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

• We cloned from Y-organs a cDNA (Cas-PMCA) encoding a plasma membrane Ca2 + ATPase.
• The Cas-PMCA transcript was widely distributed in neural and non-neural tissues.
• A phylogenetic analysis showed Cas-PMCA is most closely related to insect PMCAs.
• Cas-PMCA transcript abundance in Y-organs changes during the molting cycle.

Existing data indicate that a stage-specific increase in intracellular free Ca2 + stimulates ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca2 + 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 Ca2 + channels, Ca2 + pumps (ATPases), and Ca2 + exchangers. The family of Ca2 + 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 × 103 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 Ca2 + homeostasis in Y-organs.