Isolation and characterization of human cDNAs encoding a cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase.

Human cyclic GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2A3) cDNAs were cloned from hippocampus and fetal brain cDNA libraries. A 4.2-kb composite DNA sequence constructed from overlapping cDNA clones encodes a 941 amino acid protein with a predicted molecular mass of 105,715 Da. Extracts prepared from yeast expressing the human PDE2A3 hydrolyzed both cyclic AMP (cAMP) and cyclic GMP (cGMP). This activity was inhibited by EHNA, a selective PDE2 inhibitor, and was stimulated three-fold by cGMP. Human PDE2A is expressed in brain and to a lesser extent in heart, placenta, lung, skeletal muscle, kidney and pancreas. The human PDE2A3 differs from the bovine PDE2A1 and rat PDE2A2 proteins at the amino terminus but its amino-terminal sequence is identical to the bovine PDE2A3 sequence. The different amino termini probably arise from alternative exon splicing of the PDE2A mRNA.

Identification and characterisation of a human calmodulin-stimulated phosphodiesterase PDE1B1.

A cDNA encoding a calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine "63 kDa" calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated approximately 2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX approximately equal to 8-methoxymethyl-IBMX > vinpocetine approximately equal to zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominately in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.

Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3',5'-cyclic nucleotide phosphodiesterases.

cDNAs corresponding to two human calcium, calmodulin (CaM)-regulated 3',5'-cyclic nucleotide phosphodiesterases (PDEs) were isolated. One, Hcam1 (PDE1A3), corresponds to the bovine 61-kDa CaM PDE (PDE1A2). The second, Hcam3 (PDE1C), represents a novel phosphodiesterase gene. Hcam1 encodes a 535-amino acid protein that differs most notably from the bovine 61-kDa CaM PDE by the presence of a 14-amino acid insertion and a divergent carboxyl terminus. RNase protection studies indicated that Hcam1 is represented in human RNA from several tissues, including brain, kidney, testes, and heart. Two carboxyl-terminal splice variants for Hcam3 were isolated. One, Hcam3b (PDE1C1), encodes a protein 634 amino acids (72 kDa) in length. The other, Hcam3a (PDE1C3), diverges from Hcam3b 4 amino acids from the carboxyl terminus of Hcam3b, and extends an additional 79 amino acids. All the cDNAs isolated for Hcam3a are incomplete; they do not include the 5'-end of the open reading frame. Northern analysis revealed that both splice variants were expressed in several tissues, including brain and heart, and that there may be additional splice variants. Amino-truncated recombinant proteins were expressed in yeast and characterized biochemically. Hcam3a has a high affinity for both cAMP and cGMP and thus has distinctly different kinetic parameters from Hcam1, which has a higher affinity for cGMP than for cAMP. Both PDE1C enzymes were inhibited by isobutylmethylxanthine, 8-methoxymethyl isobutylmethylxanthine, zaprinast, and vinpocetine.

Complex expression pattern of the CEF-4 cytokine in transformed and mitogenically stimulated cells.

The transformation of chicken embryo fibroblasts (CEF) by the Rous sarcoma virus leads to the constitutive expression of the cellular gene designated CEF-4. With specific antisera, we confirmed that transformed cells actively synthesize and secrete a 6 kDa polypeptide corresponding to the CEF-4 gene product. The expression of CEF-4 was investigated by Northern and immunoprecipitation analyses. Upon activation of pp60v-src in cells infected by a ts mutant of RSV, the expression of CEF-4 was biphasic with an early transient and a late constitutive period of expression. CEF-4 was expressed in cells transformed by a variety of oncogenes, but the level of constitutive expression differed quantitatively among transformed cells. Cells transformed by v-myc alone did not express CEF-4. Unlike other members of the interleukin 8 gene family, CEF-4 was induced in response to a broad spectrum of growth factors and inflammatory agents. Dexamethasone repressed the induction of CEF-4 by lipopolysaccharides but had little or no effect on the response to serum or pp60v-src. These data emphasize the complexity of CEF-4 expression and suggest the existence of multiple levels or pathways of CEF-4 regulation in normal and transformed cells.

