cDNA encoding a novel phosphodiesterase (PDE) was isolated from a human fetal lung cDNA library and designated PDE10A. The deduced amino acid sequence contains 779 amino acids, including a putative cGMP binding sequence in the amino-terminal portion of the molecule and a catalytic domain that is 16 -47% identical in amino acid sequence to those of other PDE families. Recombinant PDE10A transfected and expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 0.26 and 7.2 M, respectively, and V max with cGMP was almost twice that with cAMP. Of the PDE inhibitors tested, dipyridamole was most effective, with IC 50 values of 1.2 and 0.45 M for inhibition of cAMP and cGMP hydrolysis, respectively. cGMP inhibited hydrolysis of cAMP, and cAMP inhibited cGMP hydrolysis with IC 50 values of 14 and 0.39 M, respectively. Thus, PDE10A exhibited properties of a cAMP PDE and a cAMP-inhibited cGMP PDE. PDE10A transcripts were particularly abundant in the putamen and caudate nucleus regions of brain and in thyroid and testis, and in much lower amounts in other tissues. The PDE10A gene was located on chromosome 6q26 by fluorescent in situ hybridization analysis. PDE10A represents a new member of the PDE superfamily, exhibiting unique kinetic properties and inhibitor sensitivity.Cyclic nucleotides cAMP and cGMP are well known as second messengers and regulate many functions in various tissues (1-4). Intracellular cAMP and cGMP concentrations are controlled via stimulation of adenyl and guanyl cyclases in response to extracellular signaling and their degradation by cyclic nucleotide phosphodiesterases (PDEs), 1 respectively. Many kinds of PDEs are involved in the metabolism of cyclic nucleotides. Based on their amino acid sequence homology, biochemical properties, and inhibitor profiles, seven PDE families have been recognized in mammalian tissues (5, 6). PDE1 is Ca 2ϩ / calmodulin-dependent, hydrolyzing both cAMP and cGMP. PDE2 is stimulated by cGMP and hydrolyzes cAMP and cGMP.PDE3 is cGMP-inhibited. PDE4 is cAMP-specific and rolipramsensitive. PDE5 is cGMP-specific. PDE6 is a photoreceptor cGMP PDE. PDE7 is cAMP-specific and rolipram-insensitive. Very recently, cDNAs encoding two kinds of novel PDEs were isolated from humans and mice (7-11). One is cAMP-specific (PDE8), and the other is cGMP-specific (PDE9). PDE7 and the two latter PDEs are insensitive to the nonspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). cDNA cloning of these novel PDEs was done by an approach using bioinformatics. A search of data bases of expressed sequence tags (ESTs) was performed using parts of PDE sequences such as the catalytic domain. The approach was shown to be effective for the isolation of novel PDE cDNAs.Genome sequencing projects are progressing in many organisms, providing us information of a variety of pathways involved in the cyclic nucleotide metabolism necessary to maintain life. In Caenorhabditis elegans, which is a small soil nematode found in temperate regions, there are five pairs of autosomal chromosomes and a...

cDNAs encoding a novel phosphodiesterase, phosphodiesterase 11A (PDE11A), were isolated by a combination of reverse transcriptase-polymerase chain reaction using degenerate oligonucleotide primers and rapid amplification of cDNA ends. Their catalytic domain was identical to that of PDE11A1 (490 amino acids) reported during the course of this study. However, the cDNAs we isolated had N termini distinct from PDE11A1, indicating two novel N-terminal variants of PDE11A. PDE11A3 cDNA encoded a 684-amino acid protein including one complete and one incomplete GAF domain in the Nterminal region. PDE11A4 was composed of 934 amino acids including two complete GAF domains and shared 630 C-terminal amino acids with PDE11A3 but had a distinct N terminus containing the putative phosphorylation sites for cAMP-and cGMP-dependent protein kinases. PDE11A3 transcripts were specifically expressed in testis, whereas PDE11A4 transcripts were particularly abundant in prostate. Recombinant PDE11A4 expressed in COS-7 cells hydrolyzed cAMP and cGMP with K m values of 3.0 and 1.4 M, respectively, and the V max value with cAMP was almost twice that with cGMP. Although PDE11A3 showed the same K m values as PDE11A4, the relative V max values of PDE11A3 were approximately one-sixth of those of PDE11A4. PDE11A4, but not PDE11A3, was phosphorylated by both cAMPand cGMP-dependent protein kinases in vitro. Thus, the PDE11A gene undergoes tissue-specific alternative splicing that generates structurally and functionally distinct gene products. Cyclic nucleotide phosphodiesterases (PDEs)1 metabolize cAMP and cGMP, which are second messengers regulating many functions in various cells and tissues. Based on their amino acid sequence homology, biochemical properties, and inhibitor profiles, many kinds of PDEs have been identified in mammalian tissues (1-3). The PDE1 family is Ca 2ϩ /calmodulindependent and hydrolyzes both cAMP and cGMP. PDE2 is stimulated by cGMP and hydrolyzes cAMP and cGMP, while PDE3 is cGMP-inhibited. The cAMP-specific and rolipram-sensitive PDEs belong to the PDE4 family. PDE5 is a cGMPbinding, cGMP-specific PDE. The photoreceptor cGMP PDEs are in the PDE6 family. PDE7 is cAMP-specific and rolipraminsensitive. PDE8 is a cAMP-specific PDE, and PDE9 is a cGMP-specific PDE (3-8). Recently, we revealed a new member of the PDE group, PDE10A, which hydrolyzes both cAMP and cGMP (9). Some of these PDEs constitute subfamilies encoded by distinct genes. In each PDE family, alternative splice variants have been reported (1, 10, 11). In many cases, different gene products and alternative splice variants in each PDE family show different expression patterns in tissues and different subcellular localization (1,(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). PDEs encoded by alternatively spliced mRNAs have been reported to differ in their regulation by some kinases including cAMP-dependent protein kinase (cAK) and cGMP-dependent kinase (cGK) and associated proteins (19, 23). Thus, cyclic nucleotide levels are controlled by a complex system. Each ...

cGMP‐binding, cGMP‐specific phosphodiesterase which is encoded by the PDE5A gene plays important roles in cardiovascular system, and is a significant target molecule of therapeutic agents. However, little is known about molecular characteristics of the human PDE5A gene. The 4.4‐kb cDNA encoding human PDE5A was isolated from lung and placenta cDNA libraries. The deduced amino acid sequence analysis demonstrated that N‐terminal amino acid sequence is dissimilar to that of rat PDE5A [Kotera, J., Yanaka, N., Fujishige, K., Imai, Y., Akatsuka, H., Ishizuka, T., Kawashima, K. & Omori, K. (1997) Eur. J. Biochem. 249, 434−442]. Human PDE5A mRNA is produced in high amounts in various tissues such as pancreas, skeletal muscle, placenta, heart, thyroid, adrenal cortex, testis, small intestine and stomach. In addition, the megakaryocyte‐like cell line Dami cells and two types of human vascular smooth muscle cells also produce the mRNA. Over 100‐kb chromosomal DNA corresponding to the human PDE5A gene was isolated and analyzed. The human PDE5A gene was revealed to contain 21 exons. Comparison of genomic organization with the rod photoreceptor phosphodiesterase β‐subunit gene (PDE6B), which is another kind of cGMP‐specific phosphodiesterase, has shown that the PDE5A and PDE6B genes are very similar in their relative exon−intron organization. In particular, the evolutionary relatedness of these genes was suggested in the catalytic domain. Furthermore, chromosomal location of the PDE5A gene was defined as being chromosome 4q26 by fluorescent in situ hybridization analysis.

Psoriasis is a common skin disease characterized by hyperplastic regenerative epidermal growth and infiltration of immunocytes. The etiology of psoriasis is unknown, although several genetic and cellular factors have been elucidated. To find new psoriasis-related genes, we have cloned cDNAs that are differentially expressed between normal and psoriatic skins. Among these clones, we have identified a new gene that codes for a new member of the type IV cytosolic phospholipase A 2 (cPLA 2 ) family. We refer to this gene as cPLA 2 ␦. It encodes a polypeptide of 818 amino acids that has significant homology with known cPLA 2 proteins in the C2 and catalytic domains. The cPLA 2 ␦ gene was mapped to the 15q13-14 chromosomal locus, near to the locus of the cPLA 2 ␤ gene, from which it is separated by a physical distance of about 220 kb. To identify the phospholipase A 2 activity of cPLA 2 ␦, we transfected COS-7 cells with His-tagged cPLA 2 ␦. The cell lysate from these cells had calcium-dependent phospholipase A 2 activity. Northern blot analysis revealed that a cPLA 2 ␦ transcript of about 4 kb is expressed in stratified squamous epithelia, such as those in skin and cervix, but not in other tissues. In situ hybridization and immunohistochemistry revealed that cPLA 2 ␦ is expressed strongly in the upper spinous layer of the psoriatic epidermis, expressed weakly and discontinuously in atopic dermatitis and mycosis fungoides, and not detected in the epidermis of normal skin; cPLA 2 ␣ is not detected in either normal or psoriatic skin. These results suggest that cPLA 2 ␦ exhibits a unique distribution pattern compared with that of known cPLA 2 subtypes, and it may play a critical role in inflammation in psoriatic lesions.

cGMP-dependent protein kinase (cGK) is a major intracellular receptor of cGMP and is implicated in several signal transduction pathways. To identify proteins that participate in the cGMP/cGK signaling pathway, we employed the yeast two-hybrid system with cGK Ialpha as bait. cDNAs encoding slow skeletal troponin T (skTnT) were isolated from both mouse embryo and human skeletal muscle cDNA libraries. The skTnT protein interacted with cGK Ibeta but not with cGK II nor cAMP-dependent protein kinase. The yeast two-hybrid and in vitro binding assays revealed that the N-terminal region of cGK Ialpha, containing the leucine zipper motif, is sufficient for the association with skTnT. In vivo analysis, mutations in cGK Ialpha, which disrupted the leucine zipper motif, were shown to completely abolish the binding to skTnT. Furthermore, cGK I also interacted with cardiac TnT (cTnT) but not with cardiac troponin I (cTnI). Together with the observations that cTnI is a good substrate for cGK I and is effectively phosphorylated in the presence of cTnT in vitro, these findings suggest that TnT functions as an anchoring protein for cGK I and that cGK I may participate in the regulation of muscle contraction through phosphorylation of TnI.