MOLECULAR-CLONING AND EXPRESSION OF A HUMAN PHOSPHODIESTERASE 4C

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated f rom the mRNA pre pared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypept ide. The sequence differed at the 5' end from the human PDE4C previous ly reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicat ing that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5' splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting u sing a PDE4C specific antiserum. The activity of cell lysates was typi cal of PDE4 being specific for cAMP and inhibitable by the selective i nhibitor, rolipram. However, the K-m for cAMP of the enzyme produced i n COS cells was 0.6 mu M compared to 2.6 mu M for the yeast 4C activit y. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 n M). This difference in rolipram sensitivity was associated with the de tection of a high affinity [H-3] R rolipram binding site on the COS ce ll 4C enzyme but not on the yeast expressed enzyme. The results indica te that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram. (C) 1997 Elsevie r Science Inc.