Functions of the N-terminal Region of Cyclic Nucleotide Phosphodiesterase 3 (PDE 3) Isoforms

Abstract

The N-terminal portion of phosphodiesterase (PDE) 3 was arbitrarily divided into region 1 (amino acids 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, and region 2 (amino acids 301-500), with a smaller hydrophobic domain (ϳ50 residues). To analyze these regions, full-length human (H)PDE3A and mouse (M)PDE3B and a series of N-terminal truncated mutants were synthesized in Sf9 cells. Activities of HPDE3A, H3A-⌬189, MPDE3B, and M3B-⌬196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-⌬321 and M3B-⌬302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-⌬397 and H3A-⌬457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-⌬510 and M3B-⌬604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-⌬196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-⌬607, H3A-⌬721, and M3B-⌬604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixazinone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with intracellular membranes, as well for self-association or aggregation during gel filtration and sensitivity to a specific inhibitor.

Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase (PDE1) Is a Pharmacological Target of Differentiation-Inducing Factor-1, an Antitumor Agent Isolated from Dictyostelium

“…Baculoviruses of the human full-length PDE3A, human PDE3A NH 2 -terminal deletion mutant (amino acids 511-1141; Ref. 19), human full-length PDE3B (20), and rat PDE3B NH 2 -terminal deletion mutant (amino acids 577-1108; Ref. 21) were gifts of Dr. V. C. Manganiello (NIH, Bethesda, MD).…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…PDE3A and PDE3B, which are products of separate genes, are widely expressed and involved in many processes. They are coexpressed in many cells but also have unique localizations (189,213,236,286,340,364). These PDEs have similar affinities for cAMP and cGMP, but the rate of cGMP hydrolysis is ϳ10% that of cAMP; cGMP can compete with and inhibit cAMP breakdown at the catalytic site.…”

“…2) PDE3 contains two regions [NH 2 -terminal hydrophobic regions (NHR1 and NHR2)] that provide for membrane association. Forms of PDE3A lacking NHR domains are largely cytosolic (89,189,340). 3) PDE4A1 contains a NH 2 -terminal domain that interacts with phosphatidic acid and provides for membrane insertion (17,50,131,153,158,297,298).…”

Isoforms of Cyclic Nucleotide Phosphodiesterase PDE3A in Cardiac Myocytes

“…NHR1 (aa Ϸ60 -255) contains transmembrane helices, whereas NHR2 (aa Ϸ340 -400) appears to be involved in the binding of PDE3 to as yet unidentified membrane proteins. rtPDE3As containing NHR1 localize exclusively to intracellular membranes in transfected cells, whereas rtPDE3As containing NHR2 but not NHR1 are found in both the cytosol and intracellular membranes of transfected cells (3,4). Between NHR1 and NHR2 are sites for phosphorylation and activation by PK-B (site "P1," Ser 292 in PDE3A ORF) and PK-A (site "P2," Ser 312 ) (5-7).…”