Institutes of 1Physiology and 2Anatomy, Zurich University, Zurich, Switzerland; and 3Genzyme Corporation, Framingham, Massachusetts
Submitted 15 September 2004 ; accepted in final form 14 February 2005
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ABSTRACT |
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proximal tubule; opossum kidney cells; phosphorylation; endocytosis
NaPi-IIa interacts with the Na+/H+ exchanger-regulatory factor-1 (NHERF1), a protein of 358 residues, the mRNA of which is detected among other tissues in kidney, proximal small intestine, and liver (11, 35). Although originally identified as a factor required for cAMP-induced inhibition of the Na+/H+ exchanger type 3 (NHE3) (37), NHERF1 has been shown to interact with different subsets of proteins, including membrane transporters and receptors, cytoplasmic proteins involved in intracellular signaling, and cytoskeletal proteins (for review, see Ref. 36).
NHERF1 is expressed in the apical membrane of different nephron segments of the kidney and colocalizes with NaPi-IIa in the BBM of proximal tubules (11). It contains two PSD-95/Drosophila disk large-1/zonula occludens-1 (PDZ) domains, sequences of about 90 amino acids that are involved in protein-protein interaction (9), and a merlin-ezrin-radixin-moesin (MERM) binding domain. The apical location of NHERF1 is dependent on the MERM-binding domain, which indicates that its subcellular distribution is mediated by binding to members of the MERM family of cytoskeletal proteins. Indeed, studies in opossum kidney (OK) cells showed that the MERM-binding domain alone shows apical expression, whereas the single PDZ domains accumulate intracellularly (15).
On the basis of in vitro assays, interaction with NaPi-IIa takes place via the first PDZ domain (PDZ1) of NHERF1 and the last three amino acids of the cotransporter (TRL), which represent a class I PDZ-binding motif S/T-X-(V/I/L), where X is any amino acid (11, 12). Moreover, in vivo experiments have shown that transfection of PDZ1 alone in OK cells has a dominant negative effect on the apical expression of the endogenous cotransporter (15). This effect is consistent with our previous finding that TRL of NaPi-IIa impairs the apical expression of the cotransporter (17). In addition, recent reports have suggested that the NaPi-IIa-NHERF1 association can also take place independently of the TRL motif (19, 22).
Interaction with NHERF1 is involved in different cellular events such as apical targeting and/or retention (26), regulation of the fate of retrieved proteins (6), and assembly of regulatory complexes (21, 28). As mentioned above, we have shown that the TRL of NaPi-IIa is involved in its apical expression (17) and that this at least partially involves binding to NHERF1 (15). This finding is supported by the phenotype of the NHERF1-knockout mouse, which are characterized by reduced expression of NaPi-IIa in BBM of proximal tubules (7, 33).
Parathyroid hormone (PTH) induces internalization of the cotransporter (for review, see Ref. 27). This effect has been studied in animal models as well as in OK cells. In both systems, PTH induces endocytosis and lysosomal degradation of NaPi-IIa (18, 29, 34). The PTH effect is mediated by receptors localized in the apical and basolateral membranes, and activation of these receptors transduces intracellular signals involving both PKA and PKC pathways (1, 34). The apical response is mediated essentially by PKC, whereas both pathways seem to be involved in basolateral signaling (34). Both signals converge, at least partially, at the level of ERK1/2 (4). Interestingly, NHERF1 and NHERF2 have been shown to interact with the PTH receptor 1 and PLC, and this interaction may condition the intracellular cascade activated after PTH treatment (25).
Herein we show that unlike NaPi-IIa, NHERF1 remained apically expressed after PTH treatment in both proximal tubules and OK cells. Moreover, the total amount of NHERF1 was not affected by PTH. Thus the hormonal treatment seems to induce the dissociation of NaPi-IIa-NHERF1 complexes. In agreement with this hypothesis, coimmunoprecipitation assays showed that the amount of NaPi-IIa associated with NHERF1 was reduced in OK cell lysates from PTH-treated samples compared with controls. PTH induced an increase in the phosphorylation state of NHERF1 in kidney slices, whereas NaPi-IIa was not phosphorylated either under basal conditions or after PTH treatment. Therefore, PTH-induced dissociation of NaPi-IIa-NHERF1 complexes takes place in the presence of increased phosphorylation of NHERF1, although further studies are required to show a cause-and-effect relationship between these phenomena.
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MATERIALS AND METHODS |
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Immunostaining. Confluent OK cultures plated on glass coverslips were preincubated with or without leupeptin (100 µg/ml) for 30 min, followed by incubation with 1-34 PTH (108 M) for 4 h. Next, the cells were processed for immunostaining with antibodies against endogenous NaPi-IIa (23) as well as with a monoclonal anti-myc antibody (Invitrogen) as previously reported (15). For actin detection, Alexa Fluor phalloidin (Molecular Probes, Eugene, OR) was added together with secondary antibodies. The coverslips were mounted with glycerol (Dako, Carpinteria, CA) containing 2.5% 1,4-diazabicyclo(2.2.2)octane (Sigma, St. Louis, MO) as fading retardant.
