title: Molecular cloning of a plasma membrane Ca²⁺ ATPase (PMCA) from Y-organs of the blue crab (Callinectes sapidus), and determination of spatial and temporal patterns of PMCA gene expression.
abstract: Existing data indicate that a stage-specific increase in intracellular free Ca(2+) stimulates ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca(2+) in cytosol is controlled mainly by proteins intrinsic to the plasma membrane and to the membranes of organelles. Several families of proteins are involved, including Ca(2+) channels, Ca(2+) pumps (ATPases), and Ca(2+) exchangers. The family of Ca(2+) pumps includes plasma membrane calcium ATPases (PMCAs). As a step toward understanding the involvement of calcium signaling in regulation of ecdysteroidogenesis, we used a PCR-based cloning strategy (RT-PCR followed by 3'- and 5'-RACE) to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative PMCA. The 4292 base pair (bp) cDNA includes a 3510 bp open reading frame encoding a 1170-residue protein (Cas-PMCA). The conceptually translated protein has a relative molecular mass of 128.8×10(3) and contains all signature domains of an authentic PMCA, including ten transmembrane domains and a calmodulin binding site. The predicted membrane topography of Cas-PMCA is as expected for an authentic PMCA protein. A phylogenetic analysis of nonredundant amino acid sequences of PMCA proteins from different species showed Cas-PMCA clusters with other arthropod PMCA proteins. An assessment of tissue distribution showed the Cas-PMCA transcript to be broadly distributed in both neural and non-neural tissues. Studies using quantitative real-time PCR revealed stage-specific changes in Cas-PMCA abundance during the molting cycle, with peak expression occurring during premolt stage D2, a pattern consistent with the hypothesis that Cas-PMCA functions to maintain cellular Ca(2+) homeostasis in Y-organs.
authors.affiliation: Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Hsiang-Yin
authors.initials: HY
authors.lastname: Chen
authors.forename: Robert D
authors.initials: RD
authors.lastname: Roer
authors.forename: R Douglas
authors.initials: RD
authors.lastname: Watson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003386:Brachyura / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D018978:Molting / Q000235:genetics; D016366:Open Reading Frames; D010802:Phylogeny; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods
chemical_list: D002147:Calmodulin; D018076:DNA, Complementary; D053499:Plasma Membrane Calcium-Transporting ATPases; D002118:Calcium