title: 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.
authors.affiliation: Department of Molecular Cell Biology, National Blood Center, H-1113 Budapest, Hungary.
authors.forename: Rita
authors.initials: R
authors.lastname: Padányi
authors.forename: Yuning
authors.initials: Y
authors.lastname: Xiong
authors.forename: Géza
authors.initials: G
authors.lastname: Antalffy
authors.forename: Krisztina
authors.initials: K
authors.lastname: Lór
authors.forename: Katalin
authors.initials: K
authors.lastname: Pászty
authors.forename: Emanuel E
authors.initials: EE
authors.lastname: Strehler
authors.forename: Agnes
authors.initials: A
authors.lastname: Enyedi
mesh_terms: D000199:Actins / Q000378:metabolism; D017398:Alternative Splicing / Q000187:drug effects / Q000502:physiology*; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity / Q000502:physiology*; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D053778:Thiazolidines / Q000494:pharmacology; D000099149:Ezrin
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D015638:Cytochalasin D; D003598:Cytoskeletal Proteins; D007527:Isoenzymes; D019384:Nucleic Acid Synthesis Inhibitors; D010750:Phosphoproteins; D053499:Plasma Membrane Calcium-Transporting ATPases; D017923:Sodium-Hydrogen Exchangers; D053778:Thiazolidines; D000099149:Ezrin; C093376:sodium-hydrogen exchanger regulatory factor; C506958:ATP2B2 protein, human; C037068:latrunculin B