For immunofluorescence imaging of kidney slices, 8- to 12-wk-old mice (NMRI, Janvier, France) were fed standard chow with free access to tap water. In some animals, 1-34 PTH was injected into the tail vein as a single 25-µg bolus. Thirty minutes after injection, mice were perfused with fixative, and the kidneys were then removed, cut into slices, mounted onto thin cork plates, and immediately frozen (4). Cryosections of 5-µm thickness were incubated with antibodies against NaPi-IIa (8) or with an affinity-purified anti-NHERF antibody (37). After incubation with secondary antibodies, sections were placed on coverslips using the mounting medium described above. Staining was analyzed using a laser confocal microscope (TCSSP; Leica, Wetzlar, Germany), and confocal sections were processed using Imaris software (Bitplane, Zurich, Switzerland).
Coimmunoprecipitation of NaPi-IIa and NHERF1 from OK cells. WT OK cells or cells stably transfected with V5-fused NaPi-IIa were grown to confluence. Where indicated, cultures were preincubated for 30 min with leupeptin (100 µg/ml), followed by incubation with 1-34 PTH (108 M) for the indicated times. Cultures were then homogenized in 5 mM HEPES and 0.5 mM EDTA, pH 7.2. Postnuclear supernatants were centrifugated at 80,000 g for 30 min. Membranes were resuspended in 25 mM HEPES (pH 7.2), and proteins were cross linked with 0.25 mM dithiobis (succinimidyl propionate) for 1 h on ice as previously reported (24). Next, membranes were repelleted and extracted in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl and 25 mM HEPES, pH 7.4) containing (in %) 1 Triton X-100, 0.5 sodium deoxycholate, 0.1 SDS, and 1 protease inhibitor cocktail. The solubilized material was precleared using incubation with normal rabbit sera (1:200 dilution) plus protein A/G-agarose beads for 2 h at 4°C. Beads were spun down, and supernatants were incubated overnight with either anti-NaPi-IIa (1:200 dilution) or anti-NHERF1 (1:100 dilution) polyclonal antibodies in the presence of protein A/G-agarose beads. After intensive washes in RIPA buffer, proteins were finally eluted using incubation in 1x loading buffer (containing 1.5 mg/ml DTT) for 5 min at 94°C. Eluted proteins were separated on 9% SDS-PAGE gel. After transfer, nitrocellulose membranes (Schleicher & Schuell, Keene, NH) were incubated overnight at 4°C with either a monoclonal anti-NHERF1 (Abcam, Cambridge, UK) or anti-V5 antibodies (Invitrogen, Carlsbad, CA), respectively. Immunoreactive signals were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, UK).
GST pull-down of NHERF1 and NaPi-IIa from mouse kidney samples. Glutathione-S-transferase (GST) pull-down experiments were performed as described previously (12). Briefly, glutathione agarose beads were coupled, for 30 min at 4°C with rotation, to either GST alone or GST fused to the NH2-terminal ezrin domain, the COOH-terminal cytoplasmic tail of NaPi-I, or the PDZ3 domain of PDZK1. The beads were then collected by performing centrifugation at 12,000 rpm for 30 s and incubated for 1 h at 4°C with either BBM (Fig. 1) or phosphorylated lysates from kidney cortex (see Fig. 5). After several washes in Tris-buffered saline (TBS) buffer, bound proteins were eluted by incubation for 2 min at 94°C with 2x loading buffer containing DTT. Eluted proteins were separated in 10% SDS-PAGE gel, transferred to nitrocellulose membranes, and incubated with antibodies against NHERF1 or NaPi-IIa.
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Ex vivo phosphorylation. NMRI mice were anesthetized by intraperitoneal injection and perfusion was performed through the left ventricle with sucrose phosphate buffer (140 mM sucrose and 140 mM NaH2PO4-NaH2PO4, pH 7.4) to remove blood from the kidneys. The kidneys were harvested, and coronal slices were prepared as previously described (4). Slices derived from one kidney were transferred into 2 ml of prewarmed (37°C) Hanks' buffer (in mM: 110 NaCl, 5 KCl, 1.2 MgSO4, 1.8 CaCl2, 4 sodium acetate, 1 sodium citrate, 6 glucose, 6 L-alanine, 1 NaH2PO4, 3 Na2HPO4, and 25 NaHCO3, pH 7.4), gassed with 5% CO2-95% O2. Samples were then incubated for 30 min with 1 mCi 32PO4 (PerkinElmer, Wellesley, MA), with or without leupeptin (200 µg/µl). Subsequently, they were incubated for an additional 30 min in the absence or presence of 107 M PTH, 104 M 8-BrcAMP, or 104 M 1,2-dioctanoyl-sn-glycerol (DOG; Sigma). We have previously shown the viability of the proximal cells within this incubation time (4). Slices were finally homogenized in binding buffer (120 mM NaCl and 50 mM Tris·HCl, pH 8) containing 0.5% Igepal CA-630 (Sigma) and 1:100 phosphatase inhibitor cocktail (Sigma), and centrifuged for 10 min at 10,000 g. Supernatants were processed for GST pull-down with either GST alone or GST fused to the COOH-terminal cytoplasmic tail of NaPi-I (to pull down NHERF1) or to the PDZ3 domain of PDZK1 (to pull down NaPi-IIa) (12) as described above.
