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J. Biol. Chem., Vol. 276, Issue 29, 26906-26915, July 20, 2001

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Dopamine Acutely Stimulates Na⁺/H⁺ Exchanger (NHE3) Endocytosis via Clathrin-coated Vesicles

DEPENDENCE ON PROTEIN KINASE A-MEDIATED NHE3 PHOSPHORYLATION^*

Ming Chang Hu, Lingzhi Fan, Ladonna A. Crowder, Zoubida Karim-Jimenez^§, Heini Murer^§, and Orson W. Moe^¶

From the ^¶ Medical Service, Department of Veterans Affairs Medical Center, Dallas, Texas 75216, the  Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856, and the ^§ Institute of Physiology, University of Zürich, Zürich 8057, Switzerland

Received for publication, December 15, 2000, and in revised form, April 27, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Dopamine (DA) is a key hormone in mammalian sodium homeostasis. DA induces natriuresis via acute inhibition of the renal proximal tubule apical membrane Na⁺/H⁺ exchanger NHE3. We examined the mechanism by which DA inhibits NHE3 in a renal cell line. DA acutely decreases surface NHE3 antigen in dose- and time-dependent fashion without altering total cellular NHE3. Although DA₁ receptor agonist alone decreases surface NHE3, simultaneous DA₂ agonist synergistically enhances the effect of DA₁. Decreased surface NHE3 antigen, caused by stimulation of NHE3 endocytosis, is dependent on intact functioning of the GTPase dynamin and involves increased binding of NHE3 to the adaptor protein AP2. DA-stimulated NHE3 endocytosis can be blocked by pharmacologic or genetic protein kinase A inhibition or by mutation of two protein kinase A target serines (Ser-560 and Ser-613) on NHE3. We conclude that one mechanism by which DA induces natriuresis is via protein kinase A-mediated phosphorylation of proximal tubule NHE3 leading to endocytosis of NHE3 via clathrin-coated vesicles.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Extracellular fluid volume and to a certain extent blood pressure in mammals are determined by the balance between sodium intake and renal sodium excretion (1, 2). As regulator of sodium excretion, the intrarenal autocrine-paracrine dopamine (DA)¹ system assumes far greater importance than circulating endocrine or neurogenic dopamine (3-6). DA is produced in the proximal tubule via decarboxylation of its precursor L-dihydroxyphenylalanine derived from the plasma and glomerular filtrate (7-9) and is then secreted into the tubular lumen where it exerts its effects on multiple nephron segments, which cumulates in inhibition of tubular sodium absorption and natriuresis. Renal DA synthesis and excretion are increased by increased dietary salt and an intravenous saline load (10-13), and blockade of DA synthesis or DA receptor significantly blunts the natriuretic response (14-18). Quantitatively, the most significant inhibition of sodium transport occurs in the proximal tubule. DA inhibits proximal tubule sodium absorption partially by hemodynamic alterations (19-22), but the major effect is directly on the tubule epithelium (23-26) via inhibition of two principal sodium transporters: the apical membrane Na⁺/K⁺ exchanger (NHE3) (27-33) and the basolateral Na⁺,K⁺-ATPase (34-38). These effects are mediated by the DA receptor where five molecular isoforms (DR₁-like receptors: DR₁ and DR₅, and DR₂-like receptors DR₂, DR₃, and DR₄) have been identified to date; all five isoforms are known to be present in the renal tubular epithelium (39-43).

