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Am J Physiol Renal Physiol 295: F1110-F1116, 2008. First published August 13, 2008; doi:10.1152/ajprenal.90336.2008
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Negative reciprocity between angiotensin II type 1 and dopamine D1 receptors in rat renal proximal tubule cells

Farah Khan, Zuzana Spicarová, Sergey Zelenin, Ulla Holtbäck, Lena Scott, and Anita Aperia

Department of Woman and Child Health, Karolinska Institutet, Astrid Lindgren Children's Hospital, SE-171 76 Stockholm, Sweden

Submitted 30 May 2008 ; accepted in final form 6 August 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling. Similarly, exposure of rat proximal tubule cells and renal tissue to angiotensin II resulted in a rapid partial internalization of D1R and abolishment of D1R signaling. D1R and AT1R were, by use of coimmunoprecipitation studies and glutathione-S-transferase pull-down assays, shown to be partners in a multiprotein complex. Na+-K+-ATPase, the target for both receptors, was included in this complex, and a region in the COOH-terminal tail of D1R (residues 397-416) was found to interact with both AT1R and Na+-K+-ATPase. Results indicate that AT1R and D1R function as a unit of opposites, which should provide a highly versatile and sensitive system for short-term regulation of sodium excretion.

AT1 receptors; Na+-K+-ATPase; calcium signaling

RENAL SODIUM EXCRETION IS bidirectionally regulated by angiotensin II (ANG II) and dopamine (13). Long-term dopamine exposure is known to decrease AT1 receptors (AT1R) in renal proximal tubular cells (7). Furthermore, studies by Zeng et al. (21) have shown that long-term stimulation of AT1R results in an upregulation of D1-like receptors (D1R). This effect was not observed in spontaneously hypertensive rats, indicating that the interaction between AT1R and D1R has an impact on blood pressure regulation.

Since sodium excretion must be regulated with great precision over a short period of time, it is important that control mechanisms are able to exert their effects within a short time frame. The aim of the current study has been to explore the short-term effects of ANG II exposure on D1R and the short-term effects of a D1-agonist on AT1R. Our approach has been to test the hypothesis that AT1R and D1R form a heteromeric signaling complex, where activation of either receptor may cause internalization and/or interruption of the signaling capacity of the other.

The studies were performed using rat proximal tubule cells, since these cells express both AT1R and D1R in both the apical and the basolateral membrane (12, 18). Previous studies from our laboratory have shown that in these cells Na+-K+-ATPase, the enzyme responsible for active sodium transport, is bidirectionally regulated by ANG II and dopamine (2).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Cells and tissue. All studies were performed using outer cortical tissue from young (3–5 wk) male Sprague-Dawley rats. Immediately after the animals were killed, 250-µm slices were taken from the outer renal cortex using a microtome. The outer 250-µm region of the rat renal cortex contains >90% proximal tubule cells (3, 4). In some protocols, the slices were lysed and used for coimmunoprecipitation studies, glutathione-S-transferase (GST) pull-down assays, or cAMP determination. In other protocols, the slices were used for preparation of primary cultures of renal proximal tubule (RPT) cells. RPT cells were prepared as described previously (14, 16). Briefly, the cells were cultured in supplemented Dulbecco's modified Eagle's medium (20 mM HEPES/24 mM NaHCO3/10 µg/ml penicillin/10 µg/ml streptomycin/10% FBS) on petri dishes or glass coverslips for 48 h in 5% CO2 at 37°C. All experiments were performed according to the Karolinska Institutet regulations concerning care and use of laboratory animals and approved by the Stockholm North ethical evaluation board for animal research.

Western blotting and immunoprecipitation. Outer cortical slices dissected from rat kidneys were kept in ice-cold buffer containing 124 mM NaCl, 26 mM NaHCO3, 10 mM D-glucose, 1.5 mM MgSO4, 1 mM n-butyric acid, 1.5 mM CaCl2, and 0.25 mM KH2PO4. The cortical slices were incubated with vehicle or ANG II (10–7 M, Sigma-Aldrich) or D1R agonist SKF81297 (10–5 M, Sigma-Aldrich) for 15 min in a buffer medium which was kept at 37°C and bubbled with 95% O2-5% CO2. After drug treatment, the medium was aspirated, and tissue slices were immediately frozen. The slices were then thawed and homogenized in RIPA buffer containing 50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 0.5% Nonidet P-40, and protease inhibitors (Roche Diagnostics), and centrifuged at 9,000 g for 20 min at 4°C. Supernatant protein concentration was measured by Bio-Rad DC protein assay (Bio-Rad). Tissue lysates (500 µg) from vehicle- or drug-treated groups were incubated for 2 h at 4°C with 3 µg of AT1R antibody or D1R antibody and with rabbit or rat IgG, respectively, as a control. Immunocomplexes were then incubated with protein G-Sepharose beads (GE Healthcare) overnight at 4°C. After the beads were washed three times with RIPA buffer, proteins were eluted with 2x Laemmli sample buffer and resolved in 7% polyacrylamide gels by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted with D1R (1:250) or the AT1R antibody (1:200) or Na+-K+-ATPase {alpha}1-subunit antibody (1:10,000). The proteins were visualized by chemiluminescence using secondary antibodies labeled with horseradish peroxidase (GE Healthcare) and ECL (GE Healthcare).

Cell surface biotinylation. Cultured RPT cells were incubated with either vehicle (Dulbecco's modified Eagle's medium), SKF81297 (10–5 M), or ANG II (10–7 M) for 15 min. The medium was then removed. Surface membrane proteins were biotinylated by exposing cells to EZ linked Sulfo-NHS-SS Biotin (Pierce), at a final concentration of 1 mg/ml in PBS at 4°C for 2 h with gentle shaking. After being washed twice with PBS and once with PBS containing 100 mM glycine to quench unreacted biotin, the cells were lysed in ice-cold lysis buffer [50 mM Tris·HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 0.5% Nonidet P-40, and protease inhibitors (Roche Diagnostics)], and the lysate was centrifuged at 14,000 g for 10 min at 4°C. After centrifugation, the supernatant was saved and protein was adjusted to the equal amount according to the Bio-Rad DC protein assay. Biotinylated proteins (450 µg) were captured with 100 µl of streptavidin-agarose beads (Pierce) overnight at 4°C with end-to-end mixing. Pellets recovered after centrifugation at 14,000 g for 10 min at 4°C were washed twice with PBS. Proteins were eluted with sample buffer and resolved by Western blotting.

D1R constructs. Constructs encoding GST fusion proteins with D1R COOH-terminal residues CT1 (L387–L416; LVY LIP HAV GSS EDL KKE EAA GIA RPL EKL) and CT2 (S417–T446; SPA LSV ILD YDT DVS LEK IQP ITQ NGQ HPT) were cloned in pDEST15 vectors by using site-specific recombination (Gateway Technology, Invitrogen). The CT1 fragment of D1R COOH-terminal cDNA was mutated at amino acid positions S397A/S398A by a site-directed mutagenesis technique using the U.S.E. Mutagenesis kit (Amersham Pharmacia). The structure of all constructs was confirmed by DNA sequencing.

GST affinity pull-down. GST affinity pull-down was used to study the interaction between the AT1R or Na+-K+_ATPase and two peptide segments corresponding to the COOH-terminal tail of D1R. GST fusion proteins were produced in Escherichia coli (BL21-A1) and purified by using glutathione-Sepharose 4B beads (Amersham Pharmacia Biosciences). The rat renal cortical lysate was prepared in lysis buffer (50 mM Tris·HCl, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, 1% Triton X-100, and protease inhibitors), and 2 mg of total protein was added to the beads in a volume of 1 ml and incubated overnight at +4°C. The beads were then washed three times with PBS+1% Triton X-100 and protease inhibitors. Proteins were eluted with 50 µl of 2x Laemmli buffer for 5 min at 100°C. Samples were then subjected to SDS-PAGE for Western blotting or Coomassie brilliant blue staining.

