Fusion Proteins as Selective Inducers of Constitutive Activity and Tools to Discriminate the Biased Signaling Profile of β2 Adrenoceptor Ligands

Date

2021-12

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Abstract

The β2AR is the 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 β2AR 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 β2AR 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 β2AR-Gαs and β2AR-βarr2 in HEK293 cells by measuring their constitutive activity, transducer recruitment, and pharmacologic modulation. Our fusion proteins show (a) steric hindrance from the reciprocal endogenous transducers, (b) constitutive activity of the β2AR for the signaling pathway activated by the tethered transducer, and (c) pharmacologic modulation by β2AR ligands. Since both fusion proteins remained functional to ligand stimuli, we quantified the pharmacological properties of affinity, efficacy, and potency for cAMP accumulation and ERK1/2 phosphorylation as surrogates of the Gs and βarr2 pathways, respectively, in selected β2AR ligands. Using these pharmacological parameters, we developed a mathematical method for direct ligand bias quantification based on the well-known transduction coefficient ratios formula (ΔΔ log(τ/KA)). Our method has the advantage of quantifying an ‘absolute’ value of signaling bias for any ligand that shows negative or positive efficacy at any signaling pathway. The term absolute is used here to highlight that a reference ligand is not required in our method as each ligand becomes its own reference. Regarding the isolated constitutive activity observed for each fusion protein, we used this feature to induce 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 β2AR stimulation. Our findings suggest that each signaling pathway of the β2AR is biased towards either the Th1 or Th2 inflammatory response regulating the immune phenotype of respiratory diseases. Our data implies our fusion proteins can be used as tools to isolate the function elicited by a unique signaling pathway in physiologically relevant cell types.

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Keywords

beta 2 adrenergic receptor, beta arrestin 2, Fusion Proteins, Biased signaling, Ligand Bias, inflammation, cytokines

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