintenance of resting membrane potential, including restoration of membrane potential following depolarization (14).
Several investigators have demonstrated dopaminergic control of the NKA in primary striatal, lung, and kidney cells (15)(16)(17)(18)(19)(20). However, in all of these studies, dopamine and other neurotransmitter receptor agonists appeared to regulate the NKA via signal transduction pathways; second messenger kinases and downstream signaling caused insertion or removal of the NKA at the plasma membrane (15, 17, 20 -23). Our current data now provide the first evidence of a direct interaction between these two proteins. Furthermore, we show that the DARs and NKA are able to functionally regulate one another via protein-protein interactions in the absence of ligands or downstream signaling events. Our results indicate that, in addition to traditional second messenger-mediated communication, the DARs and NKA can associate in a complex to provide a more rapid and immediate response to external stimuli or changing the cellular environment.
Cell Culture and Transfection-HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 50 g/ml streptomycin, 50 units/ml penicillin, and 10 g/ml gentamycin. Cells were grown at 37°C in 5% CO 2 and 90% humidity. HEK293T cells were transfected using a calcium phosphate precipitation kit (Clontech) according to the manufacturer's instructions.
Several expression constructs were used in this study. An expression construct containing only the FLAG peptide (FLAG-tag), pCMV-Tag2B, was obtained from Stratagene (La Jolla, CA). The D 2L DAR expression construct containing an amino-terminal FLAG-tag epitope in pSR-D 2L was created by our laboratory as described previously (25). An amino-terminal FLAG epitope-tagged construct for the rat D 1 DAR (26) was created and named pSFD 1 , as reported previously by our laboratory (27); additionally, a FLAG-tagged D 1 receptor truncated at amino acid 347 and missing most of the carboxyl terminus (designated FLAG-D 1 -T 0 ) was created by our laboratory as described previously (28). A red fluorescent protein (RFP)tagged rat D 2L receptor was created by our laboratory using the pSR-D 2L construct and pRFP (Evrogen, Moscow, Russia). Human NKA␣1 in pCMV was obtained from OriGene (Rockville, MD), and rat enhanced green fluorescent protein (eGFP)tagged NKA␣1 was a generous gift from Dr. Alejandro Bertorello (19). Plasmid small interfering RNA (siRNA) directed against the NKA␣1 subunit and scrambled siRNA were synthesized by Retrogen (San Diego, CA) in pSilencer 3.1-H1 vector.
Immunoprecipitation and Gel Electrophoresis-For HEK293T cells, cells were removed from culture flasks in calcium-free Earle's balanced salt solution (EBSS) and collected by centrifugation (200 ϫ g). Pelleted cells 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) with Complete-Mini protease inhibitor mixture tablets (Roche Applied Science) and incubated on ice for 1 h. The lysate was centrifuged at 30,000 ϫ g for 30 min to remove insoluble cell debris and then pre-cleared via incubation with protein G-agarose (Invitrogen) for 3 h. The agarose was removed by centrifugation (7,000 ϫ 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 via resuspension and re-pelleting in solubilization buffer (7,000 ϫ g, 5 min). The agarose was then washed twice in 1ϫ TE buffer, pH 7.4. Proteins were eluted from the beads using NuPAGE LDS sample buffer (Invitrogen) at 37°C. Agarose was removed via centrifugation, and proteins were separated on 4 -12% BisTris NuPAGE gel (Invitrogen) according to the manufacturer's instructions. For bovine corpus striatum, diced striatum was Polytron-homogenized for ϳ1 min in 10 mM Tris-HCl ϩ 1% CHAPS, pH 7.5, with Complete-Mini protease inhibitor mixture tablets and then incubated on ice for 1 h. Solubilized striatum was centrifuged at 30,000 ϫ g for 10 min to remove insoluble cell debris; the resulting supernatant was centrifuged for an additional 30 min (30,000 ϫ g). The supernatant was then pre-cleared via incubation with protein G-agarose for 3 h at 4°C; agarose was removed by centrifugation (7,000 ϫ g, 5 min) and discarded. Protein concentrations of the sample supernatants were determined using the BCA protein assay (Pierce). Cell lysate (500 g), anti-D 2S/L antibody (4 g), and Antibody Capture Affinity ligand were added according to the manufacturer's instructions to washed Catch and Release kit beads (Pierce) and incubated overnight at 4°C. Beads were washed three times with Catch and Release kit buffer, 3ϫ with Tris-HCl/CHAPS, and an additional three times with Catch and Release kit buffer. Proteins were eluted from beads according to the manufacturer's instructions, heated at 37°C for 1 h, and separated on 4 -12% BisTris NuPAGE gel.
Western Blotting and Gel Staining-Proteins separated by denaturing SDS-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-D 1 dopamine receptor (clone 1-1-F11 S.E6, catalogue number D187, Sigma), rabbit polyclonal anti-D 2S/L (catalogue number AB5084P, Chemicon Millipore, Billerica, MA), and mouse monoclonal anti-NKA␣1 (clone C464.6, catalogue number 05-369, Upstate Millipore, Billerica, MA). After washing with Tris-buffered saline containing 0.1% Tween, membranes were incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA; antirat for the D 1 , anti-rabbit for D 2 , and anti-mouse for NKA␣1). Proteins were visualized via the SuperSignal West Pico chemiluminescent substrate (Pierce) according to the manufacturer's instructions, and images were recorded on film or computer using the UVP Bioimaging Systems EpiChemi 3 Darkroom (UVP, Upland, CA). In some experiments, blots were stripped of all antibody using Restore Western blot stripping buffer (Pierce) according to the manufacturer's instructions. Removal of all primary antibody by the buffer was checked via incubation with HRP-secondary followed by ECL and film exposure. In experiments where total protein was examined, gels were stained using colloidal Coomassie SimplyBlue Safe Stain (Invitrogen).