Ratiometric imaging of intracellular calcium. RPT cells cultured on glass coverslips were loaded with the calcium-sensitive dye fura 2-AM (5 µM mixed with Pluronic acid, 0.02 µM in PBS, Molecular Probes) for 40 min at 37°C. Ratiometric imaging of intracellular calcium was performed using a heated chamber mounted on a Zeiss Axioskop 2 microscope with a x40/1.3 NA epifluorescent oil-immersion objective. Loaded cells were excited at wavelengths of 340 and 380 nm, and emission fluorescence was detected with a CCD camera (Hamamatsu ORCA-ER C4742-95) via an image-intensifier unit (Hamamatsu C9016). All experiments were performed in physiological PBS (100 mM NaCl, 4 mM KCl, 20 mM HEPES, 25 mM NaHCO3, 1.5 mM CaCl2, 1.1 mM MgCl2, 1 mM NaH2PO4, 10 mM D-glucose, pH 7.4). Cells were perfused with different agents (in PBS) with intermittent washing with PBS as indicated (see Fig. 4A). Ratio images were recorded every 5 s for 120 s for each perfusate. The data were analyzed with Metafluor software (Molecular Devices). For each glass, a single cluster of 25–35 cells was analyzed.

cAMP assay. Renal cortical slices were treated with varying concentrations of SKF81297 (10–9 to 10–4 M) for 10 min and homogenized in 0.1 M HCl. Cytosolic cAMP levels were measured by a correlate EIA direct cAMP kit (Assay Designs) following the manufacturer's protocol. SKF81297 in 10–5 and 10–6 M concentrations was found to generate optimal cAMP response. Based on this dose-response study, the 10–5 and 10–6 M concentrations were used for all protocols. For subsequent cAMP experiments, slices were treated with SKF81297 alone (10–5 and 10–6 M), ANG II (10–7 M) alone, or pretreated with ANG II for 5 min and then treated with both ANG II (10–7 M) and SKF81297 (10–5 M/10–6 M) for 10 min, and cAMP levels were measured in respective cortical lysates.

Antibodies and chemicals. The following antibodies were used: rat monoclonal anti-D1R antibody (Sigma), mouse monoclonal anti-Na+-K+-ATPase {alpha}1-subunit antibody (Upstate Biotechnology), and mouse monoclonal anti-actin antibody (BD Transduction). Rabbit polyclonal anti-human AT1R antibody (Santa Cruz Biotechnology) used was from two different lots. The antibody from lot E3006 gave two close bands, while lot D0607 gave a single band of desired size. Immunoprecipitation controls were mouse and rabbit IgG (Sigma-Aldrich). Secondary IgG antibodies were rabbit, mouse, and rat and conjugated with horseradish peroxidase (GE Healthcare). All other chemicals were obtained from Sigma-Aldrich.

Statistical analysis. Data are given as means ± SE. Statistical analysis was performed using Student's t-test. P < 0.05 was used as the level of significance.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Interaction between D1R and AT1R: identification of a motif in the COOH terminus of D1R that interacts with AT1R. To test whether D1R and AT1R are associated in the outer layer of rat renal cortex, we immunoprecipitated either AT1R or D1R and blotted the precipitates with a D1R antibody or an AT1R antibody, respectively. We found that the D1R antibody could immunoprecipitate AT1R and that the AT1R antibody could immunoprecipitate D1R from lysate of rat renal cortex (Fig. 1A). Results indicate that the D1R and AT1R are partners in the same macromolecular protein complex. At least two isoforms, D1 and D5, belong to the D1-like receptor family, which is characterized by the capacity of the receptors to assemble with Gs proteins and generate cAMP. There are no antibodies and no agonists that can completely discriminate between D1 and D5. The homology between the D1 and D5 receptor is high, except for the COOH terminus. Since the COOH-terminal tail of D1R has been shown to have binding motifs for other proteins (5, 15, 19), we tested whether AT1R may interact with the COOH terminus of D1R. For this purpose, we generated GST fused peptides that corresponded to two regions of the D1R COOH-terminal tail, the region containing L387–L416 (D1R-CT1) and the region containing S417–T446 (D1R-CT2) (Fig. 1B). In a recent paper from our laboratory, it was shown that, in the striatal region of the brain, these regions of the D1R COOH terminus interact with the NR1 subunit of the NMDA receptor, an interaction that has several functional consequences (19). Renal cortical lysate was incubated with D1R-CT1, D1R-CT2, or purified GST alone, used as a negative control. Figure 1C shows that D1R-CT1 peptide can pull down AT1R, while the D1R-CT2 peptide cannot pull down AT1R from a lysate of rat renal cortex. To further identify the amino acids critical for the D1R-AT1R interaction, a mutant D1R-CT1(SS/AA), where S397 and S398 were substituted to alanine, was used for GST pull down (Fig. 1D). The ability of the mutant D1R-CT1(SS/AA) to pull down AT1R was significantly reduced compared with wild-type D1R-CT1. We conclude from this protocol that D1R in the D1-like family of dopamine receptors and AT1R are partners in the same macromolecular protein complex.


