Fusion of the β 2 ‐adrenergic receptor with either Gαs or βarrestin‐2 produces constitutive signaling by each pathway and induces gain‐of‐function in BEAS‐2B cells

The β 2 AR is a prototypical G protein‐coupled receptor (GPCR) known to orchestrate different cellular responses by the stimulation of specific signaling pathways. The best‐established signaling pathways for the β 2 AR are the canonical Gs pathway and the alternative β arrestin 2 (βarr2) pathway. Exploring each pathway separately remains a challenging task due to the dynamic nature of the receptor. Here, we fused the β 2 AR with its cognate transducers, Gαs and βarr2, using short linkers as a novel approach for restricting the conformation of the receptor and preferentially activating one of its two signaling pathways. We characterized the behavior of our fusion proteins β 2 AR‐Gαs and β 2 AR‐βarr2 in HEK293 cells by measuring their constitutive activity, transducer recruitment, and pharmacological modulation. Our fusion proteins show (a) steric hindrance from the reciprocal endogenous transducers, (b) constitutive activity of the β 2 AR for the signaling pathway activated by the tethered transducer, and (c) pharmacologic modulation by β 2 AR ligands. Based on these characteristics, we further explored the possibility of a gain‐of‐function mechanism in the human lung non‐tumorigenic epithelial cell line, BEAS‐2B cells. This immortalized human bronchial epithelial cell line has immunomodulatory properties through cytokine release mediated by β 2 AR stimulation. Our findings suggest that each signaling pathway of the β 2 AR is biased toward either the Th1 or Th2 inflammatory response suggesting a role in regulating the immune phenotype of respiratory diseases. Our data imply that our fusion proteins can be used as tools to isolate the function elicited by a single signaling pathway in physiologically relevant cell types.