Functional crosstalk between Mas and AT1 receptors addressed by BRET-based biosensors

Abstract

G protein-coupled receptors (GPCRs) are physiologically important cell-surface proteins that sense signaling messengers such as hormones, photons and even mechanical stress, and are the targets of several prescribed drugs. An important therapeutic target in hypertension and congestive heart failure is the renin-angiotensin system (RAS). Angiotensin II (AngII), the main component of the RAS cascade, exerts most of its effects by activating the angiotensin II type 1 (AT1) receptor. RAS has also a second component, the angiotensin-converting enzyme (ACE)-2-angiotensin-(1-7)-Mas receptor axis, which counterbalances the effects of the ACE-AngII-AT1R. The functional interactions between Mas and AT1 receptors have been investigated. After co-expression of Mas, AT1R-mediated AngII-induced production of second messengers was shown to be reduced. This effect has been attributed to a constitutive hetero-oligomeric complex formed between the receptors. In contrast to such blunting of the AT1R-mediated signaling, the Mas receptor has been reported to up-regulate AT1R levels by constitutively activating Gq/11 and hence stimulating PKC-dependent phosphorylation of the AT1R C-terminal tail. Interestingly, the cellular distribution of the Mas receptor is widespread, while a G protein-uncoupled Mas receptor mutant is located predominantly at the cell membrane. Despite the clear relevance, the spatio-temporal aspects associated with the interactions between the receptors and their functional outcomes need to be better addressed, such as by the improved technologies and approaches here outlined. In this project, we propose to study the crosstalk between Mas and AT1 receptors by investigating the possibility that the constitutive activity of Mas involved in the first steps of the crosstalk occurs in intracellular compartments. Therefore, our specific aims are to: 1) evaluate the constitutive activity of the Mas receptor regarding its effects on different G proteins; 2) identify the cellular region(s)/compartment(s) from which the Mas receptor signals to promote AT1R up-regulation; 3) assess the Mas-induced mechanisms regulating AT1R levels at the cell surface. This study is of great interest to our laboratory in Brazil. It falls within the scope of an ongoing Thematic Project (FAPESP Grant #2012/20148-0), and would be a natural extension of the research started under the SPRINT Program in collaboration with Professor Michel Bouvier (FAPESP Grant #2015/50086-4).