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RESULTS AND DISCUSSION |
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NaPi-IIa and NHERF1 localize in different compartments after PTH treatment. PTH induces downregulation of NaPi-IIa in both renal proximal tubules and OK cells. This downregulation involves megalin-dependent endocytosis (2, 3) via clathrin-coated vesicles, followed by lysosomal degradation of internalized cotransporters (18, 29, 34). In the present study, we first investigated whether the expression of NHERF1 is also regulated in response to PTH treatment. For this purpose, we stained mouse kidney slices from control and PTH-treated animals with antibodies against NaPi-IIa and NHERF1. As shown in Fig. 2A, both proteins were expressed at the BBM of untreated proximal tubules. However, 45 min after incubation with PTH, the signal for NaPi-IIa was strongly reduced, whereas no significant changes for NHERF1 could be detected. The PTH effect on the distribution of both proteins was also studied in OK cells. We previously reported the production of a myc-NHERF1-expressing cell line and showed that the pattern of expression of the myc-fused NHERF1 is similar to that of the endogenous protein (15). In our present study, we used this cell line to study the localization of NaPi-IIa (green), myc-NHERF1 (red), and actin (blue) in control cultures as well as in cells treated with PTH (Fig. 2B). In all cases, lysosomal degradation was inhibited by pretreatment with leupeptin. In control cultures, NaPi-IIa and myc-NHERF1 colocalized within apical actin patches. Colocalization of both partners is evidenced by the yellow signal of the merged image; the confocal section (Fig. 2B, rectangle) shows that both proteins were indeed expressed at the apical membrane in control cultures. Four hours after addition of PTH, NaPi-IIa was detected almost exclusively in intracellular organelles (Fig. 2B). Fractionation experiments performed in rats indicated that PTH leads to a lysosomal routing of NaPi-IIa (18). Although similar experiments in OK cells have not been performed, the fact that intracellular accumulation is not detected upon PTH treatment in the absence of leupeptin suggests that the organelles are late endosomes and/or lysosomes. In contrast to NaPi-IIa, the pattern of expression of NHERF1 remained similar to that of actin, both in control cultures and in samples treated with PTH. The merged confocal image of NaPi-IIa and NHERF1 clearly shows that 4 h after PTH treatment, both proteins were located in different cellular compartments: NaPi-IIa localized intracellularly, whereas NHERF1 remained in apical patches. Although the overall expression of NHERF1 and actin seemed disturbed in the cultures treated with PTH, NHERF1 retained the same pattern of expression as actin. Taken together, these immunostaining studies in kidney samples and OK cells indicate that the PTH-induced endocytosis of NaPi-IIa takes place without detectable internalization of NHERF1.
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PTH disturbs the interaction between NaPi-IIa and NHERF1 in OK cells.
The above finding that NaPi-IIa and NHERF1 are located in different compartments after PTH treatment suggests that prior alterations occur in the interaction between both partners. To test this hypothesis, we performed coimmunoprecipitation experiments in lysates from OK cells stably expressing V5-NaPi-IIa. Cells were pretreated with leupeptin, followed by 3-h incubation in the absence or presence of PTH. As shown in Fig. 4, lysates from control samples () or samples treated with PTH (+) contained similar amounts of NHERF1 (Fig. 4A) and NaPi-IIa (Fig. 4B), because lysosomal degradation of endocytosed cotransporter was prevented by leupeptin. The NHERF antibody immunoprecipitated comparable amounts of NHERF1 from both samples (Fig. 4C). However, the amount of NaPi-IIa coimmunoprecipitated with NHERF1 was reduced in the PTH-treated sample compared with control (Fig. 4D). Quantification of three independent experiments indicated a reduction of 35% in the PTH-treated samples after coimmunoprecipitation (Fig. 4E). This finding suggests that PTH regulates (i.e., weakens) the association of NaPi-IIa and NHERF1. The apparent discrepancy between a
35% reduction of coimmunoprecipitation compared with a virtually 100% PTH-induced downregulation of NaPi-IIa may be explained by the fact that coimmunoprecipitation experiments were performed in the presence of leupeptin to prevent degradation of the cotransporter. Therefore, the total amount of cotransporter available for interaction with NHERF1 was kept higher than physiological levels and thus may mask a stronger effect of PTH on the dissociation of both proteins.
