1Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado; and 2Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daelon, Korea
Submitted 23 November 2004 ; accepted in final form 16 May 2005
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ABSTRACT |
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PDZ domains; endocytosis; degradation; epithelia
Among the best characterized membrane domains, the structure, function, and dynamic nature of the PSD in neurons is dependent on a number of PDZ domain proteins. Among these are the Shank family of PDZ proteins. The three distinct Shank genes share a high degree of homology and encode for the same general protein organization. This organization includes six ankyrin (ANK) repeats, an SH3 domain, a single PDZ domain, a polyproline-rich region (PRR), and a sterile -motif (2, 29). Shank-1 expression is limited to neurons, and, conversely, Shank-3 is broadly distributed (2). Shank-2 is not ubiquitously expressed but is found outside of neurons (14, 18, 26). Epithelial cells express a specific Shank2 spliceoform, termed Shank2E, at the apical domain of epithelial cells that, in pancreatic duct epithelial cells, binds and inhibits the Cl channel activity of cystic fibrosis transmembrane conductance regulator (CFTR) (14, 18). Shank2 is also expressed within the kidney but its distribution and function is unknown. Like the PSD, the apical domain of renal proximal tubule (PT) cells is a dynamic domain responsible for coordinating and regulating the movement of ions, solutes and water across the apical membrane. Thus it is not surprising that PDZ proteins play a central role in these pathways (13, 19, 27).
The Na+-dependent Pi cotransporter IIa (NaPi-IIa) is a PT protein known to be bound and regulated by PDZ proteins. Concentrated in the brush border membrane of the PT cells, NaPi-IIa reabsorbs 70% of the Pi from the glomerular filtrate and is essential for regulating serum Pi levels (1). NaPi-IIa activity is regulated primarily by its insertion, retention, and retrieval from the apical brush border membrane (21). The relative balance of insertion and retrieval is moderated by hormones such as parathyroid hormone and by dietary Pi levels (16, 33). In opossum kidney (OK) cells, a proximal tubular cell model, low extracellular Pi induces NaPi-IIa insertion/retention in the apical membrane, while increased extracellular Pi induces a rapid internalization and degradation of NaPi-IIa (24). The COOH tail of NaPi-IIa ends with a PDZ binding motif (-A-T-R-L), and NaPi-IIa binds specific PDZ proteins, including PDZ kidney-1 (PDZK1; a/k/a NaPi-Cap1), the ezrin, radixin, and moesin (ERM) family of actin-binding proteins binding phosphoprotein 50 (EBP50; a/k/a NHERF-1, or Na+/H+ exchanger regulatory factor-1) and NHE3 kinase-associated regulatory protein (E3KARP; a/k/a NHERF-2) (8, 9). The physiological significance of PDZ proteins in regulating NaPi-IIa activity was revealed in a transgenic EBP50- knockout mouse model. These animals had decreased serum Pi, increased urinary Pi, and lower expression of NaPi-IIa within the apical membrane of proximal tubule cells (31). While the EBP50-NaPi-IIa complex promotes NaPi-IIa activity, it is postulated that the disparate NaPi-IIa-binding PDZ domain proteins perform distinct, rather than redundant, functions.
With PDZ proteins implicated in a wide range of renal transport and signaling pathways, in the present study we sought to identify the distribution of Shank2E in the kidney and identify pathways that Shank2E might moderate. The PDZ domains of EBP50, PDZK1, and Shank2E do have similar characteristics. Each protein can bind ATP-binding cassette proteins via its PDZ domain, and the domain responsible for this binding is a type I PDZ domain. Given that EBP50 and PDZK1 also bind NaPi-IIa, the present study demonstrated and subsequently characterized a specific binding interaction between Shank2E and NaPi-IIa.
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MATERIALS AND METHODS |
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The fate of proteins within renal PT cells during acute changes in serum Pi was evaluated by moderating the Pi levels in the diet. Rats were fed a low-Pi diet (0.1% Pi, 0.6% Ca2+) for 14 days before study. Access to food was restricted to a 3-h period daily to condition rapid feeding and allow for acute changes in serum Pi levels (16). On the final day, one-half of the animals were maintained on low-Pi chow while the other half were fed a high-Pi diet (1.2% Pi, 0.1% Ca2+). After 3 h of feeding, the animals were humanely killed and either fixed for immunohistology or processed for Western blot analysis.