Mass Spectrometry (MS)-The identification of PAGE-resolved proteins by mass spectroscopy was carried out by Prot-Tech, Inc. (Norristown, PA). In brief, each protein gel band was digested "in-gel" with modified sequencing grade trypsin (Promega, Madison, WI), and the resulting peptide mixture was subjected to tandem mass spectrometry for peptide sequencing. A Finnigan ion trap mass spectrometer LCQ coupled with a high performance liquid chromatography system running a 75 m inner diameter 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 database and GenBank TM using ProtQuest software suite from ProtTech. The output of the data base search was manually analyzed and validated to verify proper protein identification.
Confocal Microscopy-HEK293T cells were plated in 100-mm tissue culture dishes (1 ϫ 10 6 cells) and transfected as described above with 1 g of rat D 2L -RFP or 0.5 g of rat NKA␣1-eGFP, either separately or co-transfected with both constructs for dual-labeling experiments. Twenty four hours post-transfection, cells were re-seeded onto poly-D-lysinecoated 35-mm glass-bottom culture dishes and maintained for an additional 48 h. Confocal microscopy experiments were performed on a Zeiss laser scanning confocal microscope (LSM 5 Pascal). Images were collected using single line excitation (488 nm for NKA␣1-eGFP or 543 nm for D 2L -RFP).
Membrane Radioligand Binding Assays-HEK293T cells were plated in 150-mm tissue culture dishes (5 ϫ 10 6 cells) and transfected as described above. Forty eight hours later, the cells were removed mechanically using EBSS. For experiments involving ouabain treatment, a final concentration of 5 mM ouabain was added to cells, and dishes were incubated at 37°C for 15 min prior to removal. Intact cells were pelleted and then lysed with 5 mM Tris-HCl and 5 mM MgCl 2 , pH 7.4. Homogenates were centrifuged at 20,000 ϫ g for 30 min. The membranes were resuspended with 50 mM Tris-HCl, pH 7.4. Membrane preparations were incubated for 90 min at room temperature with various concentrations of [ 3 H]SCH23390 (D 1 binding) or [ 3 H]methylspiperone (D 2 binding) in a reaction volume of 1 ml. Nonspecific binding was determined in the presence of 4 M (ϩ)-butaclamol. Bound ligand was separated from unbound 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 at 200,000 cells/well and cultured for 1 day prior to the experiment. For D 1 accumulation assays, cells were washed three times with 400 l of EBSS per well. Various concentrations of dopamine dissolved in stimulation buffer (Dulbecco's modified Eagle's medium, 20 M Ro-20-1724, 0.2 mM sodium metabisulfite) were added to each well in a volume of 400 l for 10 min at 37°C. For D 2 inhibition assays, cells were washed three times with 400 l of EBSS per well. Various concentrations of dopamine containing 3 M forskolin and 10 M propranolol in stimulation buffer were added to each well in a volume of 400 l for 10 min at 37°C. For experiments involving ouabain treatment, stimulation buffer (for either D 1 or D 2L experiments) was prepared with or without 5 mM ouabain. Cells were incubated at 37°C for 5 min in the ouabain or control stimulation buffer prior to dopamine addition. Dopamine solutions were prepared in appropriate stimulation buffer for the experiment. The reactions were terminated by adding 200 l of 3% perchloric acid and incubated on ice for 30 min. 80 l of 15% KHCO 3 was then added to neutralize the acid. The plates remained on ice for an additional 20 min and were centrifuged at 1,300 ϫ g for 20 min. 50 l of the supernatant from each well was transferred to a 1.2-ml reaction tube containing 250 l of purified cAMP-dependent protein kinase (PKA) in Tris/EDTA buffer and 50 l of [ 3 H]cAMP. The reaction was incubated for 90 min at 4°C. After the incubation, 250 l 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 ϫ g) for 20 min. Radioactivity in the supernatant was quantified by liquid scintillation spectroscopy at a counting efficiency of 60%. cAMP concentrations were determined using a standard assay curve of cAMP values from 0.1 to 100 pmol.
Intact Cell Binding Assays-Transfected HEK293T cells were seeded into poly(D)-lysine-coated 24-well plates at 200,000 cells/well. Twenty four hours later, cells were washed once with EBSS, and binding was performed by incubation for 15 min at 37°C in 500 l of EBSS containing increasing concentrations of [ 3 H]sulpiride with or without 5 mM ouabain. After incubation, the binding buffer was aspirated, and the plates were washed three times with EBSS. The cells were dissolved in 500 l of 1 mM EDTA containing 1% Triton X-100, transferred to vials, and quantified for radioactivity by liquid scintillation spectroscopy at a counting efficiency of 60%. Specific binding was defined as the difference between total and nonspecific binding, with the latter measured in the presence of 5 M (ϩ)butaclamol.
