1 Division of Nephrology, Hypertension, and Clinical Pharmacology, Oregon Health Sciences University, and Veterans Administration Medical Center, Portland, Oregon 97201; 2 University of Colorado Health Sciences Center, Denver, Colorado 80220; and 3 Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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The thiazide-sensitive sodium-chloride cotransporter (NCC) is expressed by distal convoluted tubule cells of the mammalian kidney. We used yeast two-hybrid screening to identify that glucose-regulated protein 58 (grp58), a protein induced by glucose deprivation, binds to the COOH terminus of the NCC. Immunoprecipitation of rat kidney cortex homogenates using a guinea pig anti-NCC antibody confirmed that grp58 associates with the NCC in vivo. Northern blots indicated that grp58 is highly expressed in rat kidney cortex. Immunofluorescence showed that grp58 protein abundance in kidney is highest in epithelial cells of the distal nephron, where it colocalizes with NCC near the apical membrane. To determine whether this interaction has a functional significance, NCC and grp58 cRNA were coexpressed in Xenopus laevis oocytes. In oocytes overexpressing grp58, sodium uptake was increased compared with control. Because oocytes express endogenous grp58, antisense experiments were performed to evaluate whether endogenous grp58 affected NCC activity in oocytes. Sodium uptake was lower in oocytes injected with both antisense grp58 cRNA and sense NCC compared with sense NCC oocytes. Western blot analysis did not show any effect of grp58 expression on processing of the NCC. These data indicate a novel, functionally important interaction between grp58 and the NCC in rat kidney cortex.
kidney tubules; distal; thiazide-sensitive sodium-chloride cotransporter; glucose-regulated protein 58; molecular chaperones; two-hybrid system techniques
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INTRODUCTION |
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THE THIAZIDE-SENSITIVE SODIUM-CHLORIDE cotransporter (NCC) is expressed at the apical membrane of the distal convoluted tubule and is the major mediator of sodium and chloride transport by this segment (2, 18, 21). Gamba et al. (5) first cloned the NCC from flounder bladder. Homologous proteins were later cloned from rats, mice, rabbits, and humans (7, 10, 26, 30). NCC protein is predicted to contain 12 membrane-spanning domains, 2 extracellular glycosylation sites, an intracellular NH2-terminal domain, and a large intracellular COOH-terminal domain (5, 10, 17). NCC is homologous to the bumetanide-sensitive Na-K-2Cl cotransporters and K-Cl cotransporters (6, 7).
Gitelman's syndrome is caused by mutations in the NCC and is an autosomal recessive disorder characterized by hypokalemic alkalosis, salt wasting, hypomagnesemia, low urinary calcium excretion, and low-to-normal blood pressure. The genetic basis of Gitelman's syndrome was first described by Simon et al. (26), who reported 18 discrete mutations that lead to the phenotype. Subsequently, many other NCC mutations were described that cause Gitelman's syndrome (25). Of the mutations reported to cause Gitelman's syndrome, many are clustered in the COOH-terminal domain of the protein (12). These include G738R, the most common Gitelman's mutation (25), which appears to cause a loss of function by disrupting protein processing (10). The NCC COOH terminus is large, is highly conserved among species, and has numerous potential regulatory regions. Truncation and mutation of the NCC COOH terminus leads to a loss of function that is associated with an impairment of proper protein processing; i.e., glycosylation is abolished (10). These observations suggest that the COOH terminus of the transporter plays a vital role in maintaining NCC function.
This study used the yeast two-hybrid system to screen for proteins that interact with the COOH terminus of the NCC. One such mouse kidney protein was shown to be glucose-regulated protein 58 (grp58), a protein involved in molecular processing. Further experiments indicated that grp58 binds to the NCC in vivo and influences functional properties of the NCC. The data suggest that grp58 makes an important contribution to normal NCC function, possibly by binding to the COOH terminus of the transport protein.
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EXPERIMENTAL PROCEDURES |
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Yeast two-hybrid analysis.
To screen for proteins that interact with the COOH-terminal
cytoplasmic domain of the NCC, a GAL4 DNA binding-domain (BD) fusion
protein comprising the entire putative COOH-terminal cytoplasmic domain
of the mouse NCC (mNCC1806-3003; GenBank
U61085) was generated by using the polymerase chain reaction (Fig.
1A). The resulting products
were cloned into pCRII and sequenced. Plasmids with inserts were
digested with EcoRI and BamHI and ligated
directionally into pAS2 (Clontech). The orientation of the
inserts was verified by end sequencing. The ability of these vectors to
generate fusion proteins that were not toxic was documented by Western
blotting proteins expressed by transformed yeast cells. The expressed
fusion proteins were blotted by using a GAL4 DNA-BD monoclonal antibody (Clontech). A protein of the expected size was observed.
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Northern blot analysis. Total RNA was extracted from kidney cortex by using a QIAgen Midi kit (QIAgen). The RNA was dissolved in RNAse-free water, and the concentration was determined by ultraviolet absorbance reading at 260 nm (DU 640, Beckman). The integrity of each sample was verified by electrophoresis on an agarose-formaldehyde gel. Aliquots of 10 µg total RNA were separated by 1% agarose-formaldehyde gel electrophoresis and transferred to a nylon membrane (Duralon UV, Strategene). RNA was fixed to the nylon membrane by cross-linking (Stratalinker, Stratagene).
A [Preparation of membrane proteins.
Kidney cortices from Sprague-Dawley rats aged 10-12 wk were
excised. The tissue was homogenized in 1:8 (wt/vol) of buffer A [(in mM) 50 Tris · HCl, 150 NaCl, 50 NaF, 10 sodium
pyrophosphate, 1 EDTA, 1 sodium vandate, and 1 phenylmethylsulfonyl
fluoride, as well as 0.01 leupeptin, 0.01 pepstatin A, 1 µg/µl
aprotinin A, 1% Nonidet P-40, 0.4% deoxycholate] and centrifuged at
1,000 g for 10 min at 4°C. The supernatant was removed and
centrifuged at 48,000 g for 20 min (4°C). The resultant
pellet was resuspended in buffer A and centrifuged at 48,000 g for 20 min (4°C). The supernatant was decanted, and the
pellet was resuspended in 1 ml buffer A (4°C). Protein
preparations were stored at 70°C for later use.
Immunoprecipitation. Protein extracts were precleared by incubation with Sepharose CL-4B beads (Amersham Pharmacia Biotech) for 2-6 h and centrifuged at 14,000 g for 10 min. The supernatant, with guinea pig anti-NCC (1:50 dilution; see below in this subsection), was added to fresh Sepharose CL-4B beads and incubated overnight at 4°C. The mixture was then centrifuged at 14,000 g for 1 min, and the supernatant was discarded. The resultant beads were washed twice with buffer A. Thirty microliters of SDS sample buffer were added to the final pellet, heated at 60°C for 10 min, and centrifuged at 14,000 g for 5 min. The supernatant was then loaded on a 7% Tris-acetate SDS gel (Novex, Carlsbad, CA), and Western blot analysis was performed.
