Sections on Nephrology and Molecular Medicine, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina
Submitted 20 September 2004 ; accepted in final form 10 January 2005
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ABSTRACT |
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protein assembly; cell surface localization
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In an attempt to identify additional proteins capable of interacting with and mediating distribution or trafficking of HK2, we used the carboxy terminus of mouse HK
2 as bait and the yeast two-hybrid method to screen a mouse cDNA library. We determined that the tetraspanin protein CD63 associates with the carboxy terminus of HK
2, but not with the carboxy terminus of other X+-K+-ATPase
-subunits. The biological relevance of these findings was confirmed by establishing stable assembly of the carboxy terminus of HK
2 with CD63 in vitro and by demonstrating that selective suppression of CD63 expression by RNA interference increases both cell surface localization of the HK
2/NK
1 complex and 86Rb+ uptake in human embryonic kidney (HEK)-293 cells. Collectively, these findings suggest that CD63 functions as a negative regulator of the colonic H+-K+-ATPase.
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MATERIALS AND METHODS |
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Screening of a mouse kidney cDNA library in pACT2 vector.
A cDNA library made from total RNA purified from mouse kidney was purchased from Clontech. For screening, one colony expressing m-CT-HK2 protein fused to GAL4-BD was amplified in 300 ml of SD medium in the absence of tryptophan until the optical density reached 0.40.5. All subsequent steps were as described above, except that competent yeast (1,500 µl) was mixed with 200 µg of mouse kidney cDNA library in pACT2 and 1,500 µg of herring sperm carrier DNA. The transformed yeast was plated on 20 petri dishes (15 cm in diameter) containing 2% agar in SD supplemented with all amino acids except tryptophan, leucine, histidine, and adenine. The dishes were incubated at 30°C for 34 days, and visible colonies were transferred to 5 ml of SD lacking the same amino acids and incubated for 2 days at 30°C. Plasmid DNA was purified as described by Hoffman (22). Briefly, the yeast was centrifuged at 1,000 g for 5 min at room temperature and then digested with 100 units of lyticase (Sigma, St. Louis, MO) for 1 h at 37°C in 100 µl of 10 mM Tris·HCl and 1 mM EDTA, pH 8.0. Twenty microliters of 10% SDS were then added, and the samples were frozen at 70°C. One hundred microliters of breaking buffer (4% Triton X-100, 1% SDS, 200 mM NaCl, 10 mM Tris·HCl, and 1 mM EDTA, pH 8.0) and 300 mg of glass beads (Sigma) were added to thawed samples and vortexed for 2 min. The proteins were extracted with 200 µl of H2O-saturated phenol/chloroform/isoamyl alcohol (24:24:1). The aqueous phase was retained, and the RNA was digested with 1 mg/ml RNAse A. The samples were diluted to a final volume of 1 ml, and the DNA was precipitated with 400 µl of 40% PEG-8000/1.5 M NaCl and resuspended. The isolated DNA was used to transform XL1blue MR Escherichia coli by electroporation, and the transfected E. coli were plated on petri dishes containing 100 µg/ml ampicillin. Colonies from these dishes were used to purify and sequence the insert (prey) in the mouse kidney library that interacted with m-CT-HK
2.
Assessment of m-CT-HK2 and m-CD63 interaction in vitro.
