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

Abstract

BACKGROUND AND PURPOSEAgonists 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 RESULTSNoradrenaline, phenylephrine, methoxamine and A61603 increased Ca 2+ 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 offtarget effects at 5-HT 1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca 2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. CONCLUSION AND IMPLICATIONSWe 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. AbbreviationsECAR, extracellular acidification rate; HEAT, 2-(β-4-hydroxyphenyl)ethylaminomethyltetralone; PTX, pertussis toxin

Functional activation of Gαq/11 protein via α1‐adrenoceptor in rat cerebral cortical membranes

“…The stimulation induced by oxymetazoline reached the E max value of 147.4 ± 34.3 %, a double of the %E max value of an endogenous ligand (−)‐adrenaline (70.3 ± 8.1 %). A similar property of this compound as an apparent “super‐agonist” has been reported as an ERK1/2 phosphorylation response in CHO‐K1 cells expressing human α 1A ‐adrenoceptor . In this report, however, it has been shown that this strange phenomenon is attributable to off‐target effects at 5‐HT 1B receptors that are endogenously expressed in the host cells.…”

Updates in the function and regulation of α1‐adrenoceptors

“…In LNCap cells, the co‐localization and co‐immunoprecipitation of α 1A ‐ adrenoceptors and β‐arrestin produced by oxymetazoline was as strong as those produced by noradrenaline, despite the weak partial agonism of oxymetazoline for Ca 2+ mobilization (Alcantara‐Hernandez et al, ). Strikingly, oxymetazoline was more efficacious and potent than noradrenaline for the phosphorylation of ERK 1/2 in LNCap cells but differed from what was observed in HEK293 cells (da Silva Junior et al, ). The superagonism of oxymetazoline for the ERK pathway in LNCap cells was unrelated to 5‐HT receptor activation and was antagonized by prazosin (Alcantara‐Hernandez et al, ).…”

“…In HEK293 cells, α 1Aadrenoceptors activated by oxymetazoline internalized at a much faster rate (~5 min) than when activated by noradrenaline (>45 min; Akinaga et al, 2013). Moreover, there was significant tachyphylaxis in the con- what was observed in HEK293 cells (da Silva Junior et al, 2017). The superagonism of oxymetazoline for the ERK pathway in LNCap cells was unrelated to 5-HT receptor activation and was antagonized by prazosin (Alcantara-Hernandez et al, 2017).…”

What makes the α1A‐adrenoceptor gene product assume an α1L‐adrenoceptor phenotype?

“…The 5-HT 1B receptor was selected mainly because it is known to be present in the human prostate as well as in several prostate cancer cell lines (Dizeyi et al, 2004;Siddiqui, Shabbir, Mikhailidis, Thompson, & Mumtaz, 2006). 5-HT 1B receptors are also endogenously expressed in CHO-K1 but not HEK293 cells (da Silva Junior et al, 2017). In CHO-K1 cells, overexpressing α 1A -adrenoceptors, antagonism of the endogenously expressed 5-HT 1B receptors with 300 nM SB216641 increased the potency of prazosin approximately fivefold in an ERK 1/2 phosphorylation assay (Table 1).…”