Rubidium-86 Ion Uptake-HEK293T cells were plated in 150-mm tissue culture dish (5 ϫ 10 6 cells) and transfected as described above. Forty eight hours later the cells were removed mechanically using EBSS. Intact cells were pelleted and then resuspended in 1.5 ml of assay buffer (5 mM HEPES-Tris, 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 5 mM glucose; 0.1 mM furosemide and 0.01 mM monensin were added just prior to experimentation) with or without 5 mM ouabain. Cells were incubated at 37°C for 10 min in assay buffer alone, then 2.5 Ci of 86 Rb ϩ in assay buffer was added, and samples were incubated for an additional 15 min at 37°C. Uptake was terminated by addition of 500 l of ice-cold 10 mM HEPES, 100 mM MgCl 2 followed by centrifugation at 3,000 ϫ g. Pellets were washed a total of three times with HEPES/MgCl 2 , and cells were then lysed with 500 l of NaOH and neutralized with 50 l of concentrated HCl. Radioactivity was analyzed via liquid scintillation spectroscopy at a counting efficiency of 60%. Protein concentration was determined using a Bradford protein assay (Bio-Rad).

RESULTS
Identification of the NKA as an Interacting Protein with Both D 1 and D 2 DARs-As part of ongoing efforts in DRIP discovery, co-immunoprecipitation assays of epitope-tagged rat D 2L receptors were coupled with MS analysis to identify D 2 DARinteracting proteins. For these experiments, HEK293T cells (which do not endogenously express the D 2 DAR) were transiently transfected with constructs expressing either a FLAG peptide (FLAG tag) alone or a FLAG-D 2L receptor. Proteins from solubilized cells were immunoprecipitated with anti-FLAG antibody-conjugated agarose, and the precipitated proteins were analyzed by SDS-PAGE. Staining with colloidal Coomassie Blue dye revealed multiple proteins that were precipitated from both transfection conditions ( Fig. 1). All visible protein bands from the FLAG-D 2L lane (Fig. 1, right lane) were excised, along with corresponding control bands (FLAG tag, Fig. 1, left lane) for comparison and selection purposes. The excised bands were digested with trypsin, and the eluted proteins were subjected to liquid chromatography-MS/MS tandem mass spectroscopy as described under "Experimental Procedures"; all identified proteins from this experiment are listed in Table 1. Proteins that were identified in both sets of immunoprecipitates were considered nonspecific interactors; no identified proteins were unique to the FLAG tag control immunoprecipitates. The NKA was identified in these experiments as a specific interacting protein with the FLAG-tagged rat D 2L receptor and was chosen for further study. Similar liquid chromatography-MS/MS experiments that were conducted with the FLAG-D 1 receptor revealed the NKA as a putative specific interacting protein with this receptor as well (29). Multiple nonoverlapping peptide fragments from both the D 2L protein ( Fig. 1, Band 13) and the NKA protein ( Fig. 1, Band 3) were identified from the FLAG-D 2L receptor bands; the sequence of these peptides is reported in Table 2. These proteins were absent from the corresponding control FLAG tag lanes (Fig. 1). The same NKA peptides, and three additional peptide fragments from the NKA, were identified from FLAG-D 1 receptor immunoprecipitated samples. These peptides, as well as identified D 1 receptor peptide sequences, are listed in Table 2.
Verification and Characterization of DAR Interactions with NKA␣1-To verify the mass spectroscopy results, the DAR-NKA interactions were investigated by co-immunoprecipitation followed by Western blot analysis. For these experiments, HEK293T cells (which endogenously express the NKA␣1 but do not natively express the D 1 or D 2 DARs) were transiently transfected with the FLAG tag alone, the FLAG-D 1 receptor, or the FLAG-D 1 receptor along with the NKA␣1. Probing the Western blots with an anti-D 1 receptor antibody showed that the D 1 receptor was present only in cells that were transfected with the FLAG-D 1 expression construct ( Fig. 2A, left panel). Interestingly, overexpression of NKA␣1 caused an apparent decrease in D 1 receptor number ( Fig. 2A, left panel, ϳ50 kDa in size). The Western blot in Fig. 2A was stripped and re-probed with an antibody against NKA␣1 ( Fig. 2A, right panel, migrating at ϳ95 kDa in size), revealing that endogenous NKA␣1 only immunoprecipitates with the D 1 receptor and not with the FLAG tag alone. Furthermore, overexpression of NKA␣1 caused a proportionate increase in the amount of NKA interacting with the D 1 receptor, despite the fact that less D 1 receptor was present in these samples.  Table 1. Band number indicates the position of bands that were determined by MS to contain NKA␣1 subunit or rat D 2L receptor peptides as reported in Table 2.

TABLE 1
Proteins identified by mass spectroscopy that co-immunoprecipitated with the D 2 dopamine receptor as shown in Fig. 1 Accession numbers for identified proteins are reported in parentheses.

Specific rat D 2L interactors Nonspecific interactors
a Band number refers to location of band on Coomassie-stained gel in Fig. 1.
The carboxyl terminus of the D 1 receptor is a common site for protein interactions (30 -32). A D 1 receptor construct truncated at amino acid 347 to lack the carboxyl terminus (T 0 ) was used to determine whether this region might be the location of the NKA-D 1 receptor interaction. Because the D 1 T 0 receptor construct does not express as well as the wild-type receptor, all co-immunoprecipitated samples had to be normalized for D 1 receptor expression by radioligand binding. After normalizing for expression levels, the samples were resolved by SDS-PAGE, and resultant Western blots were probed with anti-NKA␣1 antibody (Fig. 2B). The NKA␣1-D 1 interaction remained intact despite the removal of the D 1 receptor carboxyl terminus, indicating that this is not the region of interaction between these two proteins.
To verify the original mass spectroscopy findings of the D 2 -NKA interactions, similar co-immunoprecipitation experiments were also conducted with the D 2 receptor. In Fig. 3, HEK293T cells expressing the FLAG tag, FLAG-D 2L alone, or FLAG-D 2L along with NKA␣1 were solubilized, immunoprecipitated with anti-FLAG-agarose, and resolved by SDS-PAGE. The Western blots from these experiments were probed with antibodies against the D 2 receptor (Fig. 3, left panel, migrating as a glycosylated protein ϳ50 -65 kDa in size), then stripped, and re-probed with anti-NKA␣1 antibodies (Fig. 3, right panel, ϳ95 kDa in size). Similar to the D 1 receptor experiments, there was a decrease in D 2L receptor numbers when NKA␣1 was overexpressed in these cells. There was also an increase in NKA-D 2L association when the NKA␣1 subunit was overexpressed, despite the apparent decrease in D 2L receptor protein levels. In addition, there was no NKA immunoreactivity in the FLAG tag lane, indicating that the presence of NKA is receptorspecific in these experiments. Reverse co-immunoprecipitation assays were also performed from HEK293T cells. In these experiments, cells transfected with empty vector, FLAG-D 2L , or FLAG-D 2L along with NKA␣1 were solubilized, and antibodies against the NKA␣1 were used to immunoprecipitate the NKA-D 2L complex. The NKA␣1 and D 2L proteins were both present in Western blots from these experiments (data not shown).

IP:
Anti-FLAG Anti-FLAG Anti-FLAG Transfection: Of note, no visible change in endogenous NKA␣1 expression was observed when the FLAG-D 2L receptor was overexpressed (supplemental Fig. 1). In these experiments, cells were transfected with empty vector, FLAG-D 2L alone, NKA␣1 alone, or FLAG-D 2L along with NKAa1. Cells were solubilized, and total lysates were resolved by SDS-PAGE, and Western blots were probed with antibodies against the NKA␣1 or D 2L proteins. Importantly, D 2L DAR transfection produced no change in the expression levels of either endogenous or overexpressed NKA␣1 (supplemental Fig. 1).
Endogenous proteins from brain tissue were also investigated to ensure that the interaction between D 2 DAR and NKA␣1 was not unique to HEK293T cells. In these experiments, bovine striatum was used to enrich the D 2 receptor. The striatum was solubilized and immunoprecipitated with either anti-D 2 antibody or species-matched, pre-immune serum using the Catch and Release kit as described under "Experimental Procedures." The resultant Western blot was probed with anti-D 2 antibody and revealed a less glycosylated form of the receptor migrating at ϳ52 kDa (Fig. 4, left panel). The blot was stripped of all antibodies and re-probed with antibody against NKA␣1 (Fig. 4, right panel, migrating at ϳ95 kDa). In both Western blots, the proteins were not detected when control, pre-immune serum was used for the immunoprecipitation. These data indicate that the D 2 receptor and NKA␣1 subunit interact in a physiological setting with native levels of protein.
Confocal microscopy was employed to investigate D 2 DAR and NKA␣1 co-localization. In these experiments, HEK293T cells were co-transfected with fluorescent-tagged rat D 2L -RFP and rat NKA␣1-GFP, enabling subcellular visualization of the proteins. These experiments revealed that, in HEK293T cells, both the D 2L -RFP (Fig. 5A) and the NKA␣1-GFP (Fig. 5B) are predominantly expressed at the plasma membrane, and are similarly distributed when either fluorescent-tagged protein is expressed alone (supplemental Fig. 2). Of note, when the images are merged, it is clear that the two proteins co-localize at the cell surface (Fig. 5C). These data further support the finding of a D 2L -NKA␣1 protein complex.
Overexpression of NKA␣1 Decreases D 1 and D 2 Receptor Function and Density-To investigate the impact of NKA␣1 on DAR function and expression, HEK293T cells were transiently transfected with D 2L receptor cDNA and evaluated for cAMP levels in the presence of increasing concentrations of dopamine (Fig. 6A, solid line). In parallel experiments, cells expressing both the D 2L receptor and the NKA␣1 subunit were treated with various concentrations of dopamine, and inhibition of cAMP was evaluated (Fig. 6A, dashed line). Overexpression of the NKA␣1 subunit caused a marked inhibition of D 2L DAR function. The maximal dopamine-induced inhibition of cAMP decreased by ϳ30% in the presence of NKA␣1, and the EC 50 was significantly shifted to lower affinity. A similar decrease in efficacy and potency was observed for the D 1 receptor following overexpression of the NKA␣1 subunit (Fig. 6B). The maximum dopamine-stimulated increase in cAMP was diminished by ϳ20%, and the EC 50 was significantly shifted to a lower affinity.
Because receptor expression can impact function, the influence of NKA␣1 on DAR expression was investigated. For these experiments, total receptor density was examined in transiently transfected HEK293T cells. For both the D 1 and D 2L receptors, overexpression of the NKA␣1 subunit caused a significant decrease in total receptor density ( Fig. 7) with no significant change in DAR affinity for radioligand. Total D 2L density was reduced by ϳ80% (Fig. 7A), and D 1 density was decreased by ϳ60% (Fig. 7B) when NKA␣1 was co-expressed. As a control experiment, we co-transfected a plasmid expressing RFP, which is not predicted to interact with the DARs. Co-expression of RFP did not significantly alter the expression density of the receptors as determined using saturation radioligand binding assays for both D 1 and D 2 DARs (data not shown). These  results indicate that the diminished DAR number following NKA expression is specific to the NKA. Inhibition of NKA Function Enhances DAR Activity without Altering DAR Density-The cardiotonic steroid ouabain is known to inhibit NKA function (33). Ouabain was used to determine whether the activity or the presence of NKA was responsible for the observed decrease in DAR expression and function. An acute 15-min treatment with ouabain successfully inhibits NKA activity (22) without decreasing the expression of the protein. This short term ouabain treatment was used in all experiments to investigate the impact of NKA activity on DAR function and binding parameters. A relatively high concentration of ouabain (5 mM) was employed in these experiments for several reasons. First, the rat NKA␣1, which is more resistant to ouabain (34), was used in confocal microscopy analysis (see below). Because ouabain experiments with this NKA␣1-GFP construct require a higher dose of drug, the same ouabain concentration was employed for all assays to minimize confounding experimental variables. In addition, lower concentrations of ouabain (1 and 10 M) were also employed in DAR functional assays (data not shown), and we found that the concentration of ouabain did not significantly impact the observed D 1 or D 2 DAR functional results.
In functional assays with the D 2L receptor, ouabain treatment of cells overexpressing the NKA restored dopamine-induced cAMP inhibition to levels comparable with D 2L alone (Fig. 8A). Moreover, inhibition of endogenous NKA with ouabain enhanced D 2L function, causing a greater decrease in cAMP levels as compared with vehicle-treated D 2L (ϳ60% decrease in cAMP with ouabain treatment versus ϳ40% with vehicle; Fig. 8A). Similar results were obtained when ouabain concentrations of 1 or 10 M were used (data not shown), indicating that the potential toxicity of 5 mM ouabain treatment is   DECEMBER 26, 2008 • VOLUME 283 • NUMBER 52 not responsible for the observed decrease in cAMP levels. To ascertain whether the NKA had a similar influence on D 1 receptor function, cells expressing both the D 1 and NKA were treated with ouabain. In these experiments, inhibition of overexpressed NKA caused ϳ15% increase in maximal D 1 response as compared with cells that were not treated with ouabain (Fig. 8B). Likewise, inhibiting the endogenous NKA with ouabain also enhanced D 1 function by ϳ15% (Fig. 8B); similar results were obtained when 10 M ouabain was used (data not shown). Complete E max and EC 50 data for D 1 and D 2 receptors are presented in Table 3. These results for both D 1 and D 2 receptors suggest that endogenous NKA in HEK293T cells is able to regulate transfected DAR function. Interestingly, although ouabain treatment was able to surmount the NKA-induced decrease in E max , it did not alter the rightward shift in EC 50 that was observed for both D 1 and D 2 receptors after NKA overexpression (Table 3).

Reciprocal Regulation of Function between DARs and NKA
Total receptor binding assays were conducted in the presence and absence of ouabain to determine whether alterations in DAR density were responsible for the ouabain-induced increase in DAR function. Ouabain did not significantly increase D 1 or D 2 receptor density (Table 4); however, the treatment with ouabain was brief (15 min), and it is questionable that a significant increase in DAR number would occur in such a short time. Within this time frame, however, it might be possible to increase the surface expression of any intracellularly stored receptors, thus increasing the functional receptor pool. To investigate this possibility, the hydrophilic ligand [ 3 H]sulpiride was used to determine the number of D 2L receptors on the cell surface using intact cell binding assays (Fig. 9). No significant increase in cell-surface D 2L expression was observed after a 15-min ouabain treatment for cells with either endogenous or overexpressed NKA ( Fig. 9; summarized data in Table 4). These data imply that ouabain treatment does not increase the number of functional receptors at the cell surface.  Table 3.

and D 2 DAR regulation of cAMP production following NKA expression and ouabain treatment
cAMP accumulation assays were performed as described in Fig. 7. D 2 DAR data are reported as percent dopamine-induced inhibition following forskolin stimulation of cAMP accumulation. C.I. means confidence interval. All statistical analyses were determined by one-way ANOVA with Bonferroni's post test.  a D 2 DAR binding in membrane preparations was determined with ͓ 3 H͔methylspiperone as described in Fig. 6. b D 1 DAR binding in membrane preparations was determined with ͓ 3 H͔SCH23390 as described in Fig. 6. c Values are p Ͻ 0.01 as compared with control. d Values are p Ͻ 0.001 as compared with control. e Values are p Ͻ 0.01 as compared with ϩNKA. f D 2 DAR surface binding was determined with ͓ 3 H͔sulpiride using intact cell binding assays as described in Fig. 8. g ND means not determined.
