THE STRUCTURE OF A BOVINE LUNG CGMP-BINDING, CGMP-SPECIFIC PHOSPHODIESTERASE DEDUCED FROM A CDNA CLONE

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-speci fic phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucl eotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair P CR product was recovered and used as a probe to screen a bovine lung c DNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB -PDE was isolated. The open reading frame of this cDNA predicted an 87 5 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cG B-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequ ence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE co ntained a segment (AA 578-812) that was homologous to the putative cat alytic region conserved among all mammalian PDEs and a segment (AA 142 -526) that was homologous to the putative cGMP binding region of the c GMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the put ative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein k inase.