m-CT-HK
2 in pGBKT7 and m-CD63 in pGADT7 plasmids were used to synthesize [35S]methionine-labeled m-CT-HK
2 and m-CD63 proteins using the TnT T7-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions. After synthesis, the proteins were mixed and incubated for 1 h at room temperature. The m-CT-HK
2/m-CD63 complex was immunoprecipitated for 1 h at room temperature by the addition of 10 µl of rabbit anti-hemagglutinin polyclonal antibody (Clontech). The reaction was diluted with 400 µl of buffer (10 mM Tris·HCl, pH, 8.0, 150 mM NaCl, 1 mM PMSF, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor). Five microliters of packed agarose A (Santa Cruz Biotechnology, Santa Cruz, CA) were added and incubated for an additional 1 h at room temperature with continuous shaking. The resin was washed four times with 1 ml of 10 mM Tris·HCl, 150 mM NaCl, 1 mM PMSF, 3 mM benzamidine, 1 µg/ml soybean trypsin inhibitor, and 1% egg albumin, followed by two additional washes with the same buffer without egg albumin. Proteins were extracted with Laemmli sample buffer (27) and resolved using 10% SDS-PAGE that had been prerun for 4 h at 100 V to increase the resolution of low molecular weight proteins. The gel was fixed with 50% methanol, washed extensively with DMSO, submerged in a solution of 20% PPO in DMSO for 45 min, dried, and exposed to film for 3 days at 70°C.
Northern blot analysis. Northern blot analysis was performed as described previously by our laboratory (16) using a 32P-labeled probe based on the mouse CD63 sequence identified in the yeast two-hybrid screen.
Specific suppression of CD63 protein synthesis in HEK-293 cells by siRNA. A sense oligonucleotide 5'-TCGAGGTTCTT GCTCTACGTCCTCCTAGTACTGAGGAGGACGTAGA GCAAGAACTTTTT-3' containing an XhoI site, a 20-nucleotide sequence (GTTCTTGCTCTACGTC CTCC) corresponding to nucleotides 3352 of the open reading frame of the human CD63 (GenBank accession no. BT008095), a nonrelevant sequence (TAGTACTGA), followed by a reverse complementary sequence of human CD63 (GGAGGACGTAGAGCAAGAAC) and a polyT (TTTTT) tail, were synthesized. The reverse oligonucleotide (5'-CTAGAAAAAGTTCTTGCTCT ACGTCCTCCTCA GTACTAGGAGGACGTAGAGCAAGAACC-3') containing the 5'-overhanging end of the XbaI site (CTAGA) was also synthesized. The two oligonucleotides were purified, annealed as described previously (1), and cloned into the plasmid pSuppressorNeo (Imgenex, San Diego, CA). The sequence of the insert was verified by performing double-stranded DNA sequencing. The construct was linearized with BamHI and used (9 µg/10-cm dish) to stably transfect HEK-293 cells according to the instructions of the manufacturer. Colonies were selected using 250 µg/ml G418 (Invitrogen, Carlsbad, CA) and screened by immunolocalization of CD63 protein in the intracellular compartments with anti-human CD63 monoclonal antibody (Jackson ImmunoResearch, West Grove, PA) as described below.
Intracellular localization of CD63 in HEK-293 cells. HEK-293 cells were washed twice with PBS, fixed with 3.7% formaldehyde in PBS, washed twice with PBS, and incubated in 1% BSA plus 0.1% saponin in PBS for 10 min at room temperature. Anti-human CD63 monoclonal antibody (Jackson ImmunoResearch), diluted 1:100, was added to the cells for 30 min at room temperature. Cells were washed three times with PBS, after which rhodamine-conjugated goat anti-mouse IgG (1:1,000 dilution; Jackson ImmunoResearch) was added for 30 min at room temperature. Cells were viewed using a Zeiss Axioplan 2 fluorescence microscope equipped with rhodamine filters and recorded using a Zeiss Axiocam charge-coupled device camera. Control experiments were performed by omitting the primary antibody. Colonies growing in presence of G418 that did not express CD63 proteins were designated the experimental group (CD63 knockdown). Colonies growing in the presence of G418 but expressing levels of CD63 protein similar to those of nontransfected cells were used as controls.
Subcellular localization of HK2/NK
1 complex in control and CD63-knockdown HEK-293 cells.
