Assessing the microdomain localization-dependent role of GPCRs and effectors on signal transduction using Bioluminescence Resonance Energy Transfer (BRET)-based biosensors

Abstract

G protein-coupled receptors (GPCRs) are integral membrane proteins characterized by a topology of seven transmembrane ±-helices that mediate a large number of physiological processes after binding of a wide range of ligands. It has been shown that during signal transduction, dynamic changes can occur in the localization of receptor and effectors in plasma membrane microdomains and intracellular organelles such as endosomes. Among these microdomains, caveolar and non-caveolar lipid rafts are among the most described, despite some controversies in the literature. Such membrane domains are enriched in cholesterol and sphingolipids and are thought to contribute to signal transduction. However, a clear understanding of the mechanisms that regulate receptor localization, and of how lipid rafts act as dynamic platforms for GPCRs have remained unclear mainly due to the lack of sound technologies. Therefore, the goal of this project is to develop and characterize new bioluminescence resonance energy transfer (BRET) biosensors targeting different microdomains in order to study the role of lipid rafts and non-lipid raft microdomains in GPCR dynamics and in regulating signal transduction. We will also investigate the role of caveolae-dependent endocytosis in the phenomenon of tachyphylaxis of the AT1 receptor using biosensors of caveolae/lipid rafts after activation by AngII non-tachyphylactic analogs recently synthesized by our group in Brazil (Claudio Costa Neto's laboratory). We believe that the study of the dynamic changes in receptor localization in membrane microdomains is pivotal to understand the molecular mechanisms involved in signal transduction and receptor endocytosis, having significant contribution to the ultimate cellular responses and therefore of relevance to drug discovery.