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

BACKGROUND AND PURPOSE Agonists acting at GPCRs promote biased signalling via G alpha or G beta gamma subunits, GPCR kinases and beta-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 alpha(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 (R) using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen (R) 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 offtarget 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 alpha(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.