Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDES) - Two N-terminal, domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reti culum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both f ound almost exclusively in the ER. The N-terminal portion of PDE3 can be ar bitrarily divided into region 1 (aa 1-300), which contains a large hydropho bic domain with six predicted transmembrane helices, followed by region 2 ( aa 301-500) containing a smaller hydrophobic domain (of similar to 50 aa). To investigate the role of regions 1 and 2 in membrane association, we exam ined the subcellular localization of a series of catalytically active, Flag -tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as we ll as a series of fragments from regions 1 and 2 of MPDESB synthesized as e nhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 c ells, the localization of a mutant HPDE3A, lacking the first 189 amino acid s (aa) and therefore four of the six predicted transmembrane helices (H3A-D elta 189), was virtually identical to that of the wild type. R3B-Delta 302 (lacking region 1) and H3A-Delta 397 (lacking region 1 as well as part of r egion 2) retained, to different degrees, the ability to associate with memb ranes, albeit less efficiently than H3A-Delta 189. Proteins that lacked bot h regions I and 2, H3A-Delta 510 and M3B-Delta 604, did not associate with membranes. Consistent with these findings, region I EGFP-MPDE3B fusion prot eins colocalized with the ER, whereas region 2 EGFP fusion proteins were di ffusely distributed. Thus, some portion of the N-terminal hydrophobic domai n in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDES.