McHale M M, Cieslinski L B, Eng W K, Johnson R K, Torphy T J, Livi G P
Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Mol Pharmacol. 1991 Feb;39(2):109-13.
The low-Km cAMP-specific phosphodiesterases (PDEases) are of great pharmacological significance because of their involvement in regulating cAMP concentrations, which, in turn, are responsible for mediating the cellular response to extracellular signals such as hormones and neurotransmitters. We recently reported the isolation of a cDNA clone that encodes a human monocyte low-Km, rolipram-sensitive, cAMP PDEase (isozyme IV). We have engineered the inducible expression of this human PDEase in yeast. Cells of Saccharomyces cerevisiae contain two genes that encode cAMP PDEases. PDEase-deficient mutants are viable but exhibit specific growth arrest phenotypes associated with elevated intracellular cAMP content; these phenotypes include heat shock sensitivity and the inability to grow on acetate as a carbon source. We show that functional expression of our human cAMP PDEase in a genetically engineered PDEase-deficient strain of S. cerevisiae reverses these aberrant phenotypes. Furthermore, under conditions for growth arrest, rolipram is cytotoxic to PDEase-deficient mutants expressing the human cAMP PDEase, indicating that it is capable of inhibiting the human recombinant enzyme in vivo. This system can be used in the development of a yeast cell-based assay for isozyme-selective inhibitors of the human recombinant cAMP PDEase.
低Km的环磷酸腺苷（cAMP）特异性磷酸二酯酶（PDE酶）具有重要的药理学意义，因为它们参与调节cAMP浓度，而cAMP浓度又负责介导细胞对激素和神经递质等细胞外信号的反应。我们最近报道了一个编码人单核细胞低Km、对咯利普兰敏感的cAMP PDE酶（同工酶IV）的cDNA克隆的分离。我们已经构建了该人PDE酶在酵母中的诱导表达。酿酒酵母细胞含有两个编码cAMP PDE酶的基因。PDE酶缺陷型突变体是可行的，但表现出与细胞内cAMP含量升高相关的特定生长停滞表型；这些表型包括热休克敏感性以及无法以乙酸盐作为碳源生长。我们表明，我们的人cAMP PDE酶在基因工程改造的酿酒酵母PDE酶缺陷型菌株中的功能性表达可逆转这些异常表型。此外，在生长停滞条件下，咯利普兰对表达人cAMP PDE酶的PDE酶缺陷型突变体具有细胞毒性，表明它能够在体内抑制人重组酶。该系统可用于开发基于酵母细胞的人重组cAMP PDE酶同工酶选择性抑制剂检测方法。