Previous studies in isolated apical membrane vesicles have shown that DA inhibits proximal tubule apical membrane Na⁺/H⁺ exchange activity mainly via DR₁-like receptors (25-28, 30, 31, 33) through both PKA-dependent and PKA-independent mechanisms (27, 31). The Na⁺/H⁺ exchanger on the apical membrane of the renal proximal tubule is encoded by NHE3 (44-46), one of the seven members of the NHE gene family (47). We have shown in opossum kidney (OK) cells that the DA₁-like and DA₂-like receptors have synergistic actions on NHE3 activity and that inhibition of NHE3 activity by DA is accompanied by complex changes in NHE3 phosphorylation and dephosphorylation (33). However, the mechanisms by which DA acutely reduces NHE3 activity have not been examined. Redistribution of NHE3 transporters has been shown to mediate regulation of NHE3 activity in intact kidney (48-54), in cultured renal epithelial cells (55, 56), and in transfected fibroblasts (57-64). In addition, although NHE phosphorylation has been associated with changes in NHE3 activity (52, 65-68), and phosphorylation appears to be functionally important for regulation of NHE3 activity by pharmacologic activators of protein kinases in transfected fibroblasts (65-67), the physiologic significance of NHE3 phosphorylation is still undetermined. In this paper we characterize one mechanism by which DA acutely inhibits NHE3: the internalization of NHE3 secondary to PKA-mediated NHE3 phosphorylation and NHE3 endocytosis via clathrin-coated vesicles.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture, Agonists, Transfection, and Plasmid Constructs-- OK cells were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 1 mM sodium pyruvate, 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin and were rendered quiescent postconfluence by serum removal for 48 h prior to experimentation. Transient transfections were performed with LipofectAMINE (Life Technologies, Inc.). Transfection efficiency was monitored by cotransfection with -galactosidase and staining cells with 5-bromo-4-chloro-3-indolyl -D-galactopyranoside as well as staining with anti-epitope (c-Myc) antibody (typically 80%). Mammalian expression plasmids used in this study include: 1) C-terminal c-Myc- and hexahistidine (6His)-tagged wild type opossum (NHE3/c-Myc/6H); 2) C-terminal enhanced green fluorescent protein (eGFP)-tagged NHE3 (NHE3/eGFP); 3) C-terminal c-Myc- and 6His-tagged NHE3 with two mutated serines, S560A and S613A (NHE3^S560A/S613A/c-Myc/6H); 4) wild type dynamin (dyn^WT); 5) dominant-negative GTP binding-defective dynamin (dyn^K44A); 6) cyclic AMP binding-defective regulatory subunit of protein kinase A (RII^mut). Agonists used include dopamine (stabilized with 1.1 mM sodium ascorbate) (Sigma), the DR₁-specific agonist SKF38393 (Tocris, St. Louis, MO), the DR₂-specific agonist quinpirole (Tocris), DR₁-specific antagonist SCH23390 (Research Biochemicals, Natick, MA), the DR₂-specific antagonist sulpiride (Tocris), 8-bromo-cAMP, and the PKA inhibitor H89. Commercial antisera include anti-c-Myc (Invitrogen, Carlsbad CA) and anti-adaptin a (Santa Cruz Biotechnology, Santa Cruz, CA). To disrupt activation of PKA by cAMP by RII^mut, a 5-min pulse of 5 mM 8-bromo-cAMP was given to the cells 16 h post-transfection to allow the dominant-negative RII^mut to engage the native PKA-catalytic subunit. Experiments with dopamine were performed at 48 h post-transfection.

Measurement of Surface NHE3 Antigen and NHE3 Endocytosis and Exocytosis-- These assays were performed as described previously (56). To measure surface NHE3, OK cells were surface-labeled with biotin using a modification of the method of Gottardi after the addition of agonists (56, 69). After rinsing in PBS/calcium/magnesium (150 mM NaCl, 10 mM Na₂HPO₄, pH 7.40, 0.1 mM CaCl₂, 1 mM MgCl₂), cells were incubated with the arginine and lysine reactive of NHS-SS-biotin (2 mg/ml; Pierce) in buffer (150 mM NaCl, 10 mM triethanolamine, pH 7.4, 2 mM CaCl₂), quenched (PBS/calcium/magnesium with 100 mM glycine), and lysed in biotin-RIPA (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS). Lysates were centrifuged (109,000 × g for 25 min at 2 °C, Beckman TLX/TLA 100.3 rotor, Fullerton, CA), and protein content in the supernatant was quantified by the method of Bradford. Equal amounts of cell lysate were equilibrated with streptavidin-agarose (Pierce) at 4 °C. Beads were rinsed sequentially with solutions A (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA), B (50 mM Tris-HCl, pH 7.4, 500 mM NaCl), and C (50 mM Tris-HCl, pH 7.4), and biotinylated proteins were liberated by reduction incubation in 100 mM dithiothreitol, reconstituted in Laemmli's buffer, resolved by SDS-polyacrylamide gel electrophoresis, and electrotransferred to Imobilin. NHE3 antigen was quantified by labeling with either anti-NHE3 antiserum 5683 or for experiments with exogenous c-Myc-tagged NHE3, a monoclonal anti-c-Myc was used (Invitrogen, Carlsbad, CA). Endocytosis was measured by a protocol adapted and modified from the stage-specific MesNa-resistant and avidin-protection endocytosis assays originally described by Carter and co-workers (56, 70). OK cells were surface-labeled with NHS-SS-biotin and quenched as described above. Cells were then warmed to 37 °C in the presence of DA or vehicle to allow endocytosis to occur over 30 min. Surface biotin was then saturated with avidin (50 mg/ml PBS) and washed with biocytin (50 mg/ml PBS), or alternatively, surface biotin was cleaved with the small cell-impermeant reducing agent MesNa (50 mM in 50 mM Tris, pH 7.4). The freshly endocytosed proteins bearing biotin were protected from either avidin saturation or MesNa cleavage. Cells were then solubilized in RIPA, and biotinylated proteins were retrieved and assayed for NHE3 as described above. Avidin-protected fraction measures early and late endocytosis because avidin cannot enter the constricted necks of clathrin-coated pits. TCEP-protected fraction measures late endocytosis because complete excommunication from the exterior is required to prevent TCEP access.

Imaging of NHE3 in Live Cells-- OK cells were plated on glass coverslips and transfected with NHE3/eGFP. Cultures of transfected OK cells were maintained at 37 °C for 48 h, and fresh medium was replenished 2 h prior to the experiment. Using a fluorescence microscope, living green fluorescent cells were selected before the treatment as described previously (71). DA or vehicle was then added to the cell medium. After the stated period of incubation at 37 °C, the same selected cells were identified again, and the pattern of expression of the different cotransporters was compared with that before treatment. At least six experiments were performed for each condition to evaluate the results.

Coimmunoprecipitation-- 80% confluent OK cells were transfected with either wild type NHE3/c-Myc/6H or NHE3^S560A/S613A/c-Myc/6H. 48 h post-transfection, OK cells were treated with either vehicle or DA. After washing with ice-cold PBS, cells were lysed with ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 100 mg/ml phenylmethylsulfonyl fluoride, 4 mg/ml leupeptin, 4 mg/ml aprotinin, 10 mg/ml pepstatin). The slurry was cleared by centrifugation (109,000 × g for 25 min at 4 °C in a Beckman TLX/TLA 100.3 rotor), and the adaptin AP2 was immunoprecipitated with anti-adaptin  (1:500 dilution) and protein G-Sepharose. After washing with RIPA buffer, the antibody-antigen complex was eluted in SDS buffer (5 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 1% (w/v) -mercaptoethanol, 0.1% (w/v) SDS, 0.01% (w/v) bromphenol blue), resolved on SDS-polyacrylamide gel, and transferred to nitrocellulose membrane. NHE3 was quantified by immunoblot with anti-c-Myc.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DA Decreases Surface NHE3 Protein-- In the OK cell, DA decreases NHE3 protein in a dose-dependent (Fig. 1) and time-dependent (Fig. 2) fashion. Typical experiments are shown in Figs. 1A and 2A, and the summarized data are shown in Figs. 1B and 2B. The dose dependence (Fig. 1) of a decrease in surface NHE3 (half-maximal decrease at 10⁵ M and maximal decrease of 70% at 10⁴ M measured after 30 min DA) is similar to that of DA-induced inhibition of NHE3 activity described previously (33). The time dependence (Fig. 2) of decrease in surface NHE3 however is discrepant with changes in NHE3 activity. Whereas a decrease in NHE3 activity is evident after 5 min of DA (33), a decrease in surface NHE3 is not detectable until after 20 to 30 min. There was no change in total cellular NHE3 within the experimental period (Fig. 1A). Identical results were obtained from studying native OK NHE3 protein or with OK cells transiently expressing NHE3/c-Myc/6H (not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Effect of DA on surface and total cellular NHE3 in OK cells: dose dependence. OK cells were rendered quiescent, and the stated concentration of dopamine was added for 30 min. Monolayers were biotinylated, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot with anti-OK NHE3. n = sets of dose responses. Bars and error bars indicate mean and S.E. Panel A, typical experiment. Panel B, summary of all experiments on surface NHE3. n = 6. Asterisks indicate p < 0.05 compared with control (ANOVA).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Effect of DA on surface NHE3 in OK cells: time dependence. OK cells were rendered quiescent, and 105 M dopamine was added for the stated period of time. Monolayers were biotinylated, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot with anti-OK NHE3. n = sets of time responses. Bars and error bars indicate mean and S.E. Panel A, typical experiment. Panel B, summary of all experiments. n = 4. Asterisks indicate p < 0.05 compared with control (ANOVA).

