Translocation of arrestin induced by human A3 adenosine receptor ligands in an engineered cell line: Comparison with G protein-dependent pathways

Abstract

Structurally diverse ligands were studied in A 3 adenosine receptor (AR)-mediated β-arrestin translocation in engineered CHO cells. The agonist potency and efficacy were similar, although not identical, to their G protein signaling. However, differences have also been found. MRS542, MRS1760, and other adenosine derivatives, A 3 AR antagonists in cyclic AMP assays, were partial agonists in β-arrestin translocation, indicating possible biased agonism. The xanthine 7-riboside DBXRM, a full agonist, was only partially efficacious in β-arrestin translocation. DBXRM was shown to induce a lesser extent of desensitization compared with IB-MECA. In kinetic studies, MRS3558, a potent and selective A 3 AR agonist, induced β-arrestin translocation significantly faster than IB-MECA and Cl-IB-MECA. Non-nucleoside antagonists showed similar inhibitory potencies as previously reported. PTX pretreatment completely abolished ERK1/2 activation, but not arrestin translocation. Thus, lead candidates for biased agonists at the A 3 AR have been identified with this arrestin-translocation assay, which promises to be an effective tool for ligand screening.

Distinct Signaling Patterns of Allosteric Antagonism at the P2Y1 Receptor

“…However, the pattern of antagonism by various antagonists at this signaling pathway is unknown. Therefore, we compared the antagonism by MRS2500 and BPTU of the agonist-induced b-arrestin2 recruitment using the PathHunter protein complementation assay (DiscoverX), a widely accepted method (Gao and Jacobson, 2008;Violin et al, 2010;Goa et al, 2011Goa et al, , 2014. Figure 2e shows that MRS2500 shifts the agonist concentration-response curve to the right in a parallel manner with a slope of 0.89 6 0.05 and a K B value of 0.85 6 0.08 nM.…”

“…The b-arrestin2 recruitment to the P2Y 1 R was assessed by DiscoverX PathHunter b-arrestin assay as described previously (Gao and Jacobson, 2008;Gao et al, 2014). In this assay, the GPCR is fused in frame with the small enzyme fragment ProLink and coexpressed in U2OS cells stably expressing a fusion protein of b-arrestin2 and the larger N-terminal deletion mutant of b-galactosidase (enzyme acceptor).…”

Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor

“…The A 3 AR preferentially couples to G i/o proteins, and therefore agonist activation stimulates the canonical signal transduction pathway, inhibition of adenylate cyclase activity (Fredholm et al, 2001). However, in addition to G i/o -adenylate cyclase coupling, A 3 ARs can also modulate a number of additional G protein-dependent and G protein-independent intracellular signaling pathways (Schulte and Fredholm, 2002;Fossetta et al, 2003;Merighi et al, 2006;Gao and Jacobson, 2008). In this study, agonists were assessed for their ability to inhibit cAMP accumulation, phosphorylate ERK1/2 and Akt(Ser473) 1/2/3, increase intracellular calcium concentrations and promote cytoprotection.…”

“…Hallmarks of biased agonism include changes in the relative potency of a set of compounds across different signaling pathways, which can result in a reversal in the rank orders of potency or maximal effects (Urban et al, 2007;Kenakin et al, 2012). Although relatively unexplored, A 3 AR biased agonism and biased allosteric modulation have been observed at the A 3 AR (Gao and Jacobson, 2008;Gao et al, 2011;Verzijl and IJzerman, 2011). Previous studies, which profiled structurally distinct A 3 AR ligands in terms of cAMP accumulation and b-arrestin recruitment, identified compounds that differed with respect to their coupling to G protein-dependent and G proteinindependent pathways (Gao and Jacobson, 2008).…”

“…Although relatively unexplored, A 3 AR biased agonism and biased allosteric modulation have been observed at the A 3 AR (Gao and Jacobson, 2008;Gao et al, 2011;Verzijl and IJzerman, 2011). Previous studies, which profiled structurally distinct A 3 AR ligands in terms of cAMP accumulation and b-arrestin recruitment, identified compounds that differed with respect to their coupling to G protein-dependent and G proteinindependent pathways (Gao and Jacobson, 2008). For example, whereas the xanthine-7-riboside agonist 1,3-dibutylxanthine-7-riboside-59-N-methylcarboxamide had higher efficacy for cAMP accumulation compared with b-arrestin recruitment, -(3-iodobenzyl)adenosine had no significant effect on cAMP accumulation but were partial agonists for b-arrestin recruitment.…”