Incubation of rat adipocytes with insulin results in phosphorylation and activation of a membrane-associated cGMP-inhibited """"""""low Km"""""""" cAMP phosphodiesterase (cGI PDE). Activation of this specific cGI PDE is an important component of the antilipolytic action of insulin. We are attempting to clone the cDNA for this cGI PDE in order to increase our understanding of its structural/functional relationship and mechanisms for its cellular regulation. cGI PDE from human platelets was purified and partial peptide sequences were obtained. Based on amino acid sequences from the conserved domain in the platelet cGI PDE, mixed oligonucleotide primers were synthesized and a specific cDNA sequence (HH130) was amplified by the polymerase chain reaction from both human cardiac and rat adipose tissue cDNA libraries. The adipose tissue library was screened with the HH130 probe; two types of cDNA clones were obtained. One clone, Rat Fat 2 (1.5 kb) was similar to HH130 and the other, Rat Fat 1 (0.75 kb), was not. Both cDNA clones encoded for the cGI PDE conserved homologous sequence. In Northern blot analysis, Rat fat 1 cDNA hybridized predominantly with a single fat mRNA species, whereas Fat 2 hybridized more strongly with rat heart than rat fat mRNA species.