The guinea pig anti-mouse NCC antibody was generated by using the NCC fusion protein used previously to produce a rabbit NCC antibody (2). In preliminary experiments, it was shown to produce results identical to those obtained by using the previously characterized rabbit anti-mouse NCC antibody on Western blot and rat kidney immunocytochemistry (2).Western blot analysis. Western blots (Novex) were performed per the supplier's instructions. Gels were equally loaded with protein as determined by Bio-Rad MicroAssay and run for 1.5 h at 120 V. The protein was then transferred to a polyvinylidene difluoride microporous membrane, which was incubated at 4°C overnight in Blotto-T (PBS containing 5% wt/vol nonfat skim milk and 0.1% Tween 20). The primary antibody, either rabbit anti-NCC or rabbit anti-grp58 (Stressgen Biotechnologies,Vancouver, BC), was added at 1:1,000 dilution for 1 h at room temperature. After the blot was washed with Blotto-T, a horseradish peroxidase-conjugated donkey anti-rabbit antibody (Jackson Laboratories, West Grove, PA) was added at 1:2,000 dilution for 1 h at room temperature. The blot was then washed in Blotto-T and PBS-T (PBS with 0.1% Tween 20). The protein was detected by using ECL+ (Amersham Pharmacia Biotech) per manufacturer instructions.
Immunofluorescence. Rat kidneys were perfusion fixed and prepared for immunofluorescence as described previously (18). Immunofluorescence microscopy was carried out on semithin cryosections (0.5 µm) mounted on gelatin-coated slides. Antigen retrieval was carried out on tissue sections. This involved immersion in 0.01 M sodium citrate (pH 6) and then boiling for 10 min in a microwave. The slides were slowly cooled to room temperature, blocked with 2,4,6-trinitrobenzenesulfonic acid (TNB) buffer (100 mM Tris · HCl, 0.15 M NaCl, 0.5% blocking reagent), and incubated for 1 h in primary antibody (guinea pig anti-NCC, 1:200; rabbit anti-grp58, 1:50 in TNB). After the slides were washed five times in 2,4,6-trinitrotoluene (TNT) buffer (100 mM Tris · HCl, 0.15 NaCl, 0.05% Tween 20), secondary antibodies diluted in TNB [NCC, 1:500 Cy3-conjugated goat anti-guinea pig (Jackson Laboratories); grp58: 1:250 horseradish peroxidase-conjugated donkey anti-rabbit (Zymed, San Franscico, CA)] were added for 1 h, and the slides were then washed five times in TNT. Fluorophore tyramide amplification solution (NEN, Boston, MA) was then added for 5 min, and the slides were washed five times in TNT.
Preparation of grp58 clone. grp58 was generated from RT DNA by using the polymerase chain reaction. Mouse kidneys were excised, and total RNA was extracted by using the RNAqueous-Midi kit (Ambion, Austin, TX). The forward (5'-CAAGCGGCTGCAGATTGC-3') and reverse (5'-CCCCCCCCACAACATTCT-3') primers generated a 1,792-base pair fragment. The PCR product was ligated into pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA) and then subcloned by using XbaI and HindIII (New England Biolabs, Beverley MA) into pgh19. The grp58 clone was sequenced to ensure authenticity.
Expression of NCC and grp58 in Xenopus oocytes. NCC and grp58 DNA templates were linearized downstream of the three untranslated regions with NotI and XhoI (New England Biolabs), respectively, treated with proteinase K (mCAP mRNA Capping Kit, Stratagene, La Jolla, CA), and ethanol precipitated. The DNA was resuspended in RNase-free water, and size and quantity were determined by agarose gel electrophoresis. The cRNA was transcribed by means of the mCAP kit protocol with the addition of RNase inhibitor (Invitrogen). Transcript size and quantity were checked by RNA agarose gel electrophoresis. Oocyte harvesting, injection of cRNA into oocytes, and sodium uptake experiments were performed as previously described (10).
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RESULTS |
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Two independent clones were detected by yeast two-hybrid screening
of a mouse kidney cDNA library by using the COOH-terminal domain of the
NCC as the bait. Both clones were identical to mouse grp58 (GenBank
M73329), a glucose response protein that is a member of the protein
disulfide isomerase family. Figure 1B shows results of a
quantitative -galactosidase assay. The results indicate that
-galactosidase activity is higher in cells transformed with both
pGAD-grp58 and
pAS2-mNCC1806-3003 clones than with
pAS2, pAS2-mNCC1806-3003, or
pGAD-grp58 alone. This result excludes the possibilities
that either the NCC fusion protein (pAS2-mNCC1806-3003) or grp58 autonomously
activates reporter gene transcription.
The NCC is expressed exclusively in the kidney cortex at the apical
membrane of distal convoluted tubule cells (2, 21). To
determine whether grp58 mRNA is also expressed by the kidney, Northern
blot analysis was performed. A Northern blot (Fig.
2) showed a single dense band at ~2 kb
in lung and kidney. When a longer exposure time was used, lower level
expression was detected in other tissues.
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To determine whether grp58 associates with the NCC in the kidney in
vivo, coimmunoprecipitation experiments were performed. Rat kidney
cortex was solubilized in Nonidet P-40 and deoxycholate, and the
insoluble fraction was removed by centrifugation. The soluble fraction
was immunoprecipitated with guinea pig anti-NCC antibody, and the
immunoprecipitate was separated by SDS-PAGE and Western blotted by
using either a rabbit anti-NCC or an anti-grp58 antibody. The anti-NCC
antibody immunoprecipitated NCC, and the amount precipitated was
proportional to the amount of cortical membranes added to the reaction
mixture (Fig. 3, A and
B). There was no precipitation of grp58 or NCC from rat
kidney cortex in the absence of guinea pig anti-NCC antibody or tissue
homogenate. The anti-NCC antibody also immunoprecipitated grp58 (Fig.
3C). The specificity of this reaction was confirmed by
showing that the molecular chaperones calnexin (Fig. 3D) and
grp78 are not precipitated by the anti-NCC antibody.
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To determine cellular sites of NCC and grp58 expression in kidney
cortex, immunofluorescence microscopy was used. We confirmed that NCC
protein is located exclusively at the apical surface of the distal
convoluted tubule (2, 18, 21) (Fig.
4). grp58 was also found to be located in
an apical or subapical region of the distal convoluted tubule (Fig. 4).
In addition, fluorescence for grp58 was found in other segments of the
distal nephron. Superimposed images of NCC and grp58 fluorescence
showed extensive colocalization (Fig. 4), although NCC appeared to be
more closely associated with the apical plasma membrane and grp58 with
the subapical region.
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Figure 5 shows results of coexpression
experiments in Xenopus oocytes. Compared with rates of
sodium uptake when NCC alone was expressed, uptake by oocytes
coinjected with both NCC and grp58 cRNAs was significantly greater.
Western blots of uninjected oocytes showed that they express grp58
endogenously. Injection of grp58 cRNA increased grp58 protein
abundance, as indicated by Western blot (Fig. 5A). To
confirm that grp58 itself does not increase sodium uptake
nonspecifically, we compared sodium uptake in grp58 and water-injected
oocytes. Sodium uptake was not different in water vs. grp58-injected
oocytes [1,173 ± 287 (water) vs. 789 ± 375 cpm/h (grp58),
n = 5 experiments/15-30 oocytes per group, where
cpm is counts/min; P = not significant]. To determine
whether endogenous grp58 affects NCC activity, oocytes were injected
with antisense grp58 cRNA with or without NCC cRNA. Compared with
oocytes injected with NCC cRNA alone, rates of sodium uptake by oocytes injected with both NCC and antisense grp58 were significantly lower
(Fig. 5C). Western blot analysis (Fig. 5A)
confirmed that antisense grp58 reduced endogenous grp58 protein
abundance. To determine whether grp58 affected processing of the NCC,
oocyte membranes were probed for the NCC. We have previously shown that glycosylation of the NCC is associated with a shift in mobility on
SDS-PAGE (10). Figure 5B shows that injection
of grp58 cRNA had no effect on the relative proportion of mature
glycosylated NCC or on the absolute abundance of the protein.
