CHARACTERIZATION OF 2 RECOMBINANT PDE3 (CGMP-INHIBITED CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE) ISOFORMS, RCGIP1 AND HCGIP2, EXPRESSED IN NIH-3006 MURINE FIBROBLASTS AND SF9 INSECT CELLS

CDNAS encoding PDE3 [cGMP-inhibited cyclic nucleotide phosphodiesteras e (cGI PDE)] isoforms, cGIP1 and cGIP2, have been cloned from rat (R) and human (II) cDNA libraries. The deduced amino acid sequences of RcG IP1 and HcGIP2 are very similar in their conserved catalytic domains b ut differ in their N-terminal regulatory domains [Meacci, E., et al. ( 1992) Pi;oc. Natl. Acad. Sci. U.S.A. 89, 3721-3725; Taira, M., ct al. (1993) J. Biol. Chem. 268, 18573-18579]. cDNAs encoding both rat adipo cyte RcGIP1 and human myocardial HcGIP2 (full-length forms and truncat ed forms lacking much of the putative N-terminal domain) were expresse d in NIH 3006 fibroblasts and in Sf9 insect cells. The recombinant pro teins exhibited the expected subunit molecular mass, immunologic react ivities, and characteristics of native membrane-associated forms of th e enzymes, e.g., high affinity for cAMP (K-m), sensitivity to the sele ctive cGI PDE Inhibitors OPC 3689 and OPC 3911 and to cGMP. The full-l ength recombinants were predominantly particulate, whereas the truncat ed HcGIP2 forms were cytosolic suggesting that N-terminal domains cont ain structural determinants important for membrane association. Both f ibroblast RcGIP1 and authentic adipocyte cGI PDE were phosphorylated i i vitro by cAMP-dependent protein kinase; tryptic [P-32]peptides relea sed from rat adipocyte P-32-cGI PDE and P-32-RcGIP1 exhibited identica l electrophoretic profiles suggesting that the same peptides are phosp horylated in both.