ALTERNATIVE SPLICING OF CAMP-SPECIFIC PHOSPHODIESTERASE MESSENGER-RNATRANSCRIPTS - CHARACTERIZATION OF A NOVEL TISSUE-SPECIFIC ISOFORM, RNPDE4A8

In order to characterize the structure and regulation of members of th e cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4 fam ily), we have cloned from the rat a cDNA, pRPDE39, encoding a novel me mber of this family, which we call RNPDE4A8. Sequencing of the pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but to differ from two other PDE4A transcripts, RD1 (pRPDE8; RNPDE4A1) and pRPDE6 (RNPDE 4A5), by the presence of a unique region at its 5' end, consistent wit h alternative mRNA splicing. The pRPDE39 cDNA encodes a predicted prot ein of 763 amino acids, of which all but 21, located at the extreme am ino terminus, are found in the pRPDE6 protein. Expression of pRPDE39 i n COS cells produced a protein of 98 +/- 1.4 kDa, as determined by imm unoblotting with an antiserum specific to the carboxyl-terminal region s of all PDE4A proteins, compared to a predicted value of 87.5 Ma. RNa se protection analysis detected pRPDE39 mRNA only in testis. Immunoblo tting of testis extracts demonstrated two bands of 97 +/- 2 and 87 +/- 3 kDa, the larger of which co-migrated with the band seen in COS cell s expressing pRPDE39, COS cell expressed pRPDE39 partitioned between a high speed pellet (particulate) fraction (15% of protein; 8% of activ ity) and a cytosolic fraction. The particulate fraction had a K-m for cAMP of 3.3 +/- 0.6 mu M, and the cytosolic fraction a K-m of 5.4 +/- 2.8 mu M. The V-max values for the pRPDE39 protein, relative to the RD 1 protein, were 0.16 +/- 0.06 and 0.29 +/- 0.05 for the particulate an d cytosolic forms, respectively. The pRPDE39-encoded PDE activity coul d not be removed from the particulate fraction by high salt concentrat ions, or by nonionic detergents. The pRPDE39-encoded enzyme was inhibi ted by rolipram at an IC50 of 0.5 +/- 0.2 mu M for the particulate for m and 1.0 +/- 0.2 mu M for the cytosolic form, which are values typica l of PDE4 family members. The highly tissue-specific distribution of t he pRPDE39 mRNA suggests that the pRPDE39 protein functions to modulat e a cAMP signaling pathway that is present largely, if not exclusively , in the testis.