OK cells were used to evaluate the distribution and Pi-induced degradation of proteins. OK cells were maintained in DMEM-Ham's F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 5 mM L-glutamine at 37°C in a humidified atmosphere with 5% CO2. OK cells were cotransfected with Flag-Shank2E (2 µg) and hemagglutinin (HA)-NaPi-IIa (26 µg) or green fluorescent protein (GFP)-NaPi-IIa (2 µg) using Effectene reagent as directed by the manufacturer (Qiagen, Valencia, CA). OK cells were grown to confluence (3648 h) before the study period. In a group of studies, the degradation rate of native OK cell proteins, including Shank2E, was evaluated. Initially, OK cells were grown for 8 days to allow for consistent differentiation. The cells were then synchronized with serum-free DMEM-Ham's F-12 medium for 1216 h and endocytosis/degradation of native NaPi-IIa was then induced by preincubation in 0.1 mM Pi (low) medium for 20 h, followed by a shift to 2 mM (high) Pi medium for 4 h. Control cells were maintained in low rather than high Pi during the last 4-h period. Intracellular protein degradation was inhibited by incubating cells in 25 µM leupeptin. The high- and low-Pi media were made with DMEM high-glucose medium (Sigma, St. Louis, MO) supplemented with 1.2 mg/ml sodium bicarbonate.
Human embryonic kidney (HEK)-293 cells were used to evaluate binding of specific Shank2E domains to NaPi-IIa. These cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and 5 mM L-glutamine at 37°C in a humidified atmosphere with 5% CO2. HEK-293 cells were cotransfected at 5075% confluence using Effectene reagent, 2 µg of HA-NaPi-IIa or HA-NaPi-IIa (PDZ) and 2 µg of the individual Flag-Shank2E domain fusions proteins. Transfected cells were lysed 48 h posttransfection.
Plasmid constructs. The full-length Flag-Shank2E construct was generated by amplifying a 5' segment of Shank2E from rat cholangiocyte cDNA with XbaI ends, digesting CortBP1 (i.e., short Shank2 isoform) pCDNA 3.1() vector (5) with XbaI and subsequent ligation of the PCR product. Flag epitopes were inserted in-frame and upstream of Shank2E by NheI digestion of Shank2E pCDNA3.1, blunting of the ends with Mung Bean Nuclease (Invitrogen), and subsequent ligation of a oligonucleotide primer encoding for three Flag epitopes. Individual Flag-tagged Shank2E domain fusion proteins were generated by PCR amplification from Shank2E pCDNA3.1 template, followed by ligation into the NH2-terminal Flag-CMV-7 expression vector (Sigma). The HA-NaPi-IIa construct was developed using gene-specific primers encompassing the start and stop codons of rat NaPi-IIa (GenBank accession no. L13257). HA-NaPi-IIa (PDZ) was generated using an antisense primer that omits the COOH-terminal four amino acids. PCR products were amplified from rat kidney cDNA as previously described (18), digested with EcoRI, and ligated in-frame into the EcoRI-digested pHM6 vector (Roche, Indianapolis, IN). The GFP-NaPi-IIa construct was characterized previously (10). All constructs were verified by sequencing. Primers used are listed in Table 1.
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To isolate material for immunoprecipitation studies, crude renal cortex membranes were prepared as previously described for liver membranes (28). Briefly, perfused kidneys were cut into slabs, and cortex was trimmed away from medulla and homogenized with a Potter-Elvehjem Dounce in IB. Nuclei and large debris were pelleted (10,000 g, 15 min), the supernatant was collected, and crude membranes were pelleted (100,000 g, 2 h).
Western blotting, Far Western blotting, and immunohistochemistry.
Proteins from rat kidney, OK cells, and coimmunoprecipitation studies were assayed using Western blot analysis (4). Briefly, cells or tissues were solubilized in 5x PAGE buffer (5% sodium dodecyl sulfate, 25% sucrose, 5 mM EDTA, 50 mM Tris, pH 8.0, 5% -mercaptoethanol, and Complete Minitablet protease inhibitor cocktail; Roche), separated on a 414% gradient gel, and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). These membranes were incubated and then washed in primary and then horseradish peroxidase-conjugated secondary antibodies (1:60,000 dilution; Jackson ImmunoResearch, West Grove, PA). The antibody complexes were detected using enhanced chemiluminescence (Pierce, Rockford, IL) and captured using a photodocumentation system (UVP, Upland, CA).
