Fusion Proteins as Tools to Discriminate the Biased Signaling Profile of β 2 Adrenergic Receptor Ligands

Abstract

Introduction The β2 Adrenoceptor (β2AR) is a seven-transmembrane domain receptor that induces a cellular response by activating a unique signaling pathway dependent on the conformational state of the receptor. At least two signaling pathways have been characterized for the β2AR: the canonical Gs pathway and the β-arrestin2 (βarr2) pathway. So far, the determination of the signaling preference of multiple β2AR ligands (biased signaling) is compared to the reference β2AR endogenous ligand, epinephrine (EPI), which by convention is defined as unbiased. However, the absolute biased signaling of β2AR ligands on the basis for the distinct active conformational states of the β2AR is unknown. Thus, we used novel fusion proteins (fp) made of the β2AR receptor fused to Gαs (β2AR-Gs) or to βarr2 (β2AR-βarr2) to stabilize the β2AR conformation and characterize the pharmacological properties (affinity, efficacy, and potency) of the β2AR ligands. Our central hypothesis is that by restricting signaling to a single conformational state of the receptor, we will be able to characterize the biased signaling of β2AR ligands (independent of epinephrine). Methods For pharmacological characterization, we used HEK293 cells, a common cell line widely used to study the β2AR biased signaling. We stably transfected each fp (β2AR-Gs or β2AR-βarr2) independently into HEK293 cells. Stably transfected HEK293 cells with wild-type (WT) β2AR and untransfected HEK293 cells served as experimental controls. Using homologous time-resolving fluorescence, we measured cAMP production and ERK phosphorylation as the surrogates for β2AR signaling via Gs and βarr2, respectively. Results For the Gs protein signaling pathway, our data suggest ICI has a higher affinity for the inhibition of cAMP production by isoproterenol (ISO) in both the β2AR-βarr2 and β2AR-Gs fp (apparent pA2=10.38 and 10.16, respectively), compared to both control groups (WT β2AR pA2=9.42; untransfected HEK293 pA2=9.21). Furthermore, the ligands ISO and EPI showed a higher potency for the β2AR-Gs fp compared to the β2AR-βarr2 fp (Table-1). Regarding the βarr2 signaling pathway, EPI showed higher potency at ERK phosphorylation in cells transfected with β2AR-βarr2 vs β2AR-Gs fp (pEC50=7.0 vs 6.365). Conclusion Our preliminary results suggest that the affinity of ICI as well as the potency of EPI might change based on the conformational state of the β2AR. Ongoing binding affinity studies will further develop the absolute biased signaling profile of each β2AR ligand and reveal if the reason for the differences in potency are due to their differing affinities for the conformational states.