Identification of inhibitor specificity determinants in a mammalian phosphodiesterase

Mammalian phosphodiesterase types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP an d are essential for the regulation of this intracellular second messenger i n many cell types. Whereas these enzymes share structural and biochemical s imilarities, each can be distinguished by its sensitivity to isozyme-specif ic inhibitors. By using a series of chimeric enzymes, we have localized the region of PDE4 that confers sensitivity to selective inhibitors. This inhi bitor specificity domain lies within a short sequence at the carboxyl termi nus of the catalytic domain of the protein, consistent with the competitive nature of inhibition by these compounds, Surprisingly, the identified regi on also includes some of the most highly conserved residues among PDE isofo rms, A yeast-based expression system was used for the isolation and charact erization 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 s ensitivity. In some cases, combined point mutations contribute synergistica lly to the reduction of sensitivity (suppression of IC50). We also report t hat several mutations display differential sensitivity changes with respect to distinct structural classes of inhibitors.