Figure 1
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  		Fig. 1. D1-like receptors (D1R) interact with AT1 receptors (AT1R). A: D1R coimmunoprecipitates with AT1R and vice versa. B: glutathione-S-transferase (GST)-fused COOH-terminal segments of D1R used for GST affinity pull-down. C: Western blot of GST affinity pull-down shows immunoreactivity signal for AT1R in D1R-CT1 lane, but no signal in D1R-CT2 and GST lanes. Bottom: presence of GST and GST fusion proteins by Coomassie brilliant blue staining is shown. D: mutant D1R-CT1(SS/AA) showed reduced ability to interact with AT1R. Bottom: equal expression of wild-type and mutant GST fusion proteins and GST alone by Coomassie brilliant blue staining is shown. Representative images are shown from 3 independent experiments.

 
D1R-AT1R interaction is modulated by receptor-activating ligands. To test whether SKF81297 and ANG II may effect D1R-AT1R interaction, outer cortical slices were exposed to vehicle, SKF81297 (10–5 M), or ANG II (10–7 M) for 15 min and immunoprecipitation was carried out. The strength of D1R-AT1R interaction was significantly reduced by exposure to either SKF81297 (–40%, P < 0.05) or to ANG II (–49%, P < 0.05), as shown in Fig. 2, A and B.


Figure 2
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  		Fig. 2. Strength of D1R-AT1R interaction is modulated by receptor agonists. A: effect of SKF81297 on the coimmunoprecipitation of D1R and AT1R in the rat renal cortex. B: effect of ANG II treatment on coimmunoprecipitation of D1R and AT1R in rat renal cortex. The rat renal cortical slices were exposed to SKF81297 (10–5 M) or ANG II (10–7 M) for 15 min. Densitometric quantification of bands was done for the respective blots. The density of vehicle-treated control was taken as 100%. C: effect of SKF81297 on whole cell AT1R protein level. D: effect of ANG II on whole cell D1R protein level. The rat renal cortical slices were incubated with SKF81297 (10–5 M) or ANG II (10–7 M) for 15 min and immunoreactivity for AT1R or D1R were measured in the respective homogenates. Results are expressed as the ratio of AT1R or D1R to {alpha}-actin densities. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. control.

 
Lack of short-term effect of receptor agonists on whole cell receptor abundance. Next, we examined whether exposure to SKF81297 (10–5 M) or ANG II (10–7 M) for 15 min would have an effect on AT1R and D1R abundance in tissue from the outer renal cortex. Tissue lysate was subjected to Western blot analysis with AT1R and D1R antibodies. The membranes were then stripped off and reprobed with an anti-{alpha}-actin antibody, and the densities of the D1R and AT1R signals were normalized to the respective actin signals. As shown in Fig. 2, C and D, 15-min exposure to ANG II or SKF81297 had no effect on the abundance of whole cell AT1R and D1R, respectively.

