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

Padányi, Rita and Xiong, Yuning and Antalffy, Géza and Lór, Krisztina and Pászty, Katalin and Enyedi, Ágnes (2010) Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells. JOURNAL OF BIOLOGICAL CHEMISTRY, 285 (41). pp. 31704-31712. ISSN 0021-9258

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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.

Item Type: Article
Additional Information:
Uncontrolled Keywords: Thiazolidines/pharmacology; Sodium-Hydrogen Antiporter/genetics/*metabolism; Protein Transport/drug effects/physiology; Protein Structure, Tertiary; Plasma Membrane Calcium-Transporting ATPases/genetics/*metabolism; Phosphoproteins/genetics/*metabolism; Nucleic Acid Synthesis Inhibitors/pharmacology; Isoenzymes/genetics/metabolism; Humans; Hela Cells; Epithelial Cells/*metabolism; Dogs; Cytoskeleton/metabolism; Cytoskeletal Proteins/metabolism; Cytochalasin D/pharmacology; Cell Polarity/*physiology; Cell Membrane/*metabolism; Bicyclo Compounds, Heterocyclic/pharmacology; Animals; Alternative Splicing/drug effects/*physiology; Actins/metabolism
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia
Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3015 Molecular biology / molekuláris biológia
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 14 Jan 2014 11:18
Last Modified: 14 Jan 2014 11:18
URI: http://real.mtak.hu/id/eprint/8674

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