The possibility that either ouabain or dopamine could effectively alter the interaction between NKA and DAR was investigated by immunoprecipitating the DAR after drug treatment and probing Western blots with antibodies against the NKA␣1 or the appropriate DAR. These experiments showed no apparent change in the relative amount of NKA, endogenous or overexpressed, interacting with the DAR after either drug treatment (data not shown). In addition, confocal microscopy was used to investigate the DAR-NKA interaction after ouabain or dopamine treatment. HEK293T cells were co-transfected with rat D 2L -RFP and NKA␣1-GFP, and images were obtained before and after a 15-min treatment with 5 mM ouabain (supplemental Fig. 3) or before and after a 10-min treatment with 10 M dopamine (supplemental Fig. 4). Before ouabain treatment, both proteins clearly co-localize at the plasma membrane (supplemental Fig. 3A). Fifteen minutes after ouabain treatment, there was no change in D 2 DAR or NKA distribution (supplemental Fig. 3B), indicating that the observed increase in DAR function is not caused by either a decrease in surface expression of NKA or an increase in surface expression of D 2 DAR. Similarly, 10 min of dopamine treatment does not induce any observable changes in either NKA or D 2L DAR distribution (supplemental Fig. 4, A and B), further indicating that dopamine treatment does not disrupt the DAR-NKA interaction. Taken together, these data imply that inhibiting NKA activity or stimulating the DARs does not disrupt the DAR-NKA interactions. More importantly these results indicate that NKA association with the D 1 and D 2 DARs leads to decreased signaling through a novel mechanism that is independent of receptor density or trafficking.
siRNA Inhibition of NKA Enhances DAR Activity-Because ouabain can have toxic effects in some cells, and because ouabain treatment only examines short term inhibition of the NKA, siRNA inhibition of NKA␣1 was also utilized. HEK293T cells were co-transfected with FLAG-D 2L and empty vector, FLAG-D 2L and scrambled siRNA, or FLAG-D 2L and anti-NKA␣1 siRNA. After 48 or 72 h the FLAG-D 2L was immunoprecipitated with anti-FLAG agarose and subjected to SDS-PAGE separation, and resulting Western blots were probed with antibody against the NKA␣1. A 48-h treatment of cells with anti-NKA␣1 siRNA essentially abolished the NKA␣1-D 2L interaction (Fig. 10). Because 48 h of treatment with siRNA was more effective at eliminating the NKA-DAR interaction than 72 h of treatment (Fig. 10), the 48-h time point was used for all subsequent experiments. The amount of total NKA␣1 in whole cell lysates after siRNA treatment was also investigated. In these experiments, cells were transfected as above, and after 48 h, cell lysates were prepared and analyzed by SDS-PAGE followed by Western blotting. Probing Western blots for the NKA␣1 subunit revealed an approximate 50% decrease in total levels of NKA␣1 after inhibition with siRNA as compared with cells transfected with scrambled siRNA or empty vector (data not shown). This partial knockdown likely allows for better cell survival, as the NKA is critical for maintaining cellular ionic equilibrium. More importantly, the interaction between the NKA and the D 2L DAR was essentially eliminated by siRNA inhibition (Fig. 10), thus facilitating specific investigation of the role of the NKA in the DAR signalplex without disrupting the overall cellular mechanics.
To determine the long term impact of NKA inhibition on DAR function, HEK293T cells were transfected with D 2L receptor alone or D 2L receptor with anti-NKA␣1 siRNA and assessed for inhibition of cAMP after 48 h as described under "Experimental Procedures." Inhibiting the NKA␣1 with siRNA resulted in a 20% increase in dopamine-stimulated reduction of cAMP (Fig. 11A). The impact of siRNA-mediated NKA␣1 inhibition on the D 1 receptor was even more pronounced-an 80% increase in D 1 function was observed after siRNA treatment (Fig. 11B). Total receptor binding experiments were also conducted to assess DAR densities after siRNA treatment. Although there was no significant change in D 1 DAR density after NKA inhibition by siRNA (ϳ15% increase in D 1 DAR B max as compared with scrambled siRNA; data not shown), it is noteworthy that the D 2 DAR density was significantly decreased after anti-NKA␣1 siRNA treatment (ϳ60% decrease as com-  Table 4.   DECEMBER 26, 2008 • VOLUME 283 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 36449 pared with scrambled siRNA; data not shown). The reason for these disparate results between D 1 and D 2 receptor densities is unclear, but it is quite remarkable that, despite the pronounced decrease in D 2 density, there is still an increase in D 2 DAR functional response following siRNA inhibition of NKA␣1 expression. Because the NKA␣1 protein is still present in the cells, but diminished in the DAR signalplex, these data further implicate a role for DAR-NKA protein interactions rather than signaling to mediate the observed functional results. NKA Activity Is Modulated by DAR Expression-Endogenous NKA activity in HEK293T cells was investigated after expression of D 1 and D 2 receptors to determine the impact of the DARs on NKA function. Interestingly, expressing the D 2 receptor alone caused a significant decrease (ϳ50%) in NKA function when compared with untransfected cells as measured by rubidium-86 ion ( 86 Rb ϩ ) uptake (Fig. 12). Notably, as mentioned previously, total lysate and co-immunoprecipitation experiments demonstrated that there was no change in NKA␣1 protein expression after transfection of the D 2 receptor (supplemental Fig. 1), indicating that a decrease in NKA protein number does not mediate this decrease in function. Treatment of D 2 -expressing cells with 10 M dopamine had no significant impact on 86 Rb ϩ uptake as compared with either untransfected cells or vehicle-treated D 2 cells (Fig. 12). Because the D 2 receptor exhibits weak ligand-independent signaling (35,36), constitutive activity was investigated as a possible mechanism for the D 2 -mediated decrease in NKA activity. Cells expressing the D 2 receptor were treated with pertussis toxin to block the downstream signaling cascade, and NKA activity was measured. A 16-h pertussis toxin treatment completely eliminates D 2L -mediated cAMP regulation in the HEK293 cells (data not shown) but had no impact on NKA activity (Fig. 12), indicating that constitutive activity of the D 2 receptor was not responsible for the observed decrease in D 2 function. These data reveal that the D 2 receptor protein can decrease NKA activity in a ligand-and signal-independent manner, further suggesting a functional role for the putative DAR-NKA complex.