Whole cell lysate protein was fractionated by discontinuous sucrose gradients as described previously (28) to investigate subcellular localization of HK
2/NK
1 in transfected control and CD63-knockdown HEK-293 cells. The top fraction (lower sucrose concentration) containing the plasma membrane fraction (28, 34) was diluted 10-fold with 10 mM Tris·HCl, pH 8.0, 1 mM EDTA, 1 mM PMSF, 3 mM benzamidine, and 1 µg/ml soybean trypsin inhibitor and concentrated by centrifugation for 30 min at 4°C at 30,000 g. The pellet was resuspended, protein concentration was determined, and 50 µg of protein were deglycosylated with glycosidase F for 1 h at 37°C in the presence of 1% 3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate. Proteins were resolved on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The upper half of the membrane was probed with the anti-HK
2 polyclonal antibody, and the lower half was probed with a polyclonal antibody against rat NK
1. Equal loading of the SDS-PAGE was monitored by staining the nitrocellulose membranes with Ponceau red S.
Miscellaneous methods. HEK-293 cells were grown at 37°C in DMEM containing 10% newborn calf serum (20). 86Rb+ uptake was performed at 37°C for 15 min in presence of KCl (1 mM) and 86Rb+ (34 x 106 cpm) (8). Immunoblot analysis was performed as described previously, with the intensity of bands quantified using the Image Tools software program (10).
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RESULTS |
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HK2 is the only X+-K+-ATPase
-subunit that assembles with CD63.
All experiments described above were performed using m-CT-HK
2 and m-CD63. Results were similar when the rat carboxy terminus of HK
2 was used as bait (not shown). Additional two-hybrid screens testing whether the carboxy termini of the different
-subunits from other X+-K+-ATPases (NK
1, NK
2, NK
3, and HK
1) interacted with m-CD63 revealed that the interaction between the carboxy terminus of HK
2 and CD63 is specific and did not extend to any other members of the X+-K+-ATPase family (data not shown).
Interaction between bait and prey is required for AH109 growth in the absence of tryptophan, leucine, histidine, and adenine.
To further test the specificity of the HK2-CD63 interaction, yeast was transfected with m-CT-HK
2 in pGBKT7 and plated on SD dishes supplemented with all the nutrients, SD dishes deficient in tryptophan, SD dishes deficient in leucine, or SD dishes deficient in tryptophan, leucine, histidine, and adenine. The results presented in Table 2 demonstrate that yeast grew when all nutrients were present or when tryptophan was omitted (Table 2, lane 1). Yeast transfected with m-CD63 in pACT2 grew when all the nutrients were added or when leucine was omitted (Table 2, lane 2). Yeast cotransfected with m-CT-HK
2 in pGBKT7 and m-CD63 in pACT2 grew under all four conditions (Table 2, lane 3) as expected when bait and prey interact. Yeast cotransfected with m-M7M8 in pGBKT7 and m-CD63 in pACT2 grew in the presence of all the nutrients or when tryptophan or leucine was omitted. However, they did not grow when all four nutrients were omitted (Table 2, lane 4), demonstrating that CD63 did not interact with the
-subunit binding sequence of HK
2. As a positive control, yeast cotransfected with m-M7M8 in pGBKT7 and the extracellular domain of rat NK
1 in pGADT7 grew under all four conditions (Table 2, lane 5), demonstrating that a region of HK
2 including transmembranes 7 and 8 interacted with the carboxy terminus of NK
1 as previously demonstrated by Colonna et al. (13).
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CD63 mRNA is expressed in distal colon, renal medulla, and HEK-293 cells.
If CD63 functions as a chaperone for HK2, CD63 should be expressed in mouse inner medullary collecting duct (mIMCD3) and mouse outer medullary collecting duct (mOMCD) cells in culture and in distal colon (31). Total RNA was purified from mIMCD3, mOMCD, distal colon, and HEK-293 cells; resolved on agarose gel; transferred to a nylon membrane; and probed with the m-CD63 identified using the yeast two-hybrid system. The results displayed in Fig. 3 demonstrate that CD63 mRNA is highly expressed in mIMCD, mOMCD, and HEK-293 cells in culture and distal colon. CD63 mRNA displays the expected mobility corresponding to 1.2 kb in the four samples tested in our studies. However, one larger band was detected in mRNA isolated from mIMCD-3 or mOMCD. This larger band could represent an alternative splice variant that is expressed at low levels, a CD63 pre-mRNA species, or a cross reaction with mRNA of other homologous tetraspanin proteins.
