Factors influencing biased agonism in recombinant cells expressing the human α1A-adrenoceptor

Edilson Dantas da Silva Junior, Masaaki Sato, Jon Merlin, Natalie Broxton, Dana S. Hutchinson, Sabatino Ventura, Bronwyn A. Evans, Roger J. Summers

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Background and Purpose: Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α1A-adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. Experimental Approach: Intracellular Ca2+ mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. Key Results: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. Conclusion and Implications: We have shown that while adrenergic agonists display bias at human α1A-adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.

Original languageEnglish
Pages (from-to)2318-2333
Number of pages16
JournalBritish Journal of Pharmacology
Volume174
Issue number14
DOIs
Publication statusPublished - 1 Jul 2017

Cite this

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title = "Factors influencing biased agonism in recombinant cells expressing the human α1A-adrenoceptor",
abstract = "Background and Purpose: Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α1A-adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. Experimental Approach: Intracellular Ca2+ mobilization was monitored in a Flexstation{\circledR} using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen{\circledR} proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. Key Results: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. Conclusion and Implications: We have shown that while adrenergic agonists display bias at human α1A-adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.",
author = "{da Silva Junior}, {Edilson Dantas} and Masaaki Sato and Jon Merlin and Natalie Broxton and Hutchinson, {Dana S.} and Sabatino Ventura and Evans, {Bronwyn A.} and Summers, {Roger J.}",
year = "2017",
month = "7",
day = "1",
doi = "10.1111/bph.13837",
language = "English",
volume = "174",
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journal = "British Journal of Pharmacology",
issn = "1476-5381",
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Factors influencing biased agonism in recombinant cells expressing the human α1A-adrenoceptor. / da Silva Junior, Edilson Dantas; Sato, Masaaki; Merlin, Jon; Broxton, Natalie; Hutchinson, Dana S.; Ventura, Sabatino; Evans, Bronwyn A.; Summers, Roger J.

In: British Journal of Pharmacology, Vol. 174, No. 14, 01.07.2017, p. 2318-2333.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Factors influencing biased agonism in recombinant cells expressing the human α1A-adrenoceptor

AU - da Silva Junior, Edilson Dantas

AU - Sato, Masaaki

AU - Merlin, Jon

AU - Broxton, Natalie

AU - Hutchinson, Dana S.

AU - Ventura, Sabatino

AU - Evans, Bronwyn A.

AU - Summers, Roger J.

PY - 2017/7/1

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N2 - Background and Purpose: Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α1A-adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. Experimental Approach: Intracellular Ca2+ mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. Key Results: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. Conclusion and Implications: We have shown that while adrenergic agonists display bias at human α1A-adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.

AB - Background and Purpose: Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α1A-adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. Experimental Approach: Intracellular Ca2+ mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. Key Results: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. Conclusion and Implications: We have shown that while adrenergic agonists display bias at human α1A-adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.

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EP - 2333

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