Reciprocal Regulation of Function between DARs and NKA
Expression of the D 1 receptor caused a similar decrease in NKA function (ϳ40%) as compared with untransfected cells (Fig. 13). Conversely, NKA-mediated 86 Rb ϩ uptake was significantly increased in cells expressing the D 1 receptor when treated with dopamine (Fig. 13). To see if this effect was second messenger-mediated, we attempted to inhibit adenylyl cyclase or PKA activities; however, all tested inhibitors independently altered NKA activity. In contrast, treatment with dibutyryl-cAMP, a cAMP analog and activator of PKA, did not alter NKA activity indicating that the G␣ s signaling cascade downstream of D 1 DAR activation is not solely responsible for the increase in NKA function (data not shown). Based upon these data, it can-  not be determined whether the dopamine-stimulated increase in NKA activity (Fig. 13) is mediated by the D 1 signaling cascade or by alterations in protein-protein interactions induced by dopamine binding to the receptor.

DISCUSSION
In this study, we used mass spectroscopy analysis to identify the NKA␣1 subunit as an interacting protein in both the D 1 and D 2 DAR signalplexes. Although previous investigations have shown that dopamine can signal through its receptors to regulate NKA activity (15-17, 20 -23), this is the first demonstration that the two proteins exist in a complex. This novel discovery of a DAR-NKA interaction was confirmed by co-immunoprecipitation and Western analyses in HEK293T cells, and was further substantiated by verifying the interaction in brain tissue between native DARs and NKA. Previous studies have shown that alterations in NKA function can occur via insertion or removal of NKA at the plasma membrane (15, 17, 19 -23). However, confocal microscopy revealed that, in this system, both proteins predominantly co-localized at the plasma membrane rather than existing in separate vesicular pools implicating a mechanism apart from protein trafficking or endosomal recycling pathways as the mechanism for functional alterations in both proteins. At present, the precise location of the interaction between the NKA and DARs is unclear. Complete truncation of the carboxyl terminus of the D 1 receptor, a common site for D 1 protein-protein interactions (30 -32), did not diminish the D 1 -NKA association. As both receptor and NKA proteins have multiple transmembrane-spanning domains, it is quite possible that their interactions occur within the plasma membrane regions. Alternatively, other nonmembranous regions of the proteins may mediate their interactions. These possibilities are currently under investigation.
Functional studies of the DAR-NKA interactions in HEK293T cells revealed diminished DAR signaling in the presence of overexpressed NKA with a concomitant decrease in total DAR expression. Inhibiting the overexpressed NKA with the cardiotonic steroid ouabain restored DAR function. Interestingly, ouabain inhibition of endogenous NKA also enhanced DAR signaling, indicating that the transfected DARs can be regulated by endogenous levels of NKA. Ouabain treatment did not increase total or cell-surface DAR expression, demonstrating that the amplified DAR signal was not caused by an increase in the number of functional DARs. Moreover, confocal microscopy and co-immunoprecipitation assays indicated that ouabain treatment did not disrupt the DAR-NKA interaction or cause endocytosis or membrane insertion of either protein.
These data indicate that ouabain treatment impacts the DARs already at the plasma membrane, possibly by inducing compensatory conformational changes within the receptor structure upon ouabain binding to NKA.
To ensure that ouabain did not produce global cellular alterations as the mechanism for enhancing DAR function, siRNA treatment was employed as an alternate means to inhibit the NKA. Anti-NKA␣1 siRNA decreased total cellular NKA␣1 levels by ϳ50% but reduced the amount of NKA interacting with the D 2 DAR below detectable limits. This allowed examination of the role of NKA in the DAR signalplex over a longer period of time while still maintaining normal ionic balance in the cells. Transfection with anti-NKA␣1 siRNA did not change the total D 1 receptor density, but D 1 receptor-stimulated cAMP accumulation approximately doubled after the siRNA inhibition of NKA␣1. Interestingly, NKA␣1 siRNA treatment dramatically and significantly diminished total D 2 receptor density, but despite the reduction in D 2 receptor number, there was a significant increase in D 2 DAR function. Although the reason for the decrease in D 2 receptor number is unknown, it is clear that loss of the NKA-DAR interaction can increase the D 2 DAR signal, even when D 2 DAR numbers are reduced. These data provide further evidence that endogenous NKA interactions can modulate DAR function. Of note, these data show that the NKA-DAR interaction, and not just the presence of the NKA protein in the cell, is critical for DAR functional regulation. More importantly, these data provide the first evidence for NKA control of DAR function and support a protein interaction mechanism, rather than downstream signaling molecules, for the novel NKA modulation of DARs.
