Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting.
Plasma membrane Ca(2+)-ATPases (PMCAs) are involved in local Ca(2+) signaling and in the spatial control of Ca(2+) extrusion, but how different PMCA isoforms are targeted to specific membrane domains is unknown. In polarized MDCK epithelial cells, a green fluorescent protein-tagged PMCA4b construct was targeted to the basolateral membrane, whereas a green fluorescent protein-tagged PMCA2b construct was localized to both the apical and basolateral domain. The PDZ protein-binding COOH-terminal tail of PMCA2b was not responsible for its apical membrane localization, as a chimeric pump made of an NH(2)-terminal portion from PMCA4 and a COOH-terminal tail from PMCA2b was targeted to the basolateral domain. Deletion of the last six residues of the COOH terminus of either PMCA2b or PMCA4b did not alter their membrane targeting, suggesting that PDZ protein interactions are not essential for proper membrane localization of the pumps. Instead, we found that alternative splicing affecting the first cytosolic loop determined apical membrane targeting of PMCA2. Only the "w" form, which contains a 45-amino acid residue insertion, showed prominent apical membrane localization. By contrast, the x and z splice variants containing insertions of 14 and 0 residues, respectively, localized to the basolateral membrane. The w splice insert was the crucial determinant of apical PMCA2 localization, and this was independent of the splice configuration at the COOH-terminal end of the pump; both PMCA2w/b and PMCA2w/a showed prominent apical targeting, whereas PMCA2x/b, PMCA2z/b, and PMCA2z/a were confined to the basolateral membrane. These data report the first differential effect of alternative splicing within the first cytosolic loop of PMCA2 and help explain the selective enrichment of specific PMCA2 isoforms in specialized membrane compartments such as stereocilia of auditory hair cells.
Chicka
Michael C
MC
Department of Biochemistry and Molecular Biology, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota 55905, USA.
Strehler
Emanuel E
EE
D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008051:Lipid Bilayers / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D053499:Plasma Membrane Calcium-Transporting ATPases; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D027682:Cation Transport Proteins; D008051:Lipid Bilayers; D008164:Luminescent Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases; C506958:ATP2B2 protein, human; D000252:Calcium-Transporting ATPases; D002118:Calcium