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

Abstract

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.

Signalling in response to sub‐picomolar concentrations of active compounds: Pushing the boundaries of GPCR sensitivity

“…Interestingly, this ultra‐sensitive response followed a biphasic concentration–response relationship: An initial large contraction was observed at concentrations of 1–10 aM Ang II, followed by a plateau phase with no further increase in tone, and finally, a further gradual increase in tone from 1 pM to 0.1 μM Ang II (Goldberg et al, ). This mimics the ultra‐sensitive biphasic cAMP concentration–response curves we have observed for nine other GPCR families, which extend over a similarly wide concentration range spanning 13 orders of magnitude (Civciristov et al, ; Halls & Cooper, ). Biphasic concentration–response curves to Ang II were also observed in approximately 35% of endothelium‐intact rabbit aortic rings, with the initial threshold of contraction observed at 0.1 fM Ang II (Kuttan & Sim, ).…”

“…The AT 2 receptor was found to co‐immunoprecipitate with Gα s proteins under basal conditions (Axelband et al, ). This is particularly interesting, as we have shown that responses to femtomolar concentrations of relaxin, isoprenaline, and carbachol are all dependent on the pre‐assembly of a multi‐component protein complex (that includes G proteins) at the plasma membrane (Civciristov et al, ; Halls & Cooper, ). The precise regulation of Ca 2+ ‐ATPase activity and therefore the texture of the resultant Ca 2+ oscillations in renal proximal tubule cells play an important role in proximal fluid reabsorption (Du, Ferguson, & Wang, ).…”

“…We have recently demonstrated that the β 2 ‐adrenoceptor endogenously expressed in HEK293 cells and human cardiac fibroblasts also responds to ultra‐low concentrations of isoprenaline (Civciristov et al, ). Femtomolar concentrations of isoprenaline caused a sustained increase in cAMP and a sustained increase in nuclear ERK activity (Civciristov et al, ). This contrasted with the temporal profile of signalling observed after activation with high concentrations of isoprenaline (100 nM) as these responses were transient (Civciristov et al, ).…”

“…Activation of the muscarinic M 3 receptor endogenously expressed in HEK293 cells and human cardiac fibroblasts by carbachol causes a concentration‐dependent change in cAMP (attomolar–micromolar) with an increase in cAMP following application of concentrations as low as 10 aM (Civciristov et al, ). The response in HEK293 cells was dependent on the constitutive assembly of a protein complex associated with the M 3 receptor at the plasma membrane (Civciristov et al, ). Activation of cAMP occurred via a Gα q –Gβγ–PKC–AC 2 pathway dependent on AKAP79 (A‐kinase anchor protein 79) and β‐arrestins, and the increased cAMP was tonically opposed by PKA‐activated PDE4D3 (Civciristov et al, ).…”

“…Interestingly, in the majority of cases, these observations come from endogenous, and not overexpression, systems. Here, we aim to describe the evidence for this ultra‐sensitive signalling to reveal that this is not limited to a few family A receptors (Civciristov et al, ; Halls & Cooper, ). Rather, many prototypical and well‐studied GPCRs can respond to attomolar/femtomolar concentrations of ligand.…”