In addition to the biochemical biotin assay, we also examined the effect of DA on NHE3 by imaging live cells. OK cells were transiently transfected with NHE3/eGFP, and fluorescent microscopy on live transfected cells showed NHE3 to be a typical brush-border protein with the characteristic punctate staining (Fig. 3). The addition of DA caused a time-dependent decrease in surface NHE3 with the appearance of a characteristic intracellular staining pattern (Fig. 3). Within the 2 h of fluorescent microscopic examination, no significant decrease in total cellular NHE3 was appreciated. This is compatible with the biochemical data presented above. The addition of vehicle served as a time control where no change in NHE3 distribution was seen (Fig. 3).

View larger version (96K): [in this window] [in a new window]   Fig. 3.   Effect of DA on NHE3/eGFP in OK cells: live cell imaging. NHE3 bearing a C-terminal eGFP tag was expressed in OK cells and treated with either 105 M DA or vehicle. Fluorescent whole cell images were obtained at the indicated times. Six sets of experiments showed similar responses.

DA₁ and DA₂ Act Synergistically to Decrease Surface NHE3-- Previous studies have shown synergistic roles for DA₁ and DA₂ receptor agonism on NHE3 activity (33). We next examined the relative roles of DA₁ and DA₂ receptors on surface NHE3 antigen. DA₁ agonist alone was effective in reducing surface NHE3, whereas DA₂ alone was ineffective. The combination of DA₁ and DA₂ resulted in greater reduction of surface NHE3 than DA₁ alone. (Fig. 4, A and B). To confirm these results further, we used subtype-specific inhibitors to try to block the effect of DA on surface NHE3 (Fig. 4, C and D). DA₁ blockade abolished most of the DA-induced decrease in surface NHE3, whereas DA₂ blockade had minimal effect. These findings are similar to the previously reported synergistic effect observed with the inhibition of NHE3 activity by DA₁ and DA₂ agonists (33).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Differential and synergistic effects of DR1 and DR2 receptor agonism and antagonism. OK cells were rendered quiescent, and the agonists (105 M dopamine; DR1: 105 M SKF38393; DR2: 105 M quinpirole) and/or antagonists (DR1: 105 M SCH23390; DR2: 105 M sulpiride) were added for 30 min. Monolayers were biotinylated, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot with anti-OK NHE3. n = number of sets of experiments. Bars and error bars indicate mean and S.E. Asterisks indicate p < 0.05 compared with control (ANOVA). Panel A, typical experiment with DR1 and/or DR2 agonists. Panel B, summary of all experiments with DR1 and/or DR2 agonists. n = 5. Panel C, typical experiment with DR1 and/or DR2 antagonists. Panel D, summary of all experiments with DR1 and/or DR2 antagonists. n = 5.