Not only did NKA modify the DAR signal, but reciprocal modulation of NKA by DARs also occurred. Transfecting either the D 1 or D 2 DAR into HEK293T cells caused a marked decrease in NKA␣1 activity without diminishing the NKA protein levels. The ability of DARs to diminish NKA function in the absence of ligand and without altering NKA numbers further supports the importance of the interacting complex. Because the D 2 receptor exhibits constitutive activity (35,36), pertussis toxin was used to inhibit the G i/o signaling cascade and thus eliminate ligand-independent G-protein signaling as a potential mechanism for modulation of NKA activity. Pertussis toxin had no impact on D 2 DAR modulation of NKA activity, indicating that the D 2 protein itself, and not a second messenger, was responsible for the diminished NKA function. Dopamine binding to the D 2 receptor did not further modify NKA activity, but dopamine stimulation of D 1 receptors did significantly increase NKA function. It is possible that signaling downstream of the FIGURE 13. NKA function following D 1 DAR transfection. HEK293T cells were transfected with 5 g of either D 1 cDNA or empty pcDNA vector (UTs). Cells were washed 24 h later. 48 h post-transfection, cells were removed into assay buffer, and 86 Rb ϩ uptake, in the presence or absence of dopamine (DA), was measured as described under "Experimental Procedures." Data were normalized for individual experiments as a percentage of the E max value for UT cells alone and are expressed as mean Ϯ S.E. of five independent experiments, each conducted in duplicate. All statistical analyses were performed using one-way ANOVA with Bonferroni's post-test; *, p Ͻ 0.05; **, p Ͻ 0.01. UT raw E max values ranged from 106 to 174 pmol/mg protein/min. D 1 receptor was responsible for amplifying NKA activity. To examine this possibility, PKA and adenylyl cyclase inhibitors were used to block the G s signaling cascade. However, all tested inhibitors altered NKA function independent of the D 1 DAR. Consequently, it remains unclear whether the dopaminestimulated increase in NKA activity is mediated by the D 1 signaling cascade or by alterations in protein-protein interactions induced by dopamine binding to the receptor. It is clear from our data that neither constitutive activity of DARs nor alterations in NKA number following DAR expression are responsible for the observed decrease in NKA activity. Moreover, confocal microscopy and co-immunoprecipitation assays indicated that dopamine treatment does not disrupt the DAR-NKA interaction or cause endocytosis or membrane insertion of either protein. This is significant in that prior studies have shown that dopaminergic control of NKA function occurs via NKA insertion or removal at the plasma membrane in a tissue-specific manner (15, 17, 19, 20 -23). As such, it is likely that conformational changes occurring within the DAR-NKA protein complex mediate the observed decrease in NKA function. In addition, D 1 receptor conformational changes induced upon dopamine binding may cause compensatory conformational changes in the NKA protein that further alter NKA activity.
Taken together, these data suggest a mechanism for reciprocal modulation of function between DARs and the NKA. The presented data show that NKA, when interacting with the DAR, decreases the DAR function. Similarly, expression of DARs decreases NKA activity without decreasing NKA expression. These data suggest a model wherein the co-existence of the two proteins in the signalplex causes a reciprocal dampening of each others' function (Fig. 14A). This could be caused by structural hindrance between the two proteins, resulting in diminished G-protein coupling to the receptor and ATP hydrolysis by the NKA. Within the signalplex, a conformational change in one protein likely induces a compensatory change in neighboring proteins. For the D 1 receptor, the dopamine-binding event may induce conformational changes in the NKA promoting ATP hydrolysis and ion transport across the membrane (Fig. 14B). It is also possible that second messengers within the D 1 signaling cascade and changes in the protein-protein interactions induced by dopamine binding both play a role in enhancing NKA function. Finally, inhibition of NKA within the DAR signalplex, using either cardiotonic steroids or siRNA, enhances DAR function. Because NKA activity is already reduced while interacting with the DARs, it is likely that a mechanism secondary to NKA inhibition is underlying the observed increase in DAR activity. As such, we hypothesize that ouabain binding to the NKA induces conformational changes in the proteins, thus allowing better coupling of the DAR to G-proteins (Fig. 14C). Although this model depicts a direct interaction between just the DAR and NKA, the possibility of an additional linker or tethering protein holding the DAR and NKA in close physical proximity cannot be eliminated at this time. Regardless, the apparent significance of this DAR-NKA complex and the observed reciprocal functional modulation of each protein is quite noteworthy.
In summary, interaction between the D 1 or D 2 DARs and NKA results in a reciprocal modulation of function between the two proteins, both in the presence and absence of ligands. This is the first demonstration of an interaction between these proteins, and this complex reveals the possibility of new mechanisms for functional regulation of both DARs and NKA. Physiologically, this interaction likely provides strict and immediate control of the cellular response to external stimuli by limiting the functional capacity of each protein when they are in complex. Although in complex with one another, inhibition of the NKA by endogenous cardiotonic steroids may enhance the native DAR signal, and dopamine signaling at the D 1 receptor can activate the NKA. Within the brain, this NKA activation may be necessary for restoring resting membrane potential of neurons, and the amplified DAR signal may be critical for periods of enhanced responsiveness to dopaminergic cues. Further understanding the significance of this interaction in neuronal and other physiological environments will provide insight into both the DAR signalplex and native cellular control mechanisms. A, close proximity of NKA and DAR, via protein-protein interactions, causes conformational hindrance of G-protein coupling to the receptor. This in turn diminished the downstream signaling cascade following ligand stimulation. B, binding of dopamine (DA) to the D 1 DAR results in conformational changes to the DAR that cause compensatory conformational changes in the NKA. These changes to the pump allow enhanced transport of Na ϩ and K ϩ across the plasma membrane. C, ouabain binding to the NKA results in conformational changes to the sodium pump, enabling enhanced coupling between DAR and G-proteins. This causes an increase in the downstream signaling cascade upon dopamine stimulation.