Loss of biased signaling at a G protein-coupled receptor in overexpressed systems

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and beta-arrestins. [Sarcosine(1)Ile(4)Ile(8)]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT(1)R), fails to activate G protein in physiologically relevant models. Despite this, SII and several derivatives induce cellular signaling outcomes through beta-arrestin-2-dependent mechanisms. However, studies reliant on exogenous AT(1)R overexpression indicate that SII is a partial agonist for G protein signaling and lacks beta-arrestin-exclusive functional specificity. We investigated this apparent discrepancy by profiling changes in functional specificity at increasing expression levels of AT(1)R using a stably integrated tetracycline-titratable expression system stimulated with AngII, SII, and four other AngII analogs displaying different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all tested receptor concentrations. In contrast, beta-arrestin-biased ligands induced dose-dependent calcium signaling only at higher AT(1)R overexpression levels. Using inhibitors of G proteins, we demonstrated that both G(i) and G(q/11) mediated overexpression-dependent calcium signaling by beta-arrestin-biased ligands. Regarding beta-arrestin-mediated cellular events, the beta-arrestin-biased ligand TRV026 induced receptor internalization at low physiological receptor levels insufficient for it to initiate calcium signaling. In contrast, unbiased AngII exhibited no relative preference between these outcomes under such low receptor conditions. However, with high receptor overexpression, TRV026 lost its functional selectivity. These results suggest receptor overexpression misleadingly distorts the bias of AT(1)R ligands and highlight the risks of using overexpressed systems to infer the signaling bias of GPCR ligands in physiologically relevant contexts.