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DISCUSSION |
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The NCC is an integral membrane protein that mediates the majority of sodium and chloride transport by distal convoluted tubule cells. It is believed to comprise 12 membrane-spanning domains, a short NH2-terminal cytoplasmic domain, and a long COOH-terminal cytoplasmic domain. At present, there is little information about specific functional domains on this protein. On the basis of principles derived from other co- and countertransporters, it is likely that ion transport is mediated by the region comprising the membrane-spanning domains. In contrast, the function of the large COOH-terminal cytoplasmic domain is completely unknown. Interestingly, a large number of NCC mutations that cause Gitelman's syndrome occur in the COOH terminus, suggesting that this region plays important functional roles. In the present study, a yeast two-hybrid system search for kidney proteins that interact with the COOH-terminal domain of the NCC detected the protein grp58. Furthermore, grp58 could be immunoprecipitated by an anti-NCC antibody and colocalized with NCC in rat kidney cortex. Coexpression experiments indicate that grp58 expression increases NCC-mediated sodium uptake in oocytes. Together, these results indicate that grp58 plays an important role in NCC function.
grp58 was originally cloned by Bennett et al. (1) and
misidentified as phospholipase C-. However, further investigations indicated that this protein does not have phospholipase activity but
instead has protein disulfide isomerase activity, thiol-dependent oxido-reductase activity (8, 13), cysteine-dependent
protease activity (27), and carnitine palmitoyltransferase
activity (15). Recently, this protein was found to form
mixed disulfide bonds with cysteine-containing proteins, before
rearrangement to form intramolecular disulfide bonds (14).
grp58 forms a complex with two homologous lectins, calnexin and
calreticulin, in the endoplasmic reticulum, where, together, they
mediate retention and proper folding of glycoproteins (20,
28). grp58 has been identified in numerous species including
rodents and humans. In humans, the highest steady-state mRNA expression
was found in liver, placenta, lung, pancreas, and kidney
(9). In the present study, we confirmed expression of
grp58 mRNA species in rat kidney as well as in lung.
The present experiments show that grp58 associates with the
COOH-terminal domain of the NCC. This is indicated by the results of
the yeast two-hybrid system experiments in which the COOH-terminal domain of the NCC was used as bait. grp58 was independently identified twice by screening a mouse kidney cDNA library. A quantitative -galactosidase assay was used to confirm this interaction. Although these data indicate that the COOH-terminal domain of NCC interacts with
grp58 when expressed heterologously in yeast, they do not prove that
the two proteins associate in vivo in mammals. However, the results of
immunoprecipitation experiments using an anti-NCC antibody to
immunoprecipitate from rat kidney cortex do indicate that grp58 and the
NCC interact in rat kidney in vivo.
grp58 has been shown to associate with the molecular chaperones
calnexin and calreticulin, where it binds with proteins in a
glycosylation-dependent manner (32). This interaction
occurs in the lumen of the endoplasmic reticulum (ER), and grp58
contains a modified ER retention signal (QDEL). Thus it might be
suspected that grp58 would interact with a portion of the NCC that
extends into the lumen of the ER and that is glycosylated. Instead, the present results indicate that grp58 interacts with the COOH-terminal domain of the NCC, which is believed to be cytoplasmic
(5). Although the NCC is glycosylated, both glycosylation
sites are located on the third putative extracellular domain, far
removed from the region used as bait for the yeast two-hybrid
experiments. Furthermore, as noted above, the COOH-terminal domain of
the NCC is believed to be cytoplasmic and would not be expected to
extend into the lumen of the endoplasmic reticulum. However, more
recent data indicate that ER-associated molecular chaperones in general may also subserve functions outside the ER. grp94, a protein that is
also induced by glucose deprivation and includes a COOH-terminal ER-retention signal, has been shown by electron microscopy to exist
predominately at the membranes of endosomal/lysosomal vesicles as well
as at the apical hepatocyte membranes but not in the ER (3). Calreticulin, which can associate with grp58, has
been shown to interact with the cytoplasmic domain of the integrin -domain and may be important in signal transduction by this protein (24). Furthermore, Wakui et al. (31) showed
that a large fraction of renal grp58 is located near the apical plasma
membrane, outside of the ER.