DA Stimulates NHE3 Endocytosis via a Dynamin-dependent Clathrin-coated Vesicle Pathway-- NHE3 has been visualized in both the plasma membrane and in intracellular compartments in both native kidney tissue and culture cells (48-64). The decrease in surface NHE3 in response to DA can be caused by decreased exocytotic insertion or increased endocytotic retrieval. We quantified endocytotic rate biochemically with the MesNa protection assay. Fig. 5, A and B, shows that DA stimulated NHE3 endocytosis by 58%. When cells were kept at 4 °C to arrest trafficking, a small amount of MesNa-protected NHE3 was visible. This likely reflects incomplete cleavage of the NHS-biotin rather than endocytosis at 4 °C (Fig. 5A). This usually represents <10% of the signal. The GTPase dynamin is required for both clathrin- and caveolin-mediated endocytosis (72, 73). We next cotransfected the GTP binding-defective dominant-negative dynamin I (dyn^K44A) along with c-Myc-tagged NHE3 into OK cells and studied the effect of DA on surface NHE3 protein (Fig. 6A). Because the read-out in this assay is with the anti-c-Myc antiserum, which does not react with the native NHE3, only transfected cells were selectively studied. Whereas cells transfected with wild type dynamin (dyn^WT) showed normal down-regulation of surface NHE3 by DA, cells transfected with dyn^K44A failed to respond to DA (Fig. 6, A and B). A key component of the clathrin-coated vesicle endocytotic pathway is the family of adaptor proteins (APs) (73, 74). We next examined the association of NHE3 with the adaptor protein AP2 by coimmunoprecipitation. Fig. 7A shows that DA increased the amount of NHE3 bound to total cellular AP2. In fact, the association of NHE3 to AP2 is barely detectable without DA. In sum, our data indicate that DA shifts NHE3 from the plasma membrane to endocytic vesicles via a dynamin- and AP2-dependent process.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of DA on NHE3 endocytotic rate: MesNa protection assay. Quiescent OK were surface labeled with NHS-SS-biotin, and endocytosis was initiated in the presence of either DA or vehicle at 37 °C for 40 min. Cells were then cooled at 4 °C, and exposed biotin was cleaved with MesNa. Biotinylated proteins protected from MesNa were retrieved by streptavidin-agarose precipitation, and NHE3 was quantified by immunoblot with anti-OK NHE3. Parallel control experiments were performed at 4 °C where endocytosis was arrested. n = sets of experiments. Bars and error bars indicate mean and S.E. Asterisk indicates p < 0.05 compared with control (unpaired t test). Panel A, typical experiment. Panel B, summary of experiments at 37 °C. n = 4.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of DA on surface NHE3: dependence on dynamin. OK cells were cotransfected with NHE3/c-Myc along with either wild type dynamin (dynWT) or K44A dominant-negative dynamin (dynK44A). DA (105 M 30 min) was added. Monolayers were biotinylated, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot with anti-c-Myc. n = sets of experiments. Bars and error bars indicate mean and S.E. Asterisks indicate p < 0.05 compared with control (ANOVA). Panel A, typical experiment. Panel B, summary of all experiments. n = 4.

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Effect of DA on association of NHE3 with adaptin AP2: coimmunoprecipitation. Panel A, native NHE3. Quiescent OK cells were treated with either DA (105 M 30 min) or vehicle, lysed, and adaptin was immunoprecipitated with anti-adaptin . The immune complex was resolved by SDS-polyacrylamide gel electrophoresis and probed with either anti-NHE3 or anti-adaptin . Panel B, wild type NHE3 versus NHE3S560A S613A. OK cells were transfected with either wild type or mutant NHE3/c-Myc, treated with either DA (105 M, 30 min) or vehicle, lysed, and adaptin was immunoprecipitated with anti-adaptin a. The immune complex was resolved by SDS-polyacrylamide gel electrophoresis and probed with either anti-c-Myc or anti-adaptin . Three sets of experiments showed the same results.

