Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca 2 Pump 2B Variants in Polarized Epithelial Cells * Received for publication, July 13, 2010 Published, JBC Papers in Press, July 27, 2010, DOI 10.1074/jbc.M110.164137 Rita Pada ´nyi ‡ , Yuning Xiong § , Ge ´ za Antalffy ‡ , Krisztina Lo ´r ‡ , Katalin Pa ´ szty ¶ , Emanuel E. Strehler §1 , and A ´ gnes Enyedi ‡2 From the ‡ Department of Molecular Cell Biology, National Blood Center, H-1113 Budapest, Hungary, the § Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, and the ¶ Membrane Research Group, Semmelweis University, Hungarian Academy of Sciences, H-1067 Budapest, Hungary 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 spe- cific 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 cytocha- lasin 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 tar- geting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features. Plasma membrane Ca 2+ -ATPases (PMCAs) 3 are responsible for maintaining cellular Ca 2+ homeostasis by removing excess Ca 2+ from the cytosol. In addition to their housekeeping role, PMCAs also have more specialized tasks in controlling local Ca 2+ -mediated events. The need for specific Ca 2+ handling is reflected by the existence of over 20 different PMCA variants in mammals as follows: four separate genes code for PMCA iso- forms 1– 4, and alternative splicing of the primary transcripts further increases the number of PMCA variants (1). Earlier work demonstrated that targeting of PMCAs to spe- cific membrane compartments is affected by alternative splic- ing. Splicing at site A occurs in the region encoding the first intracellular loop where a single exon coding for 12–14 amino acids is either included or excluded in the mature transcripts, producing the “x” and “z” splice variants, respectively. In mam- malian PMCA2, two additional exons can be inserted at this site, producing splice variants “w” and “y”. The “w” form has the longest insert (45 amino acid residues) in the first intracellular loop, and this appears to target PMCA2 to the apical membrane of polarized cells (2, 3). The shorter forms (x and z) of the pump are localized mostly laterally. The alternative splice at site C affects the cytosolic C-termi- nal tail of the PMCA and produces two main splice variants, “a” and “b” (1). All b splice variants contain a PDZ-binding motif (…ETSL or …ETSV) at the C terminus that connects PMCAs to specific scaffolding and signaling proteins such as members of the membrane-associated guanylate kinase family (4, 5) or the Na + /H + exchanger regulatory factor 2 (NHERF2) (6). PMCA2 is a prominent plasma membrane Ca 2+ pump of the inner ear (7), specific regions of the brain (8), and lactating mammary gland (9). In some of these places, such as the apical membrane of lactating mammary epithelia, PMCA2w/b is expressed in exceptionally large amounts. A major role of PMCA2w/b in the mammary gland is to provide milk calcium as demonstrated in PMCA2 “knock-out” mice (10). However, it is not yet known how PMCA2w/b is recruited to and concen- trated at the apical membrane of these epithelial cells. An ear- lier study showed that the C-terminal sequence (ETSL) of PMCA2b interacts with the scaffolding protein NHERF2 (6). NHERF proteins are expressed in polarized epithelial cells where they target membrane proteins to the apical cytoskeleton via their ezrin, radixin, moesin (ERM) binding domain (11, 12). NHERF2 has been assigned to play an essential role in estab- lishing the polarity of MDCK epithelial cells (13). Furthermore, NHERF2 interacts with P2Y(1) purinergic receptors (14) as well * This work was supported, in whole or in part, by National Institutes of Health Grant NS51769 (to E. E. S.). This work was also supported in part by Hun- garian Academy of Sciences Grants OTKA K49476, CK 80283, and ETT 215/ 2009 (to A. E.) and by OTKA Postdoctoral Fellowship D48496 (to R. P.). 1 Co-senior author. To whom correspondence may be addressed: Mayo Clinic College of Medicine, 200 First St. S.W., Rochester, MN 55905. Tel.: 507-284- 9372; Fax: 507-284-2384; E-mail: strehler.emanuel@mayo.edu. 2 Co-senior author. To whom correspondence may be addressed: National Blood Center, Dept. of Molecular Cell Biology, Dio ´ szegi u. 64, H-1113 Buda- pest, Hungary. Tel./Fax: 36-1-372-4353; E-mail: enyedi@kkk.org.hu. 3 The abbreviations used are: PMCA, plasma membrane Ca 2+ pump; ERM, ezrin, radixin, moesin; FRAP, fluorescence recovery after photobleaching; MDCK, Madin-Darby canine kidney; NHERF, Na + /H + exchanger regulatory factor; DPBS, Dulbecco’s modified PBS; TRITC, tetramethylrhodamine isothiocyanate. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 41, pp. 31704 –31712, October 8, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. 31704 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 41 • OCTOBER 8, 2010 by guest on June 6, 2020 http://www.jbc.org/ Downloaded from