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. 2005 Jun;288(6):C1279-86.
doi: 10.1152/ajpcell.00463.2004. Epub 2005 Jan 12.

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to decrease [corrected] plasma membrane localization and 86Rb+ uptake

Juan Codina  1 , Jian Li, Thomas D Dubose Jr
Affiliations

CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to decrease [corrected] plasma membrane localization and 86Rb+ uptake

Juan Codina et al. Am J Physiol Cell Physiol. 2005 Jun.

Erratum in

  • Am J Physiol Cell Physiol. 2007 Apr;292(4):C1567

Abstract

The carboxy terminus (CT) of the colonic H(+)-K(+)-ATPase is required for stable assembly with the beta-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKalpha(2), we selected 84 amino acids in the CT of the alpha-subunit of mouse colonic H(+)-K(+)-ATPase (CT-HKalpha(2)) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HKalpha(2) with CD63, recombinant CT-HKalpha(2) and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKalpha(2) protein (but not CT-HKalpha(1)) coprecipitated with CD63, confirming stable assembly of HKalpha(2) with CD63. In HEK-293 transfected with HKalpha(2) plus beta(1)-Na(+)-K(+)-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKalpha(2)/NKbeta(1) and (86)Rb(+) uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKalpha(2)-NKbeta(1) complex in the cell membrane.

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Figures

Figure 1
Figure 1. Predicted topology of HKα2
The carboxy-terminus of HKα2 used to screen the mouse kidney cDNA library is represented by a dashed line and extends from the EcoRI site of HKα2 to the stop codon (84 carboxy-terminus amino acids). The bold line between transmembrane M7 and M8 of HKα2 represents the binding site to the β-subunit. N = amino-terminus, C = carboxy-terminus, M1-M10 = predicted transmembrane regions.
Figure 2
Figure 2. CD63 interacts with the carboxy-terminus of HKα2
HA-tagged m-CD63, m-CT-HKα2, and r-CT-NKα1 were synthesized and incubated, and immune complexes precipitated using an anti-hemagglutinin polyclonal antibody, as described in Materials and Methods. Panel A: lane 1, m-CT-HKα2 was incubated alone; lane 2, m-CD63 was incubated alone; lane 3, m-CT-HKα2 was incubated with m-CD63. Panel B: lane 1, r-CT-NKα1 was incubated alone; lane 2, m-CD63 was incubated alone; lane 3, r-CT-NKα1 was incubated with m-CD63. Abbreviations used in all figures are the same as in tables 1 and 2 or as indicated in the list of abbreviations. The molecular weights of the different proteins are indicated in kDa. The experiment was repeated three times with similar results.
Figure 3
Figure 3. CD63 mRNA is expressed in distal colon, renal medulla, and HEK-293 cells
Total RNA (10 μg) from mouse distal colon, mIMCD3, and mOMCD and HEK-293 cells were probed with m-CD63 cDNA. The film was exposed overnight at −70°C. The molecular weight of the mRNA is expressed in kilobases (kb). The experiment was repeated three times with similar results.
Figure 4
Figure 4. CD63 protein expression is abolished in HEK-293 cells using siRNA
Panel A: Immunocytochemical localization of endogenous CD63 protein in a G418 selected HEK-293 line that expresses CD63. The experiment was performed at room temperature in presence of saponin. Panel B: CD63 expression in a clonal line of HEK-293 transfected with siRNA targeting CD63 then selected with G418, CD63-knockdown). Panel C: The same as in panel A, but with primary antibody omitted. Panel D: The same as in panel B, but with primary antibody omitted. The anti-CD63 monoclonal antibody (primary antibody) was diluted 1:100. The immunocytochemical localization of CD63 shown in panels A to D was repeated 10 times with similar results.
Figure 5
Figure 5. Expression of HKα2 and NKβ1 protein is not altered by knocking down expression of CD63 with siRNA
Control HEK-293 cells or CD63-knockdown HEK-293 cells were co-transfected with HKα2 plus NKβ1. Forty-eight hours later, the cells were lysed, and 100 μg of protein deglycosylated with glycosidase F and resolved on a 10% SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and the stained with Ponceau S to verify equal protein loading of the lanes. The immunoblot analyses were performed using an anti-HKα2 (dilution 1:1000) (10) or anti NKβ1 antibody (dilution 1:1000) (29). For these experiments, controls were HEK-293 cells transfected with CD63 siRNA using the plasmid pSuppressorNeo and selected with G418, but expression of CD63 protein was not different than that determined in wild HEK-293 cells. The experiment was repeated 4 times with similar results and the difference between control and CD63-knockdown is not statistically significant.
Figure 6
Figure 6. Plasma membrane expression of HKα2 increases in CD63-knockdown HEK-293 cells.
Control HEK-293 cells were co-transfected with HKα2 plus NKβ1. Forty eight hours later, the cells were lysed as described in Materials and Methods. The membranes were fractionated using a discontinuous sucrose gradient (28, 34). The proteins of the different fractions were processed as described in Materials and Methods and probed with anti-HKα2 antibody (panel A) or anti-NKα1 antibody (panel C). The results demonstrated that HKα2 and NKβ1 migrate to the plasma membrane (fractions 1 and 2) or remain in the heavy sucrose fraction (fraction 6). Panels B and D: The same as in panels A and C but the experiment was performed in CD63-knockdown HEK-293 cells. Panels E and F demonstrate that the presence or absence of CD63 does not alter the pattern of migration of NKα1 in the sucrose gradient when the cells were co-transfected with NKα1/NKβ1. Immunoblot analysis performed with anti-calnexin (antibody commercially available from Santa Cruz Biotechnology, Santa Cruz, CA) demonstrated that the ER components accumulated in fraction 6. The experiment was performed three times with similar results. The quantity of HKα2 and NKβ1 that accumulates in the top of the gradient is statistically greater in CD63-knockdown HEK-293 cells vs. the controls (p<0.05).
Figure 7
Figure 7. 86Rb+-uptake by HKα2/NKβ1 complex but not by NKα1/NKβ1 complex is increased in CD63-knockdown cells.
Control HEK-293 cells (open bars) or CD63-knockdown HEK-293 cells (solid bars) were co-transfected with HKα2 plus NKβ1, NKα1 plus NKβ1, or pcDNA vector plus NKβ1. Ouabain (10 μ M) was used to block endogenous Na+,K+-ATPase of HEK-293 cells and 2 mM ouabain was used to block the transport activity of the transfected rat HKα2 or rat NKα1. Results (mean ± S.E.M.) represent the difference in 86Rb+-uptake when the experiments were performed with 10 μM ouabain vs. 2 mM ouabain. ** = 86Rb+-uptake in control HEK-293 cell is statistically different (P<0.01) from that in the CD63-knockdown cells for HKα2/NKβ1 -transfected cells. No statistical difference was observed between control and CD63-knockdown cells when the cells were co-transfected with NKα1/NKβ1 or pcDNA plus NKβ1. The experiment was repeated three times with similar results.
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