DA-stimulated NHE3 Endocytosis Is Dependent on NHE3 Phosphorylation by PKA-- To examine the role of PKA in DA-induced NHE3 endocytosis, PKA was inhibited either pharmacologically or genetically. In the presence of 10⁶ M H89, DA failed to decrease surface NHE3 (Fig. 8). As a second approach to ensure specific inhibition of PKA only, a dominant-negative PKA regulatory subunit (RII^mut) was used to sequester all catalytic PKA subunits constitutively. RII^mut binds stoichiometrically to the catalytic subunit but is devoid of both cAMP binding sites and hence does not release its pseudosubstrate inhibition even in high ambient cAMP concentrations (66, 75). In the background of RII^mut, DA was unable to decrease surface NHE3 (Fig. 8B). These data indicate that intact PKA is necessary for DA to decrease surface NHE3. Note that in these experiments, we pulsed the cells with 5 mM 8-bromo-cAMP for 5 min 16 h prior to exposure to DA or vehicle to allow RII^mut to engage and sequester native PKA-catalytic subunit. Of interest is the fact that the brief 8-bromo-cAMP pulse appears to amplify the DA effect. The mechanism for this effect is not known at present.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of DA on surface NHE3: dependence on PKA. In the background of PKA inhibition, DA (105 M, 30 min) or vehicle-treated cells were surface-labeled with biotin, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot. n = number of sets of experiments. Bars and error bars indicate mean and S.E. Asterisk indicates p < 0.05 compared with control (ANOVA). Panel A, typical experiment where PKA was inhibited by 106 M H89, and the effect of DA on native NHE3 was studied. Panel B, summary of all experiments. n = 3. Panel C, typical experiment where OK cells were transfected with NHE3/c-Myc ± a cAMP-binding defective dominant-negative regulatory subunit of PKA (RIImut). The effect of DA on NHE3/c-Myc was studied. Panel D, summary of all experiments. n = 4.

Although NHE3 is a substrate for PKA (65, 66, 76), we have not proven that NHE3 phosphorylation by PKA is necessary for DA to decrease surface NHE3. Previous studies have mapped target serines (Ser-552 and Ser-605) on the cytoplasmic domain of rat NHE3 as functionally significant PKA substrates (65, 66). These serines are situated in highly conserved regions of NHE3 (Fig. 9). To examine the role of NHE3 phosphorylation in mediating NHE3 endocytosis, we eliminated both corresponding phosphoserines in OK NHE3 (Ser-560 and Ser-613) by mutation to alanines and examined for the DA-induced decrease in surface NHE3 (NHE3 mutant NHE3^S560A/S613A). Fig. 10A shows that surface NHE3^S560A/S613A antigen is not regulated by DA. Fig. 10B summarizes the data. Fig. 10C shows the effect of DA on NHE3^S560A/S613A/eGFP in transiently transfected live OK cells. Note that the DA-induced internalization pattern seen in Fig. 3 is abolished completely with the double serine mutants. To examine at which step the serine mutations arrest endocytosis, we repeated the coimmunoprecipitation studies with AP2 using NHE3^S560A/S613A. As shown in Fig. 7B, the two serine mutations virtually abolished AP2 binding to NHE3. These studies support the model that NHE3 phosphorylation by PKA is necessary for its endocytotic retrieval.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   Alignment of NHE3 sequence. Single amino acid abbreviations of amino acids flanking rat NHE3 Ser-552 and Ser-605 in seven species. The bovine clone was a partial cDNA.

