Agonist-Directed Desensitization of the β2-Adrenergic Receptor

Abstract

The β2-adrenergic receptor (β2AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β2AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β2AR desensitization at the whole cell level.

Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors

“…3). Given that the DMR is a readout of cell signaling events under non-equilibrium condition (Deng et al, 2012; Goral, Jin et al, 2011; Goral, Wu et al, 2011), and different DMR events/pathways have different kinetics (Fang et al, 2007; Tran & Fang, 2008), these results suggest that different agonists may have distinct biased agonism. Of note, we cannot rule out the possibility that the binding kinetics, in particular on-rates, may also contribute to this.…”

“…The off-rates of agonists, in particular those that cannot get into and accumulate inside the cytoplasm of cells through a mechanism (e.g., transporter) other than the endocytosis process, are expected to have more direct impact on receptor behaviors once being internalized. As a matter of fact, the recent agonist removal studies using the perfusion approach showed that certain signaling can propagate even after agonist removal, but some require the agonist occupancy (Goral, Jin et al, 2011); and the whole cell responses of long-acting β 2 -adrenergic receptor agonists, but not the short-acting ones, are insensitive to the agonist removal, suggesting that the agonist residence time is important for its signaling (Calebiro et al, 2010; Goral, Jin et al, 2011). Nonetheless, the present study provides extra evidence supporting the binding kinetics, particularly the drug residence time, as a plausible molecular mechanism defining the whole cell phenotypic efficacy of GPCR ligands.…”

“…The rapid onset DMR responses of many agonists for almost all GPCRs tested to date suggest that receptor signaling proceeds right after agonist binding but long before reaching equilibrium binding (Fang, Li, & Ferrie, 2007); and some, but not all, of signaling events downstream the activated receptor can continue propagating after agonist removal (Goral, Jin et al, 2011; Goral, Wu, Sun, & Fang, 2011). Furthermore, the ability of several antagonists to block endogenous M 3 receptor signaling in HT-29 cells has been recently found to correlate with their residence time (the reciprocal of K off ) (Deng, Wang, Su, & Fang, 2012).…”

Fluticasone propionate/formoterol: A fixed-combination therapy with flexible dosage

“…It is known that partial agonists are generally more sensitive to the reduction of functional receptors than full agonists [44,48]. In fact, low-efficacy ligands are less able to activate the receptor and may not be sufficient to generate a full response, even when bound to all available receptors.…”

Medical emergencies: pulmonary embolism and acute severe asthma

“…Tachyphylaxis to b 2 -adrenoceptor agonists (given in isolation) occurs rapidly and cannot be overcome by administration of higher doses [86]. b 2 -adrenoceptor agonists cause tachycardia, hypokalaemia, hyperglycaemia and lactic acid production [87].…”