For Far Western blotting, immunoprecipitated wild-type (WT) or truncated (PDZ) HA-NaPi-IIa proteins were run out in duplicate on an acrylamide gel and transferred onto nitrocellulose membrane. One blot was developed using Western blot analysis with an HA antibody to confirm equivalent expression levels of HA-NaPi-IIa proteins. The second blot was incubated overnight in 5% nonfat milk in blot buffer, washed, overlaid with blot buffer containing Flag-Shank2E (300 nM overnight at 4°C), and washed. The bound Flag-Shank2E was detected using Western blot analysis with an anti-Flag antibody as described above.
For immunohistochemistry, kidneys were perfused with 4% paraformaldehyde in PBS before processing for immunofluorescence staining (4). Briefly, kidney sections (5 µm) were blocked (10% goat serum, 3% nonfat dry milk, and 0.3% Triton X-100 in PBS) and incubated overnight at 4°C with primary antibody. After being washed, the sections were incubated (60 min, room temperature) with Alexa 488- or Alexa 568-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR), washed with PBS, and mounted in 90% glycerol, and 10% PBS containing 2.5% 1,4-diazabicyclo[2.2.2]octane (Sigma). OK cells were seeded on poly-L-lysine-coated coverglass (Lab-Tek, Naperville, IL), grown to confluence, fixed (20 min; 3% paraformaldehyde in PBS supplemented with Ca2+ and Mg2+), and quenched (10 min, 20 mM glycine) before being stained as described above. Stained kidney sections or OK cells were imaged using a laser scanning confocal microscope (model LSM510; Zeiss, Thornwood, NY) with a x40 water-immersion lens objective. Confocal sections were acquired throughout the cell and summed for presentation. All image manipulations were performed using Zeiss LSM510 software.
Antibodies for Western blot and immunohistochemical analysis included Flag M2 antibody (Sigma), Shank2 (5, 14), NaPi-IIa (32), EBP50 (7), actin (Chemicon, Temecula, CA), leucine amino peptidase (7), and Na+-K+-ATPase (Upstate Biotechnology, Lake Placid, NY).
Coimmunoprecipitation. Rat renal cortex membranes, OK cells, and HEK-293 cells were solubilized in immunoprecipitation buffer (50 mM Tris, 75 mM NaCl, 0.5% Triton X-100, 0.5% deoxycholate, and protease inhibitor cocktail tablets; pH 7.4) at 25°C for 10 min, and the insoluble material was pelleted (10,000 g, 10 min). The supernatants were precleared (60 min, 4°C) with immobilized protein A/G beads (Pierce, Rockford, IL) and incubated (overnight at 4°C) with NaPi-IIa antibody or anti-FLAG M2, respectively. Immunocomplexes with the NaPi-IIa and Shank2E antibodies were recovered with immobilized protein A/G beads (60 min, 4°C). The immunoprecipitation beads and precleared beads were washed (3 x 5 min) with ice-cold PBS and eluted with 5x PAGE buffer for Western blot analysis.
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RESULTS |
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DISCUSSION |
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Shank2E binds specific proteins at the apical membrane of epithelial cells. Shank protein family members were initially characterized within the PSD of neurons (22, 34, 3). There are three distinct Shank genes; each shares significant sequence homologies and domain organizations, including a single PDZ domain. Within the PSD, Shank proteins bind a number of PSD proteins and serves as a central organizing protein in the spatial and temporal distribution of proteins at the membrane-cytosol interface. At the apical membrane of pancreatic duct epithelial cells, Shank2 binds CFTR via its PDZ domain and inhibits the cAMP-regulated Cl channel (14). Several ATP binding cassette (ABC) proteins, including CFTR and multidrug resistance protein 2 (mrp2), are bound by PDZ domain proteins. Among these ABC binding proteins, EBP50 and PDZK1 also bind NaPi-IIa via a PDZ interaction (8). Given that EBP50, PDZK1, and Shank2E each bound ABC proteins and EBP50 and that PDZK1 bound NaPi-IIa, it was hypothesized that Shank2E would also bind NaPi-IIa. The present study confirmed the hypothesis by showing native Shank2E and NaPi-IIa coprecipitated from rat renal cortex, recombinant Shank2E and NaPi-IIa coprecipitated when expressed in OK and HEK cells, and Shank2E bound directly to NaPi-IIa when NaPi-IIa had its PDZ binding motif (Figs. 3 and 4). Comparative binding studies using Shank2E domains and COOH-terminal truncations of NaPi-IIa further confirmed that the Shank2E-NaPi-IIa interaction occurred through the PDZ domain of Shank2E and the PDZ binding motif at the COOH terminus of NaPi-IIa (Fig. 4).
