Richter Wito, Jin S-L Catherine, Conti Marco
Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA 94305-5317, USA.
Biochem J. 2005 Jun 15;388(Pt 3):803-11. doi: 10.1042/BJ20050030.
Cyclic nucleotide PDE4 (phosphodiesterase 4) inhibitors are being developed as potent anti-inflammatory drugs for use in chronic lung diseases, but the complexity of the PDE4 family has hampered this process. The four genes comprising the PDE4 family, PDE4A, PDE4B, PDE4C and PDE4D, are all expressed as multiple splice variants. The most widely used criterion to identify PDE4 variants expressed endogenously is their migration on SDS/PAGE. However, when a PDE4D3-selective antibody was used for immunoprecipitation, the pattern of expression obtained did not confirm the expression predicted by SDS/PAGE. This observation, together with the recent discovery of additional PDE4D transcripts, prompted us to re-evaluate the pattern of expression of these variants. The nine rat PDE4D splice variants, PDE4D1 to PDE4D9, were cloned, their electrophoretic properties compared, and their in vivo mRNA and protein levels determined. Using this approach, we found that the pattern of distribution of the PDE4D splicing variants is more complex than previously reported. Multiple variants co-migrate in single immunoreactive bands, and variant-selective antibodies were necessary to discriminate between splice variants. Tissues that were thought to express only PDE4D3, express three closely related proteins, with PDE4D8 and PDE4D9 as the predominantly expressed forms. In addition, activation of cAMP signalling produces phosphorylation and activation of variants other than PDE4D3, and expression of PDE4D mRNA does not always correlate with the pattern of protein expression. As PDE4 inhibitors have different affinities for distinct PDE4D splicing variants, our results indicate that a better definition of the pattern of PDE4 expression is required for target validation.
环核苷酸磷酸二酯酶4（PDE4）抑制剂正被开发为用于慢性肺部疾病的强效抗炎药物，但PDE4家族的复杂性阻碍了这一进程。构成PDE4家族的四个基因，即PDE4A、PDE4B、PDE4C和PDE4D，均表达为多种剪接变体。鉴定内源性表达的PDE4变体最广泛使用的标准是它们在SDS/PAGE上的迁移情况。然而，当使用PDE4D3选择性抗体进行免疫沉淀时，获得的表达模式并未证实SDS/PAGE预测的表达情况。这一观察结果，连同最近发现的其他PDE4D转录本，促使我们重新评估这些变体的表达模式。克隆了九个大鼠PDE4D剪接变体，即PDE4D1至PDE4D9，比较了它们的电泳特性，并测定了它们在体内的mRNA和蛋白质水平。使用这种方法，我们发现PDE4D剪接变体的分布模式比以前报道的更为复杂。多个变体在单个免疫反应条带中共迁移，需要变体选择性抗体来区分剪接变体。原本认为只表达PDE4D3的组织，实际上表达三种密切相关的蛋白质，其中PDE4D8和PDE4D9是主要表达形式。此外，cAMP信号的激活会导致除PDE4D3之外的变体发生磷酸化和激活，并且PDE4D mRNA的表达并不总是与蛋白质表达模式相关。由于PDE4抑制剂对不同的PDE4D剪接变体具有不同的亲和力，我们的结果表明，为了进行靶点验证，需要更好地定义PDE4的表达模式。
