Cyclic AMP Control Measured in Two Compartments in HEK293 Cells: Phosphodiesterase K_M Is More Important than Phosphodiesterase Localization

The intracellular second messenger cyclic AMP (cAMP) is degraded by phosphodiesterases (PDE). The knowledge of individual families and subtypes of PDEs is considerable, but how the different PDEs collaborate in the cell to control a cAMP signal is still not fully understood. In order to investigate compartmentalized cAMP signaling, we have generated a membrane-targeted variant of the cAMP Bioluminiscence Resonance Energy Transfer (BRET) sensor CAMYEL and have compared intracellular cAMP measurements with it to measurements with the cytosolic BRET sensor CAMYEL in HEK293 cells. With these sensors we observed a slightly higher cAMP response to adenylyl cyclase activation at the plasma membrane compared to the cytosol, which is in accordance with earlier results from Fluorescence Resonance Energy Transfer (FRET) sensors. We have analyzed PDE activity in fractionated lysates from HEK293 cells using selective PDE inhibitors and have identified PDE3 and PDE10A as the major membrane-bound PDEs and PDE4 as the major cytosolic PDE. Inhibition of membrane-bound or cytosolic PDEs can potentiate the cAMP response to adenylyl cyclase activation, but we see no significant difference between the potentiation of the cAMP response at the plasma membrane and in cytosol when membrane-bound and cytosolic PDEs are inhibited. When different levels of stimulation were tested, we found that PDEs 3 and 10 are mainly responsible for cAMP degradation at low intracellular cAMP concentrations, whereas PDE4 is more important for control of cAMP at higher concentrations.细胞内第二信使环腺苷酸（cyclic AMP, cAMP）可被磷酸二酯酶（phosphodiesterases, PDE）降解。目前学界对PDE各家族及亚型的认知已较为充分，但不同PDE如何在细胞内协同调控cAMP信号，目前仍未完全阐明。为研究区室化cAMP信号转导，我们构建了靶向细胞膜的cAMP生物发光共振能量转移（Bioluminiscence Resonance Energy Transfer, BRET）传感器CAMYEL变体，并将其与胞质型BRET传感器CAMYEL在HEK293细胞中开展胞内cAMP检测对比。借助上述传感器，我们观察到：相较于胞质区域，细胞膜处的腺苷酸环化酶激活所引发的cAMP响应略高，这与此前荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET）传感器的研究结果一致。我们利用选择性PDE抑制剂，对HEK293细胞分级裂解物中的PDE活性进行了分析，鉴定出PDE3与PDE10A为主要的膜结合型PDE，而PDE4为主要的胞质型PDE。抑制膜结合型或胞质型PDE均可增强腺苷酸环化酶激活所引发的cAMP响应，但当同时抑制膜结合型与胞质型PDE时，我们未观察到细胞膜与胞质区域的cAMP响应增强存在显著差异。在测试不同刺激强度的实验中，我们发现：当细胞内cAMP浓度较低时，PDE3与PDE10A主要负责cAMP的降解；而当浓度升高时，PDE4则在cAMP调控中发挥更关键的作用。