Dissociation of inositol trisphosphate from diacylglycerol production in Rous sarcoma virus-transformed fibroblasts.

The metabolism of phosphatidylinositol (PI) and related intermediates was studied in uninfected and Rous sarcoma virus-(RSV) infected chicken embryo fibroblasts (CEFs). Cells infected with wild-type RSV exhibited twofold increases in steady-state concentrations of inositol trisphosphate (IP3) and inositol bisphosphate (IP2) as compared to uninfected CEFs. In addition, increased concentrations of IP3 and IP2 were observed in CEFs infected with the RSV temperature-sensitive transformation mutant NY72-4 when maintained at the permissive temperature (35 degrees C) for greater than 24 h. Slight increases were observed in the amounts of inositol lipids in RSV-transformed cells. Phosphoinositol metabolic changes were related to transformation and not to viral infection since CEFs infected with NY72-4, maintained at the nonpermissive temperature (41.5 degrees C), revealed amounts of phosphoinositols similar to that of uninfected cells. CEFs infected with a transformation-defective virus exhibited PI metabolic changes intermediate between those of transformed and nontransformed cells. NY72-4 CEF exhibited no increase in phosphoinositol concentrations before 8 h incubation at 35 degrees C, indicating that the transformation-specific changes in inositol metabolism were a delayed event. Furthermore, inositol turnover was not activated during this time. In contrast to the case of inositol metabolism, significant increases in diacylglycerol (DAG) concentrations were observed within 15-30 min after shift of NY72-4 CEFs to 35 degrees C. These findings suggest that (a) the major changes in inositol metabolism are specific for RSV-transformed cells; (b) transformation-specific changes in phosphoinositol content in RSV-infected CEFs are not an early effect of the expression of pp60v-src; and (c) increases in the DAG content of transformed cells occur before changes in inositol metabolism, indicating that DAG may be derived from other lipid sources.

Molecular analyses of gene products associated with the response of cells to mitogenic stimulation.

We have completed the molecular analysis of a ribosomal protein S6 kinase identified in unfertilized Xenopus eggs. Although some of our results support the notion that an antigenically related enzyme is present in animal cells, we are uncertain if such an enzyme accounts for all of the mitogen-stimulated phosphorylation of 40S subunits. Other experiments have led to the identification of mRNAs that are rapidly expressed in mitogen-stimulated cells. The relevance of such genes and their products is in doubt, however, until a function can be demonstrated for each of them. Preliminary experiments with the pCEF-4 gene product described here have failed to show that it acts as a mitogen for cells in culture, for example. Thus, although studies such as these may lead to the identification of novel genes, we may need to search elsewhere for a physiologically significant function.

Immunologic approaches to the study of cyclic nucleotide phosphodiesterases.

The cyclic nucleotide PDE activity in crude tissue extracts is due to the composite activity of several distinct isozymes, each having different kinetic and functional characteristics. Most of these isozymes will hydrolyze the same substrates, that is, cyclic AMP and cyclic GMP. Therefore, it is difficult to conclusively identify and quantitate any given isozyme in crude systems. Since the cyclic nucleotide PDE are present in low concentrations in cells, it is also difficult to isolate these proteins without having specific solid phase affinity probes. This chapter described the use of monoclonal antibodies as probes to specifically identify, measure, characterize, and isolate the PDE isozymes in impure preparations. Monoclonal antibodies have been produced to the cyclic-GMP-stimulated PDE, ROS PDE, and a calcium-calmodulin-dependent PDE of bovine tissues. A polyclonal antiserum has also been produced to the cyclic-GMP-stimulated PDE. Sensitive immunoprecipitation assays for the measurement of PDE activity and cyclic GMP binding have been developed that have allowed the detection of femtomole amounts of each specific PDE isozyme. None of the antibodies appear to recognize antigenic determinants on other isozymes, suggesting that the PDE are antigenically distinct and therefore different proteins. Most of the monoclonal antibodies do not appear to affect kinetic parameters of the enzymes and have been used to specifically identify, measure, and characterize each isozyme in crude systems. A monoclonal antibody to the calcium-calmodulin-dependent PDE requires calcium and calmodulin for high-affinity binding to the enzyme. This apparent conformational requirement has been used to purify the isozyme from both bovine brain and heart. These two tissues contain enzymes with differing subunit MW on SDS-polyacrylamide gel electrophoresis, although no other biochemical differences were observed.

