Fusion of the ß2-adrenergic receptor with either Gαs or ßarrestin-2 produces constitutive signaling by each pathway and induces gain-of-function in BEAS-2B cells.

The ß2AR 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 ß2AR are the canonical Gs pathway and the alternative ß arrestin 2 (ßarr2) pathway. Exploring each pathway separately remains a challenging task due to the dynamic nature of the receptor. Here, we fused the ß2AR with its cognate transducers, Gαs and ßarr2, 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 ß2AR-Gαs and ß2AR-ßarr2 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 ß2AR for the signaling pathway activated by the tethered transducer, and (c) pharmacologic modulation by ß2AR 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 ß2AR stimulation. Our findings suggest that each signaling pathway of the ß2AR 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.

A NanoBiT assay to monitor membrane proteins trafficking for drug discovery and drug development.

Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.

Hypoxia Inducible Factors as Central Players in the Pathogenesis and Pathophysiology of Cardiovascular Diseases.

Cardiovascular (CV) diseases are the major cause of death in industrialized countries. The main function of the CV system is to deliver nutrients and oxygen to all tissues. During most CV pathologies, oxygen and nutrient delivery is decreased or completely halted. Several mechanisms, including increased oxygen transport and delivery, as well as increased blood flow are triggered to compensate for the hypoxic state. If the compensatory mechanisms fail to sufficiently correct the hypoxia, irreversible damage can occur. Thus, hypoxia plays a central role in the pathogenesis and pathophysiology of CV diseases. Hypoxia inducible factors (HIFs) orchestrate the gene transcription for hundreds of proteins involved in erythropoiesis, glucose transport, angiogenesis, glycolytic metabolism, reactive oxygen species (ROS) handling, cell proliferation and survival, among others. The overall regulation of the expression of HIF-dependent genes depends on the severity, duration, and location of hypoxia. In the present review, common CV diseases were selected to illustrate that HIFs, and proteins derived directly or indirectly from their stabilization and activation, are related to the development and perpetuation of hypoxia in these pathologies. We further classify CV diseases into acute and chronic hypoxic states to better understand the temporal relevance of HIFs in the pathogenesis, disease progression and clinical outcomes of these diseases. We conclude that HIFs and their derived factors are fundamental in the genesis and progression of CV diseases. Understanding these mechanisms will lead to more effective treatment strategies leading to reduced morbidity and mortality.

Therapeutic Potential of Targeting ß-Arrestin.

ß-arrestins are multifunctional proteins that modulate heptahelical 7 transmembrane receptors, also known as G protein-coupled receptors (GPCRs), a superfamily of receptors that regulate most physiological processes. ß-arrestin modulation of GPCR function includes termination of G protein-dependent signaling, initiation of ß-arrestin-dependent signaling, receptor trafficking to degradative or recycling pathways, receptor transactivation, transcriptional regulation, and localization of second messenger regulators. The pleiotropic influence ß-arrestins exert on these receptors regulates a breadth of physiological functions, and additionally, ß-arrestins are involved in the pathophysiology of numerous and wide-ranging diseases, making them prime therapeutic targets. In this review, we briefly describe the mechanisms by which ß-arrestins regulate GPCR signaling, including the functional cellular mechanisms modulated by ß-arrestins and relate this to observed pathophysiological responses associated with ß-arrestins. We focus on the role for ß-arrestins in transducing cell signaling; a pathway that is complementary to the classical G protein-coupling pathway. The existence of these GPCR dual signaling pathways offers an immense therapeutic opportunity through selective targeting of one signaling pathway over the other. Finally, we will consider several mechanisms by which the potential of dual signaling pathway regulation can be harnessed and the implications for improved disease treatments.