Abstracts Biological Sciences

Identification and characterization of three new cyclic nucleotide phosphodiesterase gene families

by Scott Haydn Soderling

This dissertation describes the cloning and characterization of three new PDE gene families: PDE8, PDE9, and PDE10. The method by which this was accomplished was a new one for this laboratory in that each new gene family was initially identified as an EST clone by searching the dBEST database. Each new PDE has unique characteristics from that of the previously identified seven PDE gene families.The cDNA of PDE8A is predicted to encode an 823 amino acid enzyme. PDE8A hydrolytic activity is specific for cAMP hydrolysis and has a Km of 0. 15 uM cAMP. RNA expression studies indicate PDE8A is expressed at highest levels in mouse testis, followed by eye, liver, skeletal muscle, heart, 7-day embryo, kidney, ovary, and brain. In situ hybridizations indicate PDE8A gene expression in testis is regulated both temporally and spatially. PDE8A also contains a PAS domain (named for the Period, Aryl-hydrocarbon nuclear translocator, and Single-minded proteins) found many signaling molecules from diverse organisms. Molecular modeling of the PDE8 PAS domain, based on the crystal structure of other PAS domains, followed by mutagenesis and two-hybrid analysis suggests that this domain may serve a protein/protein binding function.PDE9 is predicted to consist of 534 amino acids based on a cDNA that is 1,929 bases in length. Expressed in COS cells, this PDE is specific for the hydrolysis of cGMP and has a very low Km (0.07 uM). RNA expression distribution studies using both Northern and RNA dot blot analysis suggest PDE9 is expressed at highest levels in small intestine, followed by kidney, liver, lung, and brain.PDE10A represents a new dual substrate cyclic nucleotide phosphodiesterase. Kinetic analysis indicates PDE10 has both a low Km and a low V max for cAMP compared to cGMP and therefore may function as a cAMP inhibited cGMP PDE. PDE10 contains two GAF (for cGMP binding and stimulated phosphodiesterases, Anabaena adenylyl cyclases, and Escherichia coli fhlA) domains similar to many other signaling proteins from both eukaryotic and eubacterial organisms. Binding assays using purified PDE10 and 3H cGMP indicate the PDE10A GAF domains bind cGMP with a low affinity.