G protein–coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β₂-adrenergic receptor (β₂AR) and muscarinic acetylcholine M₃ receptor (M₃R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal–regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations.

G蛋白偶联受体（GPCR）是细胞表面信号蛋白中最大的一类，几乎参与所有生理过程，并且是30％上市药物的目标。通常，在实验系统中，使用纳摩尔浓度至微摩尔浓度的配体来激活GPCR。我们通过以高空间和时间分辨率测量GPCR刺激的环状单磷酸腺苷（cAMP）的生产，检测到了从大分子到毫摩尔的多种配体浓度的GPCR反应。数学模型表明，飞摩尔浓度的配体平均激活群体中40％的细胞，前提是该细胞被一到两次结合事件激活。此外，内源性β的活化₂肾上腺素能受体（β ₂AR）和毒蕈碱型乙酰胆碱M ₃受体（M ₃R）由细胞系和人心脏成纤维细胞中飞摩尔浓度的配体引起，分别导致胞外信号调节激酶（ERK）和胞质蛋白激酶C（PKC）活性的核易位持续增加。这些反应在空间和时间上不同于那些响应于更高浓度的配体而发生的反应，并导致不同的细胞蛋白质组学特征。这种高度敏感的信号转导依赖于GPCR在质膜上形成预组装的高阶信号转导复合物。认识到GPCR对超低浓度的神经递质和激素有反应，从而挑战了已建立的药物作用范式，并提供了GPCR激活之前未曾认识到的方面，这与通常在较高配体浓度下观察到的情况截然不同。