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 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 4292base pair (bp) cDNA includes a 3510bp 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.