Functions of the N-terminal region of cyclic nucleotide phosphodiesterase 3 (PDE 3) isoforms.
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 ( approximately 50 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-Delta189, MPDE3B, and M3B-Delta196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-Delta321 and M3B-Delta302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-Delta397 and H3A-Delta457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-Delta510 and M3B-Delta604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-Delta196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-Delta607, H3A-Delta721, and M3B-Delta604 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.
Kenan
Y
Y
Pulmonary/Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Murata
T
T
Shakur
Y
Y
Degerman
E
E
Manganiello
V C
VC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D015153:Blotting, Western; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011799:Quinazolines / Q000494:pharmacology; D017384:Sequence Deletion; D012995:Solubility; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D011799:Quinazolines; C049443:lixazinone; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; C517291:PDE3B protein, human; C517289:Pde3a protein, mouse; C517293:Pde3b protein, mouse