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Impedance responses reveal ??-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 ??-adrenergic receptor (??AR) 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 ??AR signaling network revealed the differential contribution of G(s)-, G(i)- and G??-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 ??AR-promoted Ca²? mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of ?-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.

SUBMITTER: Stallaert W

PROVIDER: S-EPMC3252315 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Impedance responses reveal β₂-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles.

Stallaert Wayne W Dorn Jonas F JF van der Westhuizen Emma E Audet Martin M Bouvier Michel M

PloS one 20120105 1

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 prototypica ...[more]

PMID: 22242170

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Project description:Prolonged exposure to opioid receptor agonists triggers adaptations in the adenylyl cyclase (AC) pathway that lead to enhanced production of cyclic adenosine monophosphate (cAMP) upon withdrawal. This cellular phenomenon contributes to withdrawal symptoms, hyperalgesia and analgesic tolerance that interfere with clinical management of chronic pain syndromes. Since δ-opioid receptors (DOPrs) are a promising target for chronic pain management, we were interested in finding out if cell-based signaling profiles as generated for drug discovery purposes could inform us of the ligand potential to induce sensitization of the cyclase path. For this purpose, signaling of DOPr agonists was monitored at multiple effectors. The resulting signaling profiles revealed marked functional selectivity, particularly for Met-enkephalin (Met-ENK) whose signaling bias profile differed from those of synthetic ligands like SNC-80 and ARM390. Signaling diversity among ligands was systematized by clustering agonists according to similarities in E max and Log(τ) values for the different responses. The classification process revealed that the similarity in Gα/Gβγ, but not in β-arrestin (βarr), responses was correlated with the potential of Met-ENK, deltorphin II, (d-penicillamine2,5)-enkephalin (DPDPE), ARM390, and SNC-80 to enhance cAMP production, all of which required Ca2+ mobilization to produce this response. Moreover, superactivation by Met-ENK, which was the most-effective Ca2+ mobilizing agonist, required Gαi/o activation, availability of Gβγ subunits at the membrane, and activation of Ca2+ effectors such as calmodulin and protein kinase C (PKC). In contrast, superactivation by (N-(l-tyrosyl)-(3S)-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-l-phenylalanyl-l-phenylalanine (TIPP), which was set in a distinct category through clustering, required activation of Gαi/o subunits but was independent of the Gβγ dimer and Ca2+ mobilization, relying instead on Src and Raf-1 to induce this cellular adaptation.

Dataset Information

Impedance responses reveal ??-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles.

Publications

Impedance responses reveal β₂-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles.

Similar Datasets

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