View larger version (37K): [in this window] [in a new window]   Fig. 10.   Effect of DA on surface NHE3: dependence on NHE3 phosphorylation. OK cells were transfected with either wild type NHE3 or NHE3 harboring two point mutations (S560A and S613A). DA- (105 M, 30 min) or vehicle-treated cells were surface labeled with biotin, and surface proteins were retrieved from the cell lysate by streptavidin precipitation. NHE3 protein abundance was quantified by immunoblot with anti-c-Myc. n = number of sets of experiments. Bars and error bars indicate mean and S.E. Asterisk indicates p < 0.05 compared with control (ANOVA). Panel A, typical experiment. Panel B, summary of all experiments. n = 3. Bars and error bars indicate mean and S.E. Asterisk indicates p < 0.05 compared with control (unpaired t test). Panel C, either wild type NHE3/eGFP or NHE3/eGFP bearing two serine mutations (S560A and S613A) was expressed in OK cells and treated with either 105 M DA or vehicle. Fluorescent whole cell images were obtained of whole live cells. Three sets of experiments showed similar responses.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In addition to its role in maintaining sodium homeostasis under normal conditions (3-18), defects in the renal paracrine/autocrine DA axis have been demonstrated and postulated to contribute to certain forms of polygenic hypertension in both rodents (77-86) and humans (87-92). In the proximal tubule where 50-60% of NaCl and water is reabsorbed, DA inhibits the basolateral membrane Na⁺,K⁺-ATPase (34-38), which is the primary driving force for transepithelial Na⁺ absorption. In addition, DA also inhibits two apical Na⁺-coupled transporters, the Na⁺-coupled inorganic phosphate transporter (92-96) and the Na⁺/H⁺ exchanger NHE3 (27-33). In addition to transcellular Na⁺ flux, inhibition of NHE3 also reduces the driving force for paracellular NaCl transport (97). In concert, these mechanisms potently inhibit proximal tubule NaCl reabsorption.

NHE3 is regulated acutely by a variety of hormones (98-99). The acute response of NHE3 to DA follows a biphasic response similar to that of parathyroid hormone, described previously in rat kidney (52) and in OK cells (56). Although inhibition of NHE3 activity is evident within 5 min of DA application (33), decreases in surface NHE3 antigen are not detectable until after 20 to 30 min. The mechanism of the immediate reduction of NHE3 activity is not known. This paper focuses on the second phase of the response which involves decreases in surface NHE3 antigen.

During the first hour after DA application, there is no decrease in total cellular NHE, indicating that the reduction in surface NHE3 is caused entirely by redistribution of NHE3 protein. The decrease of plasma membrane NHE3 is secondary to stimulation of endocytosis as evident by the increase in MesNa-protected fraction of NHE3. Endocytotic retrieval is a well described mode of internalization of plasma membrane proteins, ligand receptor complexes, and extracellular fluid phase constituents. Four categories of pinocytotic processes have been proposed: macropinosome, noncoated vesicles, caveolae, and clathrin-coated vesicles (100). The current data suggest involvement of both the caveolae and clathrin-coated vesicle pathway for NHE3. Two components will be considered: endocytosis of NHE3 under base-line conditions and endocytosis of NHE3 in response to DA.

The presence of a MesNa-protected NHE3 signal in the absence of DA indicates the existence of base-line NHE3 endocytosis. Intact dynamin function is absolutely necessary for the formation and discharging of clathrin-coated vesicles (101, 102). In addition, dynamin is also involved in other trafficking events such as caveolin-mediated endocytosis (103, 104) and budding from the Golgi complex (105). Dyn^K44A is expected to block all dynamin-dependent processes (102). The net effect of dyn^K44A in the absence of DA was higher levels of surface NHE3 protein (Fig. 6, A and B). Adaptin is believed to be the mediator that directs and initiates assembly of the clathrin triskeleton and coated pit in the vicinity of the protein targeted to be retrieved (72, 102, 106). The total absence of NHE3-adaptin association in the absence of DA suggests that the clathrin-coated pathway is not involved with base-line NHE3 retrieval.

The DA-induced increase in NHE3 endocytosis has two associated features: it is completely dependent on dynamin, and it involves formation of an NHE3·AP2 adaptin complex. In concert, these findings suggest that DA-induced NHE3 endocytosis proceeds via the clathrin-coated vesicle pathway. A similar association of Na⁺/K⁺-ATPase with adaptin AP2 has been described in response to DA (106). Based on the current immunoprecipitation data, one cannot distinguish whether the adaptin complex binds directly to NHE3 or whether other immediate proteins are involved. The primary sequence of the cytoplasmic domain of NHE3 potentially harbors all four of the endocytotic sorting signals identified to date (107-112): 1) tyrosine-based, 2) dileucine-based, 3) near C-terminal phosphoserine-rich domain, and 4) ligand-induced phosphoserine related to ubiquitination. The fact that the double serine mutant NHE3 completely failed to bind to AP2 irrespective of the addition of DA suggests that NHE3 phosphorylation is necessary for the NHE3-AP2 association. Several facts remain elusive. It is unknown whether phosphorylation of Ser-560 and Ser-613 is sufficient for NHE3-AP2 association. It is conceivable that factors other than NHE3 phosphorylation are required. It is also unknown whether NHE3 binds directly to AP2. The fact that AP2 and clathrin can form cages in vitro with liposomes suggests that membrane proteins may be irrelevant (113, 114).