Identification of cGMP-stimulated cyclic nucleotide phosphodiesterase in lung tissue with monoclonal antibodies.

Monoclonal antibodies (CGS-1, 2, 3, and 4) and rabbit antisera have been produced against bovine heart cyclic GMP-stimulated cyclic nucleotide phosphodiesterase. The monoclonal antibodies were all of the IgG1 subclass and had relatively high affinities (kd values, 1-5 X 10(-10) M), making them suitable for many sensitive immunological and analytical procedures. Most of the monoclonal antibodies did not inhibit the enzyme, which allowed a rapid and specific assay for this form of phosphodiesterase to be developed. All of the activity could be adsorbed from purified preparations of cGMP-stimulated phosphodiesterase by each of the antibodies indicating antigenic homogeneity of the cGMP-stimulated phosphodiesterase. Neither the monoclonal antibodies nor the antisera inhibited cGMP-binding to the phosphodiesterase. Substrate specificity, Electrophoresis, and cyclic GMP binding studies carried out on immunoadsorbed material indicated that a major part of the cyclic nucleotide phosphodiesterase activity present in bovine lung was due to the cyclic GMP-stimulated form. The presence of this enzyme was difficult to identify by other more common procedures and has not been previously demonstrated in this tissue. These studies also indicated that the cGMP-stimulated phosphodiesterase was immunologically distinct from the other major cGMP-binding proteins present in lung extract and that it could be separated from these binding proteins by chromatography on DEAE-cellulose. Finally, the data suggest that none of the other peaks of cyclic nucleotide phosphodiesterase activity, seen upon DEAE-cellulose fractionation of lung extracts, contain the immunological determinants present on the cGMP-stimulated form which are recognized by the antibodies.

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from bovine tissues.

A cGMP-stimulated cyclic nucleotide phosphodiesterase has been purified to near homogeneity from bovine adrenal and heart tissues. The purification procedure utilizes chromatography on DEAE-cellulose and cGMP affinity resin. The procedure can be completed within 2 days and is easily adapted to large scale. To obtain pure enzyme, an 8,000-9,000-fold increase in specific activity was required in adrenal tissues and 15,000-30,000-fold in cardiac muscle. A single band of protein having an apparent Mr = 105,000-107,000 was seen on sodium dodecyl sulfate gel electrophoresis. At equilibrium, native polyacrylamide gradient gel electrophoresis revealed a single major band having an apparent Mr = 240,000. Cyclic GMP binding and phosphodiesterase activity co-migrated with the protein band on native polyacrylamide gradient gels. The enzyme bound cGMP with high affinity reaching a maximum binding of 1.02 mol of cGMP bound/mol of enzyme dimer. Titration curves of the binding data indicated at least two classes of binding sites with 10% maximal binding occurring at 7 nM and 90% maximal binding at 4 microM cGMP. Kinetic analysis indicated the enzyme can hydrolyze both cAMP and cGMP with similar maximal rates. The nucleotide concentration at half-maximal velocity were 30 and 10 microM for cAMP and cGMP, respectively. The hydrolyses of both nucleotides exhibit positive homotropic cooperativity with Hill coefficients of 1.9 for cAMP and 1.3 for cGMP. The rate of cAMP hydrolysis by the purified enzyme when measured at 10 microM cAMP was enhanced 5- to 6-fold by low levels of cGMP.