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DISCUSSION |
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CD63, a member of the tetraspanin superfamily of proteins, contains four hydrophobic transmembrane sequences, a large extracellular region of 95 amino acids between transmembrane segments 3 and 4, and is heavily glycosylated. This protein is expressed in numerous tissues and cells in culture (24, 30). In the present study, we have shown that CD63 is expressed in established cell lines of renal medullary origin known to express HK2 (31). In endothelial cells, CD63 has been shown to traffic from the plasma membrane to late endosomes and then to Weibel-Palade bodies to recycle to the plasma membrane (25, 35). CD63 also has been proposed as a molecular modulator of transporter function (5, 6, 36). Therefore, one implication of the CD63-HK
2 interaction revealed by our study is that CD63 may participate importantly in the internalization of HK
2 from the apical membrane. Nevertheless, a complete understanding of the participation of CD63 in the intracellular trafficking of HK
2 requires further studies.
Several recent studies have suggested that interacting proteins may play a key role in the migration of ion transporters to or from the plasma membrane. Staub et al. (33) demonstrated that Nedd4 is required for the internalization and proteasomal degradation of the amiloride-sensitive epithelial Na+ channel. Hebert (21) demonstrated that Bartter syndrome was required for migration of ClC-Ka and ClC-Kb channels to the plasma membrane in the thick ascending limb and in marginal cells of the inner ear. Na+/H+ exchanger NHE3 requires glycophorin A to migrate successfully to the apical membrane of the -intercalated cell in the renal medulla (2, 23). AE1 interacts with kanadaptin (7). Finally, mutations in these interacting proteins can manifest abnormalities in transport function. Therefore, the growing appreciation of such protein-protein interactions suggests a widespread phenomenon in transport physiology.
Our studies are consistent with the possibility that CD63, by interacting with the carboxy terminus of HK2, facilitates internalization of the HK
2/NK
1 complex from the cell surface. Specifically, when CD63 expression is inhibited by siRNA in transfected cells, the accumulation of HK
2/NK
1 protein at the cell surface and 86Rb+ uptake are both increased. A somewhat analogous mechanism of interaction has been advanced in the studies of Duffield et al. (17). These investigators demonstrated that CD63 association with the
-subunit of the gastric H+-K+-ATPase (HK-
G) facilitated internalization of the gastric H+-K+-ATPase heterodimer. In the present study, however, we demonstrate that CD63 does not interact with the carboxy terminus of HK
1. The biological implications of CD63 interacting with NK
G or HK
2 is not yet fully appreciated.
Our studies demonstrate that the interaction of CD63 with the carboxy terminus of HK2 regulates membrane expression of CD63 and transport function as monitored by 86Rb+ uptake. Because all members of the tetraspanin protein share a large number of common properties (30), it is logical to speculate that other tetraspanin proteins may interact with HK
2 or other
-subunits of the X+-K+-ATPase family. It is also possible that HK
2 affects the function of CD63. If CD63 is regulated by HK
2, such regulation may occur in the distal colon, where both HK
2 (10) and CD63 are expressed abundantly (see Fig. 3). In the renal medulla, HK
2 is expressed at low levels in animals with a normal plasma K+ concentration. In contrast, HK
2 is upregulated robustly by chronic K+ depletion (10). Therefore, if HK
2 regulates the activity of CD63 in the renal medulla, it seems likely that this phenomenon would be observed during chronic hypokalemia.
In conclusion, our results are in agreement with and extend recent findings indicating that CD63 participates in the internalization of certain members of the H+-K+-ATPase family. We have found that CD63 specifically interacts with the carboxy terminus of HK2, but not with other members of the X+-K+-ATPase family of membrane transport proteins. This interaction serves to reduce plasma membrane expression of the HK
2/NK
1 complex, suggesting a mechanism by which CD63 functions as a negative modulator of H+-K+-ATPase.
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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.
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