Meeting Abstract
11.4 Jan. 4 Cyclic Nucleotide Phosphodiesterase from Crustacean (Procambarus clarkii) Y-Organs: Molecular Cloning, Heterologous Expression, and Functional Link to Calcium Signaling and Regulation of Ecdysteroidogenesis. NAKATSUJI, T.*; WATSON, R.D.; Univ. of Alabama, Birmingham; Univ. of Alabama, Birmingham terunakatsuji@hotmail.com
Crustacean molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ/sinus gland complex of the eyestalks, regulates molting by inhibiting the synthesis of ecdysteroids by Y-organs. Our previous results suggest that molt cycle-associated changes in cyclic nucleotide phosphodiesterase (PDE) activity affect the ability of MIH to stimulate cGMP accumulation and suppress ecdysteroidogenesis in Y-organs of the crayfish Procambarus clarkii. We report here (a) molecular cloning of a cDNA (PcPDE1) encoding PDE from P. clarkii Y-organs, (b) expression of the recombinant PcPDE1 protein in Escherichia coli, and (c) studies revealing the functional link of PcPDE1 to calcium signaling and MIH-mediated regulation of ecdysteroid production in Y-organs. Sequence analysis of the cloned cDNA revealed a 2061 base pair open reading frame. Analysis of the deduced amino acid sequence showed that PcPDE1 contains putative calcium/calmodulin binding and catalytic domains, suggesting that PcPDE1 is a calcium/calmodulin dependent PDE (PDE1). Recombinant PcPDE1 was expressed in E. coli. The refolded recombinant enzyme had PDE catalytic activity, and that activity was enhanced by addition of calcium and calmodulin. Incubating Y-organs with a calcium ionophore (ionomycin) increased glandular PDE activity three-fold and decreased MIH-induced suppression of ecdysteroid production in Y-organs. Quantitative real-time PCR revealed expression of the PcPDE1 gene in Y-organs was highest during middle premolt, a stage when the responsiveness of Y-organs to MIH is lowest. The combined results suggest that PcPDE1 is a key enzyme linking cellular calcium signaling to MIH-mediated regulation of ecdysteroid production in Y-organs. Grant sponsor: National Science Foundation, IBN-0213047 (RDW).
