Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term “buffered diffusion.” With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.

细胞仅使用少数第二信使（如 cAMP）将大量细胞外信号传递给特定的细胞反应。为了解释信号特异性，有人建议使用 cAMP 降解磷酸二酯酶 (PDE) 将 cAMP 限制在不同的细胞区室中。然而，快速 cAMP 扩散和缓慢 PDE 活动的测量速率使得 cAMP 区室化基本上不可能。使用荧光光谱，我们表明，与早期数据相反，生理浓度的 cAMP 主要与 cAMP 结合位点结合，因此是固定的。结合和解除结合导致 cAMP 动力学大大降低，我们称之为“缓冲扩散”。由于大部分 cAMP 被缓冲，PDE 可以创建低 cAMP 浓度的纳米级域。使用 FRET-cAMP 纳米尺，我们直接绘制了 PDE 分子周围的纳米级 cAMP 梯度以及由此产生的 cAMP 依赖性蛋白激酶 (PKA) 下游激活的区域。我们的研究表明，时空 cAMP 信号受到纳米尺寸域的精确控制，这些域由 PDE 形成，这些域对下游效应器的激活进行门控。