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To study the phosphorylation state of NHERF1 in OK cells, we used a cell line stably expressing myc-tagged NHERF1; as negative controls, parallel experiments were performed with cells transfected with empty plasmid (pcDNA). Upon incubation in the presence of 32P, NHERF1 was immunoprecipitated with a monoclonal myc antibody. After SDS-PAGE, the total amount of immunoprecipitated protein (Fig. 5A, top) and the incorporation of 32P (Fig. 5A, bottom) were quantified. NHERF1 was detected as a phosphoprotein under basal conditions (Fig. 5A, bottom). The overall phosphorylation state was not affected by treatment with 1-34 PTH or by independent activation of PKA or PKC (data not shown). However, these data do not rule out partial changes that could be masked because of the high basal phosphorylation signal. Three serine residues located between the second PDZ domain and the MERM-binding domain have been reported to undergo phosphorylation: Ser279, Ser289, and Ser301 (Fig. 5B). The residues at positions 279 and 301 are phosphorylated by the cyclin-dependent kinase Cdc2 during the cellular cycle (14), whereas Ser289 is phosphorylated by the G protein-coupled receptor kinase GRK6A and is constitutively phosphorylated in some cell lines (13). Therefore Ser289 was mutated to alanine (S289A), and its pattern of phosphorylation was analyzed. As shown in Fig. 5A, there was a dramatic decrease in 32P incorporation on S289A compared with WT. This suggests that in OK cells, Ser289 is responsible for the bulk of constitutive phosphorylation of NHERF1, similar to human embryonic kidney HEK-293 cells (13). This reduction in phosphorylation was associated with a shift in the apparent molecular weight (Fig. 5A, top) as reported previously (13, 31). Quantification of the ratio 32P to myc provided by three independent experiments indicated that the level of phosphorylation of the S289A mutant remained unchanged after PTH treatment or after independent activation of PKA or PKC (Fig. 5C). As shown in Fig. 5D, the mutated NHERF1 showed the same pattern of expression as WT-NHERF1 when transfected in OK cells, because it was detected in actin-containing apical patches. Furthermore, degradation of NaPi-IIa proceeded according to a time course similar to that observed in cell lines expressing either WT or S289A myc-NHERF1 (Fig. 5E), suggesting that endocytosis of NaPi-IIa is not affected by the presence of the mutated NHERF1.
To study the phosphorylation state of NHERF1 in kidney, cortical slices were incubated for 30 min with 32P, followed by an additional 30-min incubation in the presence or absence of 1-34 PTH. After homogenization, NHERF1 was pulled down with the COOH-terminal tail of NaPi-I (SLC17A1; see Ref. 32) fused to GST. We had to use this construct, owing to the failure of the COOH-terminal tail of NaPi-IIa to pull down NHERF1 (unpublished observations). NaPi-I is also expressed in the apical membrane of renal proximal tubules (27). The residues required for interaction with NHERF1 are identical in NaPi-I and NaPi-IIa (TRL), and yeast trap assays have confirmed that the COOH-terminal tail of NaPi-I interacts with NHERF1 (11); as negative controls, pull-down assays were performed with GST alone. After SDS-PAGE, the amount of protein pulled down (Fig. 6A, top) and the incorporation of 32P (Fig. 6A, bottom) were analyzed. NHERF1 was readily detected using Western blot analysis in the pull-down assays performed with GST fused to the COOH-terminal tail of NaPi-I, whereas no signal was detected in the negative control (Fig. 6A, top). Furthermore, NHERF1 was detected as a phosphoprotein under basal conditions, and its overall phosphorylation state increased upon incubation of kidney slices with PTH (Fig. 6A, bottom). Quantification of the ratio of 32P to pulled-down NHERF1 obtained in seven independent experiments indicated that the level of phosphorylation almost doubled after PTH treatment (Fig. 6B). To distinguish which kinase was responsible for the increase in phosphorylation induced by PTH, kidney slices were incubated with 8-BrcAMP (to specifically activate PKA) or with DOG (to activate PKC), and samples were processed as described above. As shown in Fig. 6 (C, bottom) activation of either kinase led to stimulation of NHERF1 phosphorylation; Fig. 6D shows the quantification of the ratio 32P to pulled-down NHERF1 obtained in three independent experiments. To our knowledge, this study is the first to report constitutive as well as regulated phosphorylation of NHERF1 in the kidney.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* N. Déliot and N. Hernando contributed equally to this work.
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