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Molecular cloning and expression of a human phosphodiesterase 4C
R J Owens1, S Lumb, K Rees-Milton
1Celltech Therapeutics Ltd., Slough, UK.
Abstract:
A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5' end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating 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 using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. 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 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.
Insights
Researchers identified a novel splice variant of human phosphodiesterase 4C (PDE4C2) from glioblastoma cells. This variant shows different rolipram sensitivity, suggesting multiple active conformations for PDE4 enzymes.
Area of Science:
- Molecular Biology
- Enzymology
- Neuroscience
Background:
- Phosphodiesterase 4 (PDE4) enzymes regulate cyclic nucleotide signaling.
- Glioblastoma cells express various signaling molecules.
- Splice variants can exhibit distinct functional properties.
Purpose of the Study:
- To isolate and characterize a novel human phosphodiesterase 4C (PDE4C) splice variant.
- To investigate the functional differences between splice variants.
- To explore the conformational flexibility of PDE4C.
Main Methods:
- cDNA isolation from U87 glioblastoma cell mRNA.
- Gene sequencing and ORF analysis.
- Transfection into COS 1 and yeast cells.
- Western blotting with PDE4C specific antiserum.
- Enzyme activity assays (cAMP hydrolysis).
- Inhibition studies with rolipram.
- Ligand binding assays with [3H] R rolipram.
Main Results:
- A novel PDE4C splice variant (PDE4C2) was identified with a unique 5' end.
- Expressed PDE4C2 in COS 1 and yeast cells produced an 80 kD polypeptide.
- Enzyme activity was specific for cAMP and inhibited by rolipram.
- COS cell-expressed PDE4C2 showed higher affinity for cAMP (lower Km) and greater sensitivity to R-rolipram compared to yeast-expressed enzyme.
- High-affinity R-rolipram binding was detected on COS cell PDE4C2 but not yeast-expressed enzyme.
Conclusions:
- The human PDE4C gene produces at least two distinct 5' splice variants.
- PDE4C splice variants can adopt different active conformations.
- Conformational differences influence substrate affinity and inhibitor sensitivity, particularly concerning rolipram interactions.