Bolger G B, McPhee I, Houslay M D
Veterans Affairs Medical Center, Salt Lake City, Utah, USA.
J Biol Chem. 1996 Jan 12;271(2):1065-71. doi: 10.1074/jbc.271.2.1065.
In order to characterize the structure and regulation of members of the cAMP-specific phosphodiesterase (PDE) family (Type IV PDEs; PDE4 family), we have cloned from the rat a cDNA, pRPDE39, encoding a novel member of this family, which we call RNPDE4A8. Sequencing of the pRPDE39 cDNA shows it to be encoded by the rat PDE4A gene, but to differ from two other PDE4A transcripts, RD1 (pRPDE8; RNPDE4A1) and pRPDE6 (RNPDE4A5), by the presence of a unique region at its 5' end, consistent with alternative mRNA splicing. The pRPDE39 cDNA encodes a predicted protein of 763 amino acids, of which all but 21, located at the extreme amino terminus, are found in the pRPDE6 protein. Expression of pRPDE39 in COS cells produced a protein of 98 +/- 1.4 kDa, as determined by immunoblotting with an antiserum specific to the carboxyl-terminal regions of all PDE4A proteins, compared to a predicted value of 87.5 kDa. RNase protection analysis detected pRPDE39 mRNA only in testis. Immunoblotting of testis extracts demonstrated two bands of 97 +/- 2 and 87 +/- 3 kDa, the larger of which co-migrated with the band seen in COS cells expressing pRPDE39. COS cell expressed pRPDE39 partitioned between a high speed pellet (particulate) fraction (15% of protein; 8% of activity) and a cytosolic fraction. The particulate fraction had a Km for cAMP of 3.3 +/- 0.6 microM, and the cytosolic fraction a Km of 5.4 +/- 2.8 microM. The Vmax values for the pRPDE39 protein, relative to the RD1 protein, were 0.16 +/- 0.06 and 0.29 +/- 0.05 for the particulate and cytosolic forms, respectively. The pRPDE39-encoded PDE activity could not be removed from the particulate fraction by high salt concentrations, or by nonionic detergents. The pRPDE39-encoded enzyme was inhibited by rolipram at an IC50 of 0.5 +/- 0.2 microM for the particulate form and 1.0 +/- 0.2 microM for the cytosolic form, which are values typical of PDE4 family members. The highly tissue-specific distribution of the pRPDE39 mRNA suggest that the pRPDE39 protein functions to modulate a cAMP signaling pathway that is present largely, if not exclusively, in the testis.
为了表征环磷酸腺苷（cAMP）特异性磷酸二酯酶（PDE）家族（IV型PDE；PDE4家族）成员的结构和调控，我们从大鼠中克隆了一个cDNA，即pRPDE39，它编码该家族的一个新成员，我们将其称为RNPDE4A8。pRPDE39 cDNA的测序表明它由大鼠PDE4A基因编码，但与另外两个PDE4A转录本RD1（pRPDE8；RNPDE4A1）和pRPDE6（RNPDE4A5）不同，其5'端存在一个独特区域，这与选择性mRNA剪接一致。pRPDE39 cDNA编码一个预测的763个氨基酸的蛋白质，除了位于极端氨基末端的21个氨基酸外，其余氨基酸都存在于pRPDE6蛋白质中。用针对所有PDE4A蛋白质羧基末端区域的抗血清进行免疫印迹分析，结果显示在COS细胞中表达的pRPDE39产生了一种98±1.4 kDa的蛋白质，而预测值为87.5 kDa。核糖核酸酶保护分析仅在睾丸中检测到pRPDE39 mRNA。睾丸提取物的免疫印迹显示有两条带，分子量分别为97±2 kDa和87±3 kDa，其中较大的一条带与在表达pRPDE39的COS细胞中看到的带迁移情况相同。COS细胞中表达的pRPDE39分布在高速沉淀（颗粒）部分（占蛋白质的15%；占活性的8%）和胞质部分之间。颗粒部分对cAMP的Km值为3.3±0.6 microM，胞质部分的Km值为5.4±2.8 microM。相对于RD1蛋白质，pRPDE39蛋白质在颗粒形式和胞质形式下的Vmax值分别为0.16±0.06和0.29±0.05。高盐浓度或非离子去污剂不能从颗粒部分去除pRPDE39编码的PDE活性。pRPDE39编码的酶被咯利普兰抑制，颗粒形式的IC50为0.5±0.2 microM，胞质形式的IC50为1.0±0.2 microM，这些值是PDE4家族成员的典型值。pRPDE39 mRNA高度组织特异性的分布表明，pRPDE39蛋白质的功能是调节一个cAMP信号通路，该通路如果不是仅存在于睾丸中，也是主要存在于睾丸中。
