Shepherd Malcolm, McSorley Theresa, Olsen Aileen E, Johnston Lee Ann, Thomson Neil C, Baillie George S, Houslay Miles D, Bolger Graeme B
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, Uk.
Biochem J. 2003 Mar 1;370(Pt 2):429-38. doi: 10.1042/BJ20021082.
We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.
我们已分离出编码磷酸二酯酶4B4（PDE4B4）的cDNA，它是一种具有新特性的新型环磷酸腺苷（cAMP）特异性磷酸二酯酶（PDE4）亚型。PDE4B4的氨基酸序列表明它由PDE4B基因编码，但与先前分离出的PDE4B1、PDE4B2和PDE4B3亚型不同，它有一个由17个氨基酸组成的新的N端区域。PDE4B4包含“长”PDE4亚型所特有的上游保守区域1（UCR1）和UCR2调节单元。核糖核酸酶保护实验表明，PDE4B4 mRNA在肝脏、骨骼肌和大脑的各个区域优先表达，这与其他已知的PDE4B长亚型PDE4B1和PDE4B3的组织分布模式不同。PDE4B4 cDNA在COS7细胞中的表达在变性条件下产生了一种85 kDa的蛋白质。对重组的、COS7细胞表达的PDE4B4进行亚细胞分级分离表明，该蛋白质定位于细胞质中，这通过对转染了绿色荧光蛋白-PDE4B4嵌合体的活COS7细胞进行共聚焦显微镜分析得到证实。PDE4B4对cAMP表现出的米氏常数（K(m)）为5.4 μM以及相对于长PDE4B1亚型的最大反应速度（V(max)）为2.1。PDE4B4被典型的PDE4抑制剂咯利普兰[4-[3-(环戊氧基)-4-甲氧基苯基]-2-吡咯烷酮]抑制，其半数抑制浓度（IC(50)）为83 nM。用福斯可林处理COS7细胞以提高cAMP水平，会导致PDE4B4的激活，这与UCR1内Ser-56位点上PDE4B4的磷酸化有关。PDE4B4独特的组织分布和细胞内定位表明，该亚型可能在调节特定细胞类型中的cAMP水平方面具有独特的功能作用。
