Research ArticleGPCR SIGNALING

Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations

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Science Signaling  09 Oct 2018:
Vol. 11, Issue 551, eaan1188
DOI: 10.1126/scisignal.aan1188

Ultrasensitivity of GPCRs

Most analyses of signaling through G protein–coupled receptors (GPCRs) are performed using nanomolar or micromolar concentrations of ligand. Civciristov et al. found that femtomolar concentrations of ligand activated signaling by the endogenous β2-adrenergic receptor (β2AR) and muscarinic acetylcholine receptor M3 (M3R) in several cell types. Such ultralow concentrations of ligand stimulated signaling that was qualitatively distinct from that elicited by high concentrations and depended on activation of preassembled GPCR complexes. In contrast, high concentrations of ligand elicited signaling through GPCRs that were not part of complexes in addition to those in preassembled complexes. Such qualitative differences in signaling elicited by different ligand concentrations suggest that low doses of GPCR-targeting drugs could have therapeutic effects and may have implications for the mechanisms of action and side effects of these drugs.


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.

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