PDZ domain proteins regulate NaPi-IIa activity. Moderating NaPi-IIa activity in the renal proximal tubule is the primary physiological mechanism for regulating extracellular Pi concentrations. While the activity of many other membrane proteins is heavily influenced by phosphoregulation and recycling at the membrane, NaPi-IIa activity is regulated primarily by controlling the delivery, retention, and retrieval of the protein to the apical membrane with little or no apparent regulation of activity by phosphorylation or recycling (21). Instead, endocytosed NaPi-IIa is directly sorted and targeted for lysosomal degradation (12, 25). PDZ domain proteins have emerged as pivotal entities in coordinating the activity of NaPi-IIa (9). After truncation of the COOH-terminal PDZ binding motif, NaPi-IIa still distributes to the apical membrane but also accumulates within intracellular compartments (11). In EBP50 knockout (EBP50/) mice, there is a marked decrease in NaPi-IIa at the apical membrane of proximal tubule cells, a significant increase in urinary Pi levels, and diminished NaPi-IIa activity in the apical membrane of PT cells in response to decreased serum Pi levels (31, 35). This indicates EBP50 is important for promoting NaPi-IIa activity through NaPi-IIa delivery or retention within the apical membrane of PT cells. In contrast, OKH cells, an OK cell clone deficient in EBP50 expression, has diminished parathyroid hormone-dependent inhibition of NaPi-IIa activity that is restored after EBP50 expression (17). A prominent difference between rat PT cells and OK cells was also observed in the present study. In OK cells, acute exposure to elevated Pi levels increased the internalization and degradation of NaPi-IIa (Fig. 6). In rat PT cells, elevated Pi levels also induced internalization (Fig. 7), but there was no significant loss of Shank2E (Fig. 8). This difference is not likely accounted for by the broader expression of Shank2E in the cells of the kidney cortex. While other cell types do appear to express Shank2 (Fig. 2), Shank2 expression is highest in the PT cells, and PT cells make up the majority of the cortical mass. Additional functional studies will elucidate the specific roles of PDZ domain proteins in moderating the distribution and activity of NaPi-IIa.
PDZ domain proteins have distinct roles in regulating NaPi-IIa activity.
It is likely that the different NaPi-IIa-binding PDZ proteins do not have redundant functions but instead perform distinct roles in the regulated delivery, retention, and recovery of NaPi-IIa. First and foremost, the development of phosphouremia in EBP50/ mice clearly indicates the inability of the other PDZ domain proteins to compensate for the loss of EBP50 function. Furthermore, analysis of three NaPi-IIa-binding PDZ domain proteins shows that they are markedly different with regard to their structural organization. EBP50 comprises two PDZ domains and an ERM binding motif, and PDZK1 comprises four PDZ domains with no other noted domains. In contrast, Shank2E has a single PDZ domain but also has six ANK repeats, an SH3 domain, a PRR, domain, and a sterile motif. Finally, the present study demonstrated a disparate response of Shank2E and EBP50 to a high-Pi environment. In OK cells, EBP50 levels were unaffected by the high-Pi environment, but Shank2E levels were significantly decreased (Fig. 6).
Shank2E parallels the endocytosis and degradation of NaPi-IIa. The present study suggests Shank2E may be linked to the internalization and degradation of NaPi-IIa (Figs. 6 and 7). Previous studies showed that Shank2 can bind dynamin II (23). Dynamin II, a broadly expressed isoform of dynamin, participates in the pinching off and internalization of endocytic vesicles. Thus Shank2E has established links to the endocytic machinery. In OK cells incubated under low-Pi conditions, Shank2E showed a comparatively high rate of degradation that was not observed with NaPi-IIa (Fig. 5). When OK cells were shifted into high-Pi conditions, Shank2E and NaPi-IIa underwent marked and parallel increases in degradation (Fig. 6). These biochemical measurements were mirrored by observations in the intact rat. Acute switching to a high-Pi diet induced Shank2E to redistribute from the apical domain to the cell interior of PT cells (Fig. 7). This Shank2E redistribution was paralleled by the known redistribution of NaPi-IIa away from the apical membrane (Fig. 7) While additional studies are clearly required, the observations made in the present study support the hypothesis that Shank2E plays a pivotal role in segregating and trafficking NaPi-IIa into the lysosomal degradation pathway.
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GRANTS |
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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.
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