The acute inhibition of NHE3 and Na⁺/K⁺-ATPase activities by DA is dependent on both DR₁ and DR₂ receptors (33, 36, 37). The DA-induced acute decrease in surface NHE3 is also dependent on both DR₁ and DR₂ receptors. DR₁ alone appears to be sufficient to incur an effect, but DA₂ alone is not. However, simultaneous DA₁ and DA₂ receptor activation produces a synergistic effect. This pattern is identical to that described for decreases in NHE3 activity (33). The signaling pathways mediating NHE3 inhibition by DA are still controversial. The contribution of the DA₁-gas-adenylyl cyclase-protein kinase A axis is the only undisputed pathway (27, 31), although non-PKA cascades are most certainly involved (31, 33, 110). In this paper, we focused only on the PKA pathway. There is a difference in PKA dependence between the acute inhibition of NHE3 activity and acute decrease in NHE3 surface antigen. Although there appears to be a PKA-independent component of acute inhibition of NHE3 activity by DA, the DA-induced decrease in NHE3 surface protein is blocked completely by either pharmacologic inhibition or pseudosubstrate inhibition by the regulatory subunit of PKA. The downstream target of PKA in mediating NHE3 endocytosis may be diverse, but at least one of the required events for NHE3 endocytosis is phosphorylation of NHE3 by PKA. Two serines shown previously to be PKA substrates in the rat homolog (65, 66) are well conserved across multiple mammalian NHE3s. The functional significance of NHE3 phosphorylation has been shown previously in NHE3-transfected fibroblasts subjected to pharmacologic activation of kinases (65-67). The current data are the first demonstration that NHE3 phosphorylation is important for hormonal regulation in a renal epithelial cell line. PKA phosphorylation of NHE3 is required at a very early step in the endocytotic pathway.

In summary, we propose the following model of acute regulation of plasma membrane NHE3 trafficking in response to DA in renal epithelia. At base line, a constitutive NHE3 endocytotic rate exists via the caveolae pathway. Upon stimulation by DA, one critical signaling pathway is PKA activation. Phosphorylation of plasma membrane NHE3 by PKA is a necessary event for assembly of AP2 around NHE3 followed by invagination, severance, and discharge of a clathrin-coated vesicle sending NHE3 to the endosomal compartment. This cascade of events is fundamental to understanding mechanisms of DA-induced natriuresis in mammalian Na⁺ homeostasis.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Robert Alpern and Dr. Michel Baum for their helpful discussions and Dr. Sandy Schmid and Dr. Stan McKnight for providing us with reagents.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK-48482 and DK-54396 (to O. W. M.), by the Department of Veterans Affairs Research Service (to O. W. M.), American Heart Association Texas Affiliate Grant 98G-052 (to O. W. M.), and Swiss National Science Foundation Grant 3100-46523.96 (to H. M.).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.

To whom correspondence should be addressed: Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8856. Tel.: 214-648-3152; Fax: 214-648-2071; E-mail: orson.moe@utsouthwestern.edu.

Published, JBC Papers in Press, April 27, 2001, DOI 10.1074/jbc.M011338200

	ABBREVIATIONS

The abbreviations used are: DA, dopamine; NHE3, Na⁺/H⁺ exchanger; PKA, protein kinase A; OK, opossum kidney; 6His, hexahistidine; eGFP, enhanced green fluorescent protein; WT, wild type; RII^mut, cAMP binding-defective regulatory subunit of protein kinase A; PBS, phosphate-buffered saline; Mes, 4-morpholineethanesulfonic acid; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; AP2, adaptor protein 2; ANOVA, analysis of variance.

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