Dataset Information

Factors influencing biased agonism in recombinant cells expressing the human α_1A -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 α_1A -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-HT_1B 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 α_1A -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.

SUBMITTER: da Silva Junior ED

PROVIDER: S-EPMC5481649 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Publications

Factors influencing biased agonism in recombinant cells expressing the human α<sub>1A</sub> -adrenoceptor.

da Silva Junior Edilson Dantas ED Sato Masaaki M Merlin Jon J Broxton Natalie N Hutchinson Dana S DS Ventura Sabatino S Evans Bronwyn A BA Summers Roger J RJ

British journal of pharmacology 20170610 14

<h4>Background and purpose</h4>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 α<sub>1A</sub> -adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in respon ...[more]

PMID: 28444738

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Project description:Two splice isoforms of rabbit alpha(1a)-adrenergic receptor (AR), (named alpha(1a)-OCU.2-AR and alpha(1a)-OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (alpha(1a)-OCU.1-AR). Although they have the identical splice position with human alpha(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. Among these rabbit alpha(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of alpha(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; alpha(1a)-OCU. 1-AR was expressed most abundantly in those tissues. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg(-1) protein in alpha(1a)-OCU.1-AR, 1200 fmol mg(-1) in alpha(1a)-OCU.2-AR and 570 fmol mg(-1) in alpha(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC(50) values of 6. 19 for alpha(1a)-OCU.1-AR, 6.49 for alpha(1a)-OCU.2-AR and 6.58 for alpha(1a)-OCU.3-AR, respectively. Noradrenaline caused a concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in the CHO clones with pEC(50) values of 6. 14 for alpha(1a)-OCU.1-AR, 7.25 for alpha(1a)-OCU.2-AR and 7.70 for alpha(1a)-OCU.3-AR, respectively. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit alpha(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but alpha(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of alpha(1a)-ARs may give the characteristic functional profile.

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Project description:Background and purposeMesenteric and carotid arteries from the alpha(1B/D)-adrenoceptor knockout (alpha(1B/D)-KO) were employed to isolate alpha(1A)-adrenoceptor pharmacology and location and to reveal these features in the wild-type (WT) mouse.Experimental approachFunctional pharmacology by wire myography and receptor localization by confocal microscopy, using the fluorescent alpha(1)-adrenoceptor ligand BODIPY FL-Prazosin (QAPB), on mesenteric (an 'alpha(1A)-adrenoceptor' tissue) and carotid (an 'alpha(1D)-adrenoceptor' tissue) arteries.Key resultsAlpha(1B/D)-KO mesenteric arteries showed straightforward alpha(1A)-adrenoceptor agonist/antagonist pharmacology. WT had complex pharmacology with alpha(1A)- and alpha(1D)-adrenoceptor components. alpha(1B/D)-KO had a larger alpha(1A)-adrenoceptor response suggesting compensatory up-regulation: no increase in fluorescent ligand binding suggests up-regulation of signalling. alpha(1B/D)-KO carotid arteries had low efficacy alpha(1A)-adrenoceptor responses. WT had complex pharmacology consistent with co-activation of all three subtypes. Fluorescent binding had straightforward alpha(1A)-adrenoceptor characteristics in both arteries of alpha(1B/D)-KO. Fluorescent binding varied between cells in relative intracellular and surface distribution. Total fluorescence was reduced in the alpha(1B/D)-KO due to fewer smooth muscle cells showing fluorescent binding. WT binding was greater and sensitive to alpha(1A)- and alpha(1D)-adrenoceptor antagonists.Conclusions and implicationsThe straightforward pharmacology and fluorescent binding in the alpha(1B/D)-KO was used to interpret the properties of the alpha(1A)-adrenoceptor in the WT. Reduced total fluorescence in alpha(1B/D)-KO arteries, despite a clear difference in the functionally dominant subtype, indicates that measurement of receptor protein is unlikely to correlate with function. Fewer cells bound QAPB in the alpha(1B/D)-KO suggesting different cellular phenotypes of alpha(1A)-adrenoceptor exist. The alpha(1B/D)-KO provides robust assays for the alpha(1A)-adrenoceptor and takes us closer to understanding multi-receptor subtype interactions.

Dataset Information

Factors influencing biased agonism in recombinant cells expressing the human α_1A -adrenoceptor.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Publications

Factors influencing biased agonism in recombinant cells expressing the human α<sub>1A</sub> -adrenoceptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

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

Background and purpose

Experimental approach

Key results

Conclusion and implications

Publications

Factors influencing biased agonism in recombinant cells expressing the human α<sub>1A</sub> -adrenoceptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Factors influencing biased agonism in recombinant cells expressing the human α_1A -adrenoceptor.