Plasma Membrane Ca2+ ATPase Isoform 2b Interacts Preferentially with Na+/H+ Exchanger Regulatory Factor 2 in Apical Plasma Membranes

• Published in: The Journal of biological chemistry Vol. 277; no. 12; pp. 10506 - 10511
• Main Authors: DeMarco, Steven J., Chicka, Michael C., Strehler, Emanuel E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22-03-2002; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Biotinylation; Brain - metabolism; Calcium - metabolism; Calcium-Transporting ATPases - chemistry; Calcium-Transporting ATPases - metabolism; Cation Transport Proteins; Cell Membrane - enzymology; Codon; COS Cells; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - metabolism; DNA, Complementary - metabolism; Dogs; Epithelial Cells - metabolism; Gene Library; Glutathione Transferase - metabolism; Green Fluorescent Proteins; Humans; Immunoblotting; Luminescent Proteins - metabolism; Microscopy, Fluorescence; Phosphoproteins; Plasma Membrane Calcium-Transporting ATPases; Plasmids - metabolism; Precipitin Tests; Protein Binding; Protein Isoforms; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - metabolism; Sodium-Hydrogen Exchangers; Two-Hybrid System Techniques
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111616200

Spatial and temporal regulation of Ca2+ signaling require the assembly of multiprotein complexes linking molecules involved in Ca2+ influx, sensing, buffering, and extrusion. Recent evidence indicates that plasma membrane Ca2+ ATPases (PMCAs) participate in the control of local Ca2+ fluxes, but the mechanism of multiprotein complex formation of specific PMCAs is poorly understood. Using the PMCA2b COOH-terminal tail as bait in a yeast two-hybrid screen, we identified the PSD-95, Dlg,ZO-1 (PDZ) domain-containing Na+/H+exchanger regulatory factor-2 (NHERF2) as an interacting partner. Protein pull-down and coimmunoprecipitation experiments using recombinant PMCA2b and PMCA4b as well as NHERF1 and NHERF2 showed that the interaction of PMCA2b with NHERF2 was specific and selective. PMCA4b did not interact with either of the NHERFs, and PMCA2b selectively preferred NHERF2 over NHERF1. Green fluorescent protein-tagged PMCA2b was expressed at the apical membrane in Madin-Darby canine kidney epithelial cells, where it colocalized with apically targeted NHERF2. Our study identifies NHERF2 as the first specific PDZ partner for PMCA2b not shared with PMCA4b, and demonstrates that PMCA splice forms differing only minimally in their COOH-terminal residues interact with unique PDZ proteins. NHERFs have been implicated in the targeting, retention and regulation of membrane proteins including the β2-adrenergic receptor, cystic fibrosis transmembrane conductance regulator, and Trp4 Ca2+channel, and NHERF2 is now shown to also interact with PMCA2b. This interaction may allow the functional assembly of PMCA2b in a multiprotein Ca2+ signaling complex, facilitating integrated cross-talk between local Ca2+ influx and efflux.