ANG II and SKF81297 exposure has an effect on the plasma membrane expression of D1R and AT1R, respectively. To examine the plasma membrane expression of AT1R and D1R, surface biotinylation assays were performed, using RPT cells. As shown in Fig. 3A, exposure of RPT cells to SKF81297 caused a significant (55%) decrease in plasma membrane expression of AT1R. Exposure to ANG II caused a significant (23%) decrease in the plasma membrane expression of D1R (Fig. 3B). Exposure to SKF81297 increased the plasma membrane expression of D1R (+36%, P < 0.05), as shown in Fig. 3C. Exposure to ANG II did not have any effect on the plasma membrane expression of AT1R (data not shown).


Figure 3
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  		Fig. 3. Effects of ANG II and SKF81297 on receptor abundance in cell surface membrane. A: effect of SKF81297 on the abundance of cell surface membrane AT1R in renal proximal tubule (RPT) cells. B: effect of ANG II on the abundance of cell surface membrane D1R in RPT cells. C: effect of SKF81297 on the abundance of cell surface membrane D1R in RPT cells. The cells were exposed to SKF81297 (10–5 M) or ANG II (10–7 M) for 15 min. Cell surface expression of receptors was studied using biotinylation technique. Densitometric quantification of bands was done. The density of vehicle-treated control was taken as 100%. Values are means ± SE of 3 independent experiments. *P < 0.05 vs. control.

 
SKF81297 interrupts AT1R signaling and ANG II interrupts D1R signaling. Next, we examined the functional significance of D1R-AT1R interaction. AT1R are known to couple to Gq proteins, and activation of AT1R triggers activation of PLC, generation of inositol 1,4,5-trisphosphate (IP3), activation of the IP3 receptor (IP3R), and, as a result, release of calcium from the intracellular stores (11, 20, 22). RPT cells were loaded with the calcium-sensitive dye fura 2, and ratiometric recordings of intracellular calcium were carried out. After recordings of basal intracellular Ca2+ concentration ([Ca2+]i) for 90 s, ANG II and/or SKF81297 was applied to the cells via a perfusion chamber.

Exposure of cells to ANG II (10–9 M) resulted in a transient calcium peak in virtually all (90–100%) cells. After being washed with PBS, the cells were exposed to SKF81297 (10–5 M). No effect on [Ca2+]i was observed. The cells were then coexposed to SKF81297 (10–5 M) and ANG II (10–9 M). Again, no effect on [Ca2+]i was observed. Finally, the cells were washed with PBS for 120 s and exposed to ANG II alone. All cells that had previously responded following exposure to ANG II alone now responded again with a transient calcium peak. Experimental data from one typical recording is shown in Fig. 4A. This study was repeated 12 times (in 4 independent experiments) with similar results. Similar results were obtained when the experiment was repeated using 10–7 M ANG II concentrations (data not shown). The second exposure to ANG II resulted in a second calcium peak with a somewhat lower intensity; this could be due to a slight desensitizing of the AT1R.


Figure 4
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  		Fig. 4. SKF81297 abolishes the ANG II-mediated calcium response and ANG II abolishes the SKF81297-mediated cAMP response. A: RPT cells were treated with the indicated agents, and intracellular Ca2+ concentration ([Ca2+]i) responses were measured. [Ca2+]i responses from all cells in a representative recording are shown. The results were consistent in 4 different experiments. Each experiment was performed in triplicate on a batch of cells derived from at least 3 rats. B: treatment-induced change in the [Ca2+]i level was calculated for each responding cell as the percent change in the [Ca2+]i [arbitrary units (a.u.)] value before and after each treatment. Bars represent the means ± SE from 332 cells. C: renal cortical slices were exposed to ANG II (10–7 M) alone or SKF81297 alone (10–5 or 10–6 M) for 10 min or pretreated with ANG II (10–7 M) for 5 min and then treated with both ANG II (10–7 M) and SKF81297 (10–5 or 10–6 M) for 10 min, and cAMP levels were measured in the tissue lysates. Values are means ± SE of 3 separate experiments. *P < 0.05 vs. control. #P < 0.05 vs. respective SKF81297-treated groups.

