Department of Physiology, Anatomy and Genetics and BHF Centre of Research Excellence, University of Oxford, Oxford, UK.
Sci Rep. 2017 Oct 26;7(1):14090. doi: 10.1038/s41598-017-13021-y.
cAMP is a ubiquitous second messenger responsible for the cellular effects of multiple hormones and neurotransmitters via activation of its main effector, protein kinase A (PKA). Multiple studies have shown that the basal concentration of cAMP in several cell types is about 1 μM. This value is well above the reported concentration of cAMP required to half-maximally activate PKA, which measures in the 100-300 nM range. Several hypotheses have been suggested to explain this apparent discrepancy including inaccurate measurements of intracellular free cAMP, inaccurate measurement of the apparent activation constant of PKA or shielding of PKA from bulk cytosolic cAMP via localization of the enzyme to microdomains with lower basal cAMP concentration. However, direct experimental evidence in support of any of these models is limited and a firm conclusion is missing. In this study we use multiple FRET-based reporters for the detection of cAMP and PKA activity in intact cells and we establish that the sensitivity of PKA to cAMP is almost twenty times lower when measured in cell than when measured in vitro. Our findings have important implications for the understanding of compartmentalized cAMP signalling.
cAMP 是一种普遍存在的第二信使，通过激活其主要效应物蛋白激酶 A（PKA），负责多种激素和神经递质的细胞效应。多项研究表明，在几种细胞类型中，cAMP 的基础浓度约为 1μM。这个值远高于报道的 cAMP 浓度，该浓度需要达到 PKA 的半最大激活，范围在 100-300nM 之间。已经提出了几种假设来解释这种明显的差异，包括细胞内游离 cAMP 的不准确测量、PKA 的表观激活常数的不准确测量，或通过将酶定位到具有较低基础 cAMP 浓度的微区来屏蔽 PKA 与胞质溶胶中 bulk cAMP 的相互作用。然而，支持这些模型中任何一个模型的直接实验证据是有限的，因此缺乏明确的结论。在这项研究中，我们使用了多种基于 FRET 的报告分子来检测完整细胞中的 cAMP 和 PKA 活性，并确定在细胞内测量时，PKA 对 cAMP 的敏感性比在体外测量时低近二十倍。我们的发现对理解有分隔的 cAMP 信号转导具有重要意义。
