title: Impedance responses reveal β₂-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles.
abstract: The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the β₂-adrenergic receptor (β₂AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the β₂AR signaling network revealed the differential contribution of G(s)-, G(i)- and Gβγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca²⁺ in the impedance response and led to the discovery of a novel β₂AR-promoted Ca²⁺ mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of β-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity.
authors.affiliation: Department of Biochemistry, Université de Montréal, Montréal, Quebec, Canada.
authors.forename: Wayne
authors.initials: W
authors.lastname: Stallaert
authors.forename: Jonas F
authors.initials: JF
authors.lastname: Dorn
authors.forename: Emma
authors.initials: E
authors.lastname: van der Westhuizen
authors.forename: Martin
authors.initials: M
authors.lastname: Audet
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D016000:Cluster Analysis; D000242:Cyclic AMP / Q000378:metabolism; D017097:Electric Impedance; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008024:Ligands; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
chemical_list: D008024:Ligands; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP; D048049:Extracellular Signal-Regulated MAP Kinases; D020962:Heterotrimeric GTP-Binding Proteins; D007545:Isoproterenol; D002118:Calcium