Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Sci Signal. 2018 Oct 9;11(551):eaan1188. doi: 10.1126/scisignal.aan1188.
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 β-adrenergic receptor (βAR) and muscarinic acetylcholine M receptor (MR) 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（GPCRs）是细胞表面信号蛋白中最大的一类，参与几乎所有的生理过程，并且是 30%上市药物的靶点。通常，实验系统中使用纳摩尔到微摩尔浓度的配体来激活 GPCRs。我们通过测量 GPCR 刺激的环腺苷酸（cAMP）产生，以高时空分辨率检测到 GPCR 对广泛的配体浓度的反应，从飞摩尔到毫摩尔。数学模型表明，只要一个细胞被一到两个结合事件激活，配体的皮摩尔浓度平均激活了群体中 40%的细胞。此外，细胞系和人心肌成纤维细胞中皮摩尔浓度的内源性β肾上腺素能受体（βAR）和毒蕈碱乙酰胆碱 M 受体（MR）配体的激活分别导致细胞外信号调节激酶（ERK）和胞质蛋白激酶 C（PKC）活性的核易位持续增加，分别。这些反应在空间和时间上与对更高浓度配体的反应不同，导致了不同的细胞蛋白质组特征。这种高度敏感的信号依赖于 GPCR 在质膜上形成预先组装的、更高阶的信号复合物。认识到 GPCR 对神经递质和激素的超低浓度有反应，这挑战了药物作用的既定模式，并提供了 GPCR 激活的一个以前未被重视的方面，与通常观察到的更高浓度配体有很大的不同。
