Role of Distinct β2 Adrenergic Receptor Pathways in Mediating Inflammation
Authors: Rojas Emilio Yaroslav Lucero Garcia , McConnell Bradley K. , Bond Richard A. 
Published: April 2020

Chronic treatment of inflammatory diseases (i.e. asthma) with β2‐adrenergic receptor (β2AR) agonists can paradoxically lead to increased inflammation and poor therapeutic outcomes. The β2AR has at least two signaling pathways: the canonical Gs pathway and the β‐arrestin2 (βarr2) pathway. Both pathways are suggested to be involved in inflammatory disorders that are regulated by immunomodulatory cells, such as the airway epithelium. However, the inflammatory response of the epithelium to β2AR ligands that preferentially (relative to epinephrine) activate one of these pathways (biased signaling) remains unknown. Thus, we will use novel fusion proteins made of the β2AR receptor fused to Gαs (β2AR‐Gs) or to βarr2 (β2AR‐βarr2) to restrict signaling to a single pathway, to characterize the inflammatory profile of β2AR ligands, relative to their pharmacological properties (affinity, efficacy, and potency). Our central hypothesis is that by restricting signaling to a single conformational state of the receptor, we will be able to correlate the signaling of β2AR ligands with their pharmacological properties. Our long‐term goal is to understand how the distinct β2AR pathways mediate the inflammatory response in the epithelium. For pharmacological characterization, we used HEK293 cells. This cell line has been used to generate most of the previous data on β2AR biased signaling and allows for comparisons for experimental rigor. We stably transfected each fusion protein (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. For the Gs protein signaling pathway, all transfected cells showed increased constitutive activity compared to untransfected HEK293 cells. β2AR‐βarr2 transfected cells, however, showed the highest sensitivity to ICI 115,881 (ICI), a β2AR inverse agonist/antagonist for both pathways, at decreasing constitutive activity. Conversely, for the βarr2 signaling pathway, only β2AR‐βarr2 transfected cells showed constitutive activity. Furthermore, both signaling pathways (Gs or βarr2) could be modulated by isoproterenol (ISO), an agonist for both pathways. Thus, while the chimeric receptor is constitutively active, the fusion proteins are still able to respond to ligands, allowing us to explore the ‘absolute bias’ of β2AR ligands. Our data suggest ICI has a higher affinity for the inhibition of cAMP production by ISO in both the β2AR‐βarr2 and β2AR‐Gs fusion proteins (apparent pA2=10.38 and 10.16, respectively), compared to both control groups (WT β2AR pA2=9.42; untransfected HEK293 pA2=9.21). Furthermore, ISO showed a higher potency for the β2AR‐Gs fusion protein (pEC50=10.12) compared to cells expressing the β2AR‐βarr2 fusion protein (pEC50=8.98). We conclude that our fusion proteins can be used to generate a gain‐of‐function phenotype for studying our long‐term goals and can be useful tools to explore the signaling bias of β2AR ligands.