Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells

Abstract

The membrane localization of the plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.

FIGURE 1.

NHERF2 enhances apical localization of PMCA2w/b. GFP-PMCA2w/b was transiently expressed in MDCK cells without (A) or with (B) NHERF2. PMCA localization was detected by confocal microscopy. Cells were stained with anti-NHERF2 antibody (panel B2) or basolateral Na⁺/K⁺-ATPase α-subunit antibody (panel B5). Panels B1 and B4 show the distribution of the GFP signal; panels B3 and B6 are merged images of panels B1 and B2 and panels B4 and B5, respectively. Note co-localization of the GFP-PMCA2w/b and NHERF2 signals in the apical membrane in the merged image panel B3, and the separation of green (GFP-PMCA2w/b, apical) and red (Na⁺/K⁺-ATPase, basolateral) fluorescence in the merged image panel B6. Arrows in xz sections on the top of each panel indicate the apical location where the xy sections were taken. The bar graph in C shows the ratio of mean PMCA fluorescence intensities of equal regions of interest in apical versus middle sections of cells expressing GFP-PMCA2w/b (2wb), C-terminally truncated GFP-PMCA2w/bΔ6 (2wbΔ6), or either of these pumps together with NHERF2 (2wb/NHERF and 2wbΔ6/NHERF). Fluorescence quantification was performed as described under “Experimental Procedures.” Removing the C-terminal residues from PMCA2w/b suppresses the effect of NHERF2 on the redistribution of the pump toward the apical region (compare the last two bars in C). Values represent the mean ± S.D. of calculations from 15 to 20 cells of three independent experiments. ***, p < 0.05 versus 2wb.

FIGURE 2.

NHERF2 anchors PMCA2w/b to the actin cytoskeleton beneath the apical membrane of polarized MDCK cells. A and B, MDCK cells co-expressing GFP-PMCA2w/b (green) and NHERF2 were formaldehyde- fixed, permeabilized with Triton X-100, and stained with anti-NHERF2 antibody (red) and either TRITC-labeled phalloidin (blue in A panels) or anti-ezrin antibody (blue in B panels). In panels A5–A8 and B5–B8, cells were treated with 2.5 μm cytochalasin D for 10 min and in panels A9–A12 and B9–B12 with 1 μm latrunculin B for 30 min before the addition of formaldehyde. The apical sections of typical cells expressing the appropriate constructs are shown. Panels A4, A8, A12, B4, B8, and B12 show merged images, where white indicates overlap of the green, red, and blue staining. C, Pearson's co-localization coefficients of the GFP-PMCA2w/b (2wb), NHERF2 (NH), ezrin, and phalloidin (ph) signals in MDCK cells without (−Cyto) or with (+Cyto) cytochalasin D treatment were determined as described under “Experimental Procedures.” Values represent the mean ± S.D. of calculations from 15 to 20 cells of three independent experiments. D, schematic showing anchorage of PMCA2w/b to the apical actin cytoskeleton by NHERF2 via ezrin. PMCA2w/b binds via its C-terminal ETSL sequence to the PDZ protein NHERF2, which interacts through its ERM domain with ezrin.

FIGURE 3.

NHERF2 greatly reduces the lateral mobility of GFP-PMCA2w/b in MDCK cells. Confocal images of GFP fluorescence at the apical surface of live MDCK cells expressing GFP-PMCA2w/b alone or together with NHERF2 are as indicated. The circled region of interest with a diameter of 2.6 μm was bleached by a high intensity argon laser beam at 488 nm. The fluorescence recovery within the region of interest was followed by scanning every 3 s for 5 min. The fluorescence at each time point after bleaching was normalized to the pre-bleach intensity and corrected for acquisition bleaching. A, black lines indicate the recovery curves of GFP-PMCA2w/b in the absence (triangles) and presence (diamonds) of NHERF2. Gray lines indicate the recovery curves of the C-terminally truncated GFP-PMCA2w/bΔ6 mutant in the absence (open circles) and presence (asterisks) of NHERF2. B shows confocal images of FRAP experiments performed on MDCK cells after 1 μm latrunculin B treatment for 30 min at 37 °C. In the graph, control curves are shown in gray (GFP-PMCA2w/b without NHERF2 (triangles) and with NHERF2 (diamonds)). Black lines indicate the recovery curves of GFP-PMCA2w/b in the absence (circles) and presence of NHERF2 (squares) after latrunculin (Lat) treatment. The solid lines through the data are exponential fits to the averaged data of five experiments ± S.E. Scale bars, 5 μm.

FIGURE 4.

NHERF-2 reduces the recycling of PMCA2w/b. Cells transfected with the proteins indicated on top of each panel were surface-biotinylated on ice and then washed and collected (input) or incubated at 37 °C for the indicated times to allow endocytosis of PMCA. After glutathione stripping, cells were lysed, and biotinylated proteins were recovered on streptavidin beads. Wild type PMCA2w/b (left panel), NHERF2-associated PMCA2w/b (middle panel), and the truncated PMCA2w/bΔ6 (right panel) were resolved and visualized by Western blotting (arrows), using anti-PMCA antibody 5F10. Glutathione stripping immediately after biotinylation efficiently removed all surface-biotinylated proteins (lanes labeled 0 min). Incubation of cells at 37 °C for 10 or 20 min allowed the recycling of PMCA. Endocytosis of the truncated PMCA2w/bΔ6 was more pronounced (65 ± 6% of input proteins were recovered after 20 min) than that of the wild type PMCA2w/b (26 ± 9% recovery after 20 min) or the NHERF2-associated PMCA2w/b (10 ± 1% recovery after 20 min). Quantified data represent the average of three independent experiments.

FIGURE 5.

Co-transfection with NHERF2 alters the localization of PMCA2z/b but not PMCA2x/b in polarized MDCK cells. GFP-PMCA2z/b (A panels) or GFP-PMCA2x/b (B panels) was co-expressed with NHERF2 in MDCK cells, and the localization of the two proteins was determined by confocal microscopy. NHERF2 was visualized using anti-NHERF antibody (A2 and B2, red). Cells were also stained with an antibody against the basolateral Na⁺/K⁺-ATPase α-subunit (A5 and B5, red). Merged images are shown on the right (A3, A6, B3, and B6). Apical xy sections are shown in the en face views in A1–A6 and B1–B3, whereas subapical xy sections are shown in B4–B6. Corresponding xz sections are shown on top of each image. GFP-PMCA2z/b and NHERF2 localization partially overlap at the apical membrane (A1–A3), in contrast to the complete separation of GFP-PMCA2x/b and NHERF2 in the lateral and apical domains, respectively (B1–B3). Both GFP-PMCA2z/b (xz sections in A4–A6) and GFP-PMCA2x/b (B4–B6) show substantial co-localization with the basolateral marker Na⁺/K⁺-ATPase α-subunit. C, fluorescence quantification was performed as described under “Experimental Procedures.” Values represent the mean ± S.E. of 15–20 cells from three independent experiments.

FIGURE 6.

Scheme illustrating the effect of NHERF2 on the cellular distribution of PMCA2b A-splice variants. Black arrows indicate the targeting of PMCA2b variants (W, PMCA2w/b; Z, PMCA2z/b; X, PMCA2x/b) in polarized MDCK cells. Under control conditions (left panel), only PMCA2w/b shows prominent targeting to the apical plasma membrane (apical PM). Upon NHERF2 expression (right panel), apical PMCA2w/b is greatly enhanced, and PMCA2z/b is recruited to the apical membrane (indicated by the thick arrows), whereas PMCA2x/b is unaffected and stays in the lateral membrane.