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 Gs-, Gi- 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.
Highlights
G protein-coupled receptors (GPCRs) are the most abundant class of cell surface receptors, responding to various types of endogenous stimuli, including hormones, neurotransmitters and odorants, and are the largest family of therapeutic targets [1,2]
We demonstrate that impedance responses provide an integrative assessment of the cellular consequence to GPCR stimulation, representing a holistic readout of the various signaling events elicited in real-time
The observation that elements of the impedance response that were blocked upon Gs or Gi inactivation following chronic treatment with cholera toxin (CTX) and pertussis toxin (PTX), respectively, could not be fully recapitulated by acute pharmacological inhibition of cAMP or ERK1/2 pathways suggests the contribution of additional G protein-dependent signaling
Summary
G protein-coupled receptors (GPCRs) are the most abundant class of cell surface receptors, responding to various types of endogenous stimuli, including hormones, neurotransmitters and odorants, and are the largest family of therapeutic targets [1,2]. Even more striking is the inversion of efficacy observed for some b-adrenergic receptor ligands, which act as inverse agonists for cAMP production yet behave as agonists for the activation of the ERK1/2 MAPK pathways [11,12] Harnessing such ligand functional selectivity could represent a promising avenue in drug development, as the design of compounds that selectively modulate a pathway involved in a given pathology without collateral effects on other pathways could provide therapeutic benefit with a decreased risk of side effects. A string of recent studies has explored the use of such integrative assays to study GPCR pharmacology using label-free, cell-based technologies developed to monitor real-time changes in higher-order cellular events such as morphology, viability, adhesion and mass distribution [16,17,18,19,20]. We performed an unsupervised clustering analysis that permitted a rapid and systematic classification of compounds based on the impedance signatures obtained in both heterologous and primary cell cultures, demonstrating that impedance responses represent signatures that are predictive of the global signaling profiles of b-adrenergic ligands previously shown to display functional selectivity [10,11]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
