Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists

• Published in: Communications biology Vol. 7; no. 1; p. 417
• Main Authors: Cullum, Sean A., Platt, Simon, Dale, Natasha, Isaac, Oliver C., Wragg, Edward S., Soave, Mark, Veprintsev, Dmitry B., Woolard, Jeanette, Kilpatrick, Laura E., Hill, Stephen J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 05.04.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 631/154/436/2387; 631/92/436/2388; 96; 96/1; 96/10; 96/35; 96/95; Adrenergic receptors; Biomedical and Life Sciences; Biosensors; Cell surface; Cell surface receptors; Cyclic AMP; G protein-coupled receptors; Glycosylation; Inverse agonists; Life Sciences; Mechanical stimuli; N-Terminus; Propranolol; Site-directed mutagenesis
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-024-06128-2

The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce β 2 -adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant β 2 -adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the β 2 -adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A 2A -adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists. Use of a cAMP biosensor to study real-time mechano-sensitive enhancement of β2-adrenoceptor constitutive activity and its inhibition by β2-inverse agonists in HEK 293 cells.