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.
Highlights
We used GFP-tagged plasma membrane Ca2؉ATPase isoform 2 (PMCA2) constructs throughout this study because previous work had shown that the localization of N-terminally GFP-tagged Plasma membrane Ca2ϩ-ATPases (PMCAs) faithfully reflects the localization of the corresponding untagged pumps in polarized MDCK cells [2]
Quantitative analysis revealed that the ratio of apical to lateral fluorescence intensity of the GFP signal increased 2-fold when Naϩ/Hϩ exchanger regulatory factor 2 (NHERF2) was co-expressed with GFP-PMCA2w/b (Fig. 1C)
A truncated mutant (GFP-PMCA2w/b⌬6) lacking the PDZ-binding C-terminal tail showed no change in the ratio of apical to lateral localization when co-expressed with NHERF2 (Fig. 1C)
Summary
Reagents and Antibodies—FuGENE 6 transfection reagent was obtained from Roche Applied Science, and LipofectamineTM was from Invitrogen. After three washes in DPBS, cells were incubated for 1 h at room temperature with Alexa Fluor 488-, Alexa Fluor 594-, or Alexa Fluor 633-conjugated goat anti-mouse and anti-rabbit or Alexa Fluor 594-conjugated goat anti-chicken secondary antibodies at a dilution of 1:250. The ratio of mean PMCA fluorescence intensities of equal regions of interest in apical versus middle sections of cells was determined. Cells were lysed by Lysis Buffer containing 0.1 mM PefablocTM, 10 g/ml leupeptin, 20 g/ml aprotinin on ice. The cell extract was centrifuged at 10,000 ϫ g for 5 min, and the supernatant was incubated with immobilized NeutrAvidinTM gel beads (Pierce) for 60 min at room temperature with end-over-end mixing to precipitate the biotinylated proteins. The precipitates were resolved by SDS-PAGE and transferred onto nitrocellulose, followed by Western blotting
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