Identification of inhibitor specificity determinants in a mammalian phosphodiesterase.
Mammalian phosphodiesterase types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger in many cell types. Whereas these enzymes share structural and biochemical similarities, each can be distinguished by its sensitivity to isozyme-specific inhibitors. By using a series of chimeric enzymes, we have localized the region of PDE4 that confers sensitivity to selective inhibitors. This inhibitor specificity domain lies within a short sequence at the carboxyl terminus of the catalytic domain of the protein, consistent with the competitive nature of inhibition by these compounds. Surprisingly, the identified region also includes some of the most highly conserved residues among PDE isoforms. A yeast-based expression system was used for the isolation and characterization of mutations within this area that confer resistance to the PDE4-specific inhibitor rolipram. Analysis of these mutants indicated that both conserved and unique residues are required for isoform-specific inhibitor sensitivity. In some cases, combined point mutations contribute synergistically to the reduction of sensitivity (suppression of IC50). We also report that several mutations display differential sensitivity changes with respect to distinct structural classes of inhibitors.
Atienza
J M
JM
Department of Biological Chemistry and the Molecular Biology Institute, UCLA School of Medicine, Los Angeles, California 90095, USA.
Susanto
D
D
Huang
C
C
McCarty
A S
AS
Colicelli
J
J
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D017931:DNA Primers; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity