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 doma in with six predicted transmembrane helices, and region 2 (amino acids 301- 500), with a smaller hydrophobic domain (similar to 50 residues). To analyz e 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-Delta 189, MPDE3B, and M3B-Delta 196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-Delta 321 and M3B-Delta 302, containing regio n 2, were recovered essentially equally in particulate and cytosolic fracti ons. H3A-Delta 397 and H3A-Delta 457, lacking both hydrophobic domains, wer e predominantly cytosolic. H3A-Delta 510 and M3B-Delta 604, lacking both re gions 1 and 2, were virtually completely cytosolic. M3B-Delta 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-Delt a 607, H3A-Delta 721, and M3B-Delta 604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixaz inone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with in tracellular membranes, as well for self-association or aggregation during g el filtration and sensitivity to a specific inhibitor.