Meeting Abstract

P2.134  Saturday, Jan. 5  Molecular Cloning of a cDNA Encoding a Putative Plasma Membrane Calcium ATPase from Y-Organs of the Blue Crab (Callinectes sapidus) CHEN, H.-Y.*; ROER, R.D.; WATSON, R.D.; University of Alabama at Birmingham; University of North Carolina at Wilmington; University of Alabama at Birmingham elgase@uab.edu

Existing data indicate that a stage-specific increase in intracellular free Ca⁺⁺ is involved in regulating (stimulating) ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca⁺⁺ 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⁺⁺ channels, Ca⁺⁺ pumps (ATPases), and Ca⁺⁺ exchangers. The family of Ca⁺⁺ pumps includes plasma membrane calcium ATPases (PMCAs). As a step toward understanding the involvement of calcium signaling in regulation of ecdysteroidogenesis, we have 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 an estimated molecular weight of 128.8 and contains all signature domains of an authentic PMCA, including phosphorylation, ATP-binding, and calmodulin-binding domains. An analysis of membrane topography predicted Cas-PMCA to have ten transmembrane domains and two large intracellular loops, a pattern consistent with the structure of known PMCA proteins. An assessment of tissue distribution showed the Cas-PMCA transcript to be broadly distributed in both neural and non-neural tissues. We anticipate that using quantitative real-time PCR to measure the abundance of the Cas-PMCA transcript in Y-organs during a molting cycle will provide insight into the possible involvement of PMCA in Ca⁺⁺-mediated regulation of ecdysteroidogenesis.