 
D1R, when activated, are known to stimulate adenylyl cyclases and increase cAMP generation (17). Renal cortical slices were treated with SKF81297 alone (10–5 and 10–6 M) or with ANG II (10–7 M) alone for 10 min, or pretreated with ANG II (10–7 M) for 5 min and then treated with both ANG II (10–7 M) and SKF81297 (10–5 M/10–6 M) for another 10 min. Exposure to SKF81297 alone increased cAMP levels significantly. In the presence of ANG II, the cAMP response to SKF81297 was abolished (Fig. 4B). ANG II alone had no effect on cAMP levels.

D1R and AT1R interact with Na+-K+-ATPase. Since D1R and AT1R have been shown to exert a bidirectional control of proximal tubular Na+-K+-ATPase activity (1, 2), we wanted to examine whether there exists an interaction between these receptors and Na+-K+-ATPase. As shown in Fig. 5, A and B, Na+-K+-ATPase coimmunoprecipitates with both D1R and AT1R. To test whether the motif in D1R that interacts with AT1R is also responsible for the interaction with Na+-K+-ATPase, GST pull-down assays were performed. We used the GST-D1R fusion proteins corresponding to two regions of the COOH terminus of D1R, D1R-CT1 and D1R-CT2 (see Fig. 1B). As shown in Fig. 5C, Na+-K+-ATPase interacts with the GST-tagged wild-type D1R-CT1 peptide, but to a significantly lesser extent with the GST-tagged mutant D1R-CT1(SS/AA) (Fig. 5D). No interaction was detected with D1R-CT2 peptide. GST alone was used as a negative control.


Figure 5
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  		Fig. 5. D1R and AT1R interact with Na+-K+-ATPase. A and B: Na+-K+-ATPase coimmunoprecipitates with D1R and with AT1R. C: Western blot of GST affinity pull-down shows that the D1R-CT1, a peptide corresponding to amino acids 387-416 in D1R COOH terminus, interacts with Na+-K+-ATPase. D: ability of the mutant D1R-CT1(SS/AA), where S397 and S398 are substituted for alanines, to interact with Na+-K+-ATPase was markedly reduced. A strong immunoreactivity signal for Na+-K+-ATPase can be seen in wild-type D1R-CT1 and a very weak signal in mutant D1R-CT1(SS/AA). No signal was seen for GST. Bottom (C and D): equal expression of wild-type and mutant GST fusion proteins and GST alone by Coomassie brilliant blue staining.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Salt homeostasis has to be regulated with great precision over a short period of time. Dopamine and ANG II play a major role in the renal control of sodium excretion and homeostasis. Here, we show that there are counterregulatory effects of AT1R and D1R occurring on a time scale of minutes and that these effects are due to a dynamic, reciprocal, regulation of receptor signaling and subcellular localization. Our results support and extend the concept that there is a negative interaction between D1R and AT1R and provide new information about the mechanism for AT1R-D1R interaction.

The coimmunoprecipitation and pull-down studies imply that a physical interaction may be required for the counterregulation to occur. The D1R was found to coimmunoprecipitate with AT1R and vice versa. To test the specificity of this finding, GST pull-down assays were performed. Since in G protein-coupled receptors, the COOH terminus is the most likely segment to interact with other proteins (5), we tested the ability of two segments of the COOH terminus of the D1R to interact with the AT1R. We found that the L387–L416 segment, but not the S417–T446 segment, interacted with the AT1R. It has previously been shown by us and others that the L387–L416 segment interacts with the NR1 subunit of the NMDA receptor and that this interaction has several functional consequences (15, 19). Replacement of the amino acid residues S397/S398 to alanines was shown to significantly attenuate the interaction between the L387–L416 segment with the NR1 subunit in tissue from the striatal region of the brain. Here, we found that the mutant L387–L416 segment pulled down significantly less AT1R than the wild-type L387–L416 segment, using tissue from the outer renal cortex. The functional consequences of the mutation remain to be elucidated, but the fact that the mutation had such an impact on the interaction provides strong support to the specificity of the pull-down study.