In the present experiments, grp58 was detected near the luminal
membrane of distal convoluted tubule cells. Colocalization experiments
indicated high-level grp58 expression by segments that express the NCC,
but grp58 expression was not limited to NCC-positive nephron segments.
This indicates that grp58 is expressed by the distal convoluted tubule
as well as other distal nephron segments. The present results are
consistent with observations made by Ohtani et al. (19),
in which grp58 (identified at that time as phospholipase C-) was
localized to the apical membrane of porcine distal tubules. In the
present study, colocalization experiments showed that areas of NCC
expression overlap with the signal for grp58. However, the majority of
grp58 appeared to be in a subapical distribution, whereas NCC is
primarily associated with the plasma membrane. Previous results by
others have indicated that the NCC may also be located in a subapical
pool (21). One potential role of grp58 might be in
regulating insertion and removal of NCC from the apical plasma membrane.
The present experiments indicate that grp58 increases functional expression of NCC in oocytes. Western blots of uninjected oocytes indicated that they express grp58. Overexpression of grp58 with NCC, by coinjection, increases NCC activity. Reduction of grp58 expression with a grp58 antisense RNA reduced NCC activity. Western blot confirmed that injection of grp58 sense cRNA increased grp58 protein expression, whereas injection of grp58 antisense cRNA reduced steady-state levels of endogenous grp58. The mechanism by which grp58 increases functional activity of the NCC is not evident from the present experiments. Because grp58 may function as a molecular chaperone, we evaluated whether overexpression or reduced expression of grp58 would alter NCC processing. Western blots did not show an effect of grp58 to alter the ratio of glycosylated (mature) to unglycosylated (immature) NCC, but it remains possible that the amount of mature NCC is affected by grp58 expression, because small changes in mature NCC are not likely to be detectable by Western blot. Alternatively, grp58 may influence the amount of NCC expressed at the plasma membrane, as discussed above. This is consistent with the previously documented ability of grp58 to interact with scaffolding proteins (4), as part of a complex involved in recruiting signal transducer and activator of transcription 3 to the cytokine receptor complex, and the subsequent transit to the nucleus (16).
Gitelman's syndrome is an autosomal recessive disease characterized by salt wasting, hypokalemia, hypomagnesemia, and hypocalciuria (23). It is caused by mutations in the NCC (11, 12, 22, 26) that lead to dysfunction of the protein (10). Many of the mutations that have been reported to cause Gitelman's syndrome affect the COOH-terminal domain of the protein (12). Some of these mutations disrupt protein processing and appear to activate the "quality control" mechanism of the ER (10). It is possible that some of these mutations activate the quality control mechanism by disrupting interactions with molecular chaperones, such as grp58, although the present results do not provide direct support for this model. Furthermore, it is likely that other molecular chaperones participate in generating a fully folded and functional NCC and in assisting in its movement from the Golgi apparatus to the plasma membrane. Evidence of a subapical pool of NCC (21) and of physiologically driven alterations in NCC activity in vivo (29) raises the possibility that grp58 may contribute to apical insertion of the NCC in response to physiological need.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants RO1-DK-51496 and 5-P50-HL-55007 and funds from the American Heart Association and the Department of Veterans Affairs. This work was conducted, in part, during the tenure of D. H. Ellison as an Established Investigator of the American Heart Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. H. Ellison, Div. of Nephrology, Hypertension and Clinical Pharmacology, Oregon Health Sciences Univ., Suite 262, 3314 S.W. US Veterans Hospital Rd., Portland, OR 97201 (E-mail: ellisond{at}ohsu.edu).
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.
10.1152/ajprenal.0028.2001
Received 2 February 2001; accepted in final form 24 October 2001.
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