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

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 Ca 2+ 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 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 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 Ca 2+ 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.