Inhibition of G protein coupling is likely to render the receptor more susceptible to internalization processes and may therefore explain the rapid internalization of the AT1R following agonist occupancy of D1R and vice versa. Short-term exposure of renal cortical slices to the D1 agonist SKF81297 resulted in a robust, 55%, loss of AT1R from the plasma membrane and short-term exposure of the outer renal cortex to ANG II resulted in a less pronounced, but still significant, loss of the D1R from the plasma membrane. These effects must be due to a redistribution of receptors from the plasma membrane to the cytoplasm, since the total number of receptors was not decreased at this time point. The redistribution and internalization of receptors may be mediated by phosphorylation-dephosphorylation reactions triggered by the agonist-occupied receptors (8, 9).

In our protocols, cells and tissue were exposed to the agonists for 2–15 min. The total abundance of AT1R was not significantly reduced following this short-term exposure of renal cortical slices to SKF81297, nor was the total abundance of D1R significantly reduced following short-term exposure of renal cortical slices to ANG II. The decreased power of interaction between D1R and AT1R following short-term exposure to either receptor agonist cannot therefore be explained by a loss of whole cell receptor protein. Previous studies from the group of Felder and Jose (21) has shown that long-term, generally 24-h, exposure to D1R or AT1R agonists decreases the total abundance of the reciprocal receptor (21).

Activation of D1R caused an immediate and reversible uncoupling of AT1R from its signaling pathway, and the signaling from the D1R was also rapidly abolished following activation of AT1R. Notably, there was a complete uncoupling of the receptors from their signaling pathway despite the fact that >40% of the receptors remained in the plasma membrane. These findings reveal a hitherto unknown mechanism for rapid interaction between the dopamine and the angiotensin systems in the kidney. Since our studies showed that D1R and AT1R are partners in a protein complex, it is possible that the effects of ligand occupation on the signaling from the reciprocal receptor can be attributed to protein-protein interaction. Spatial factors have been shown to be of importance for specific and rapid receptor interactions. In the brain, the D1R can form heterodimers with the D2 dopamine receptor and this heteromeric receptor complex has been reported to have profound effects on receptor signaling (10).

Exposure of renal cortical tissue to a D1 agonist increased the abundance of D1R in the plasma membrane. This is in agreement with a previous study where the subcellular distribution of D1R was studied with confocal imaging and subcellular fractionation (6). Most G protein-coupled receptors are internalized and desensitized following occupation by their cognate ligand. There is presently no explanation for the apparently paradoxical response of renal D1R to ligand occupation, but we speculate that this effect on a renal tubular receptor, which has a natriuretic effect, may be physiologically relevant, since a sustained effect on sodium excretion is often desirable.

In conclusion, we provide evidence that the D1R and AT1R can form a heteromeric signaling complex in the kidney and that ligand binding of either receptor within this complex will change the functional state of its partner on a time scale of minutes. Several sodium transporters, including the pump, Na+-K+-ATPase, have been reported to be inhibited by dopamine and activated by angiotensin (1). Na,-K+-ATPase was here shown to be a partner in the D1R/AT1R protein complex. Taken together, these data imply that AT1R and D1R regulate renal sodium excretion via a yin-yang mechanism, which can appropriately respond in a timely fashion to changes in dietary sodium intake. As a consequence, more attempts should be made to develop low-dose combination therapy of AT1R antagonists and D1R agonists in the treatment of sodium-dependent hypertension.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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This study has been supported by the Swedish Research Council, the Persson Family Foundation, and the Märta and Gunnar V. Philipson Foundation.


   ACKNOWLEDGMENTS
 
We thank Eivor Zettergren Markus for experimental assistance and Dr. Gerald F. DiBona for fruitful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Aperia, Dept. of Woman and Child Health, Karolinska Institutet, Astrid Lindgren Children's Hospital, Q2:09, SE-171 76 Stockholm, Sweden (e-mail: anita.aperia{at}ki.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
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 REFERENCES
 

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