Identification of molecular partners from Angiotensin II receptor type 1 (AT1) involved with the activation of a new signaling pathway

• Grant number: 14/20906-7
• Support type: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): January 01, 2015
• Effective date (End): December 31, 2018
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: Claudio Miguel da Costa Neto
• Grantee: Larissa de Bortoli Teixeira
• Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 12/20148-0 - Development of new ligands/drugs with selective agonism action (biased agonism) for receptors of the renin-angiotensin and kallikrein-kinin systems: new properties and new biotechnological applications, AP.TEM
• Associated scholarship(s): 16/10525-1 - Functional crosstalk between Mas and AT1 receptors addressed by BRET-based biosensors, BE.EP.PD

Abstract

G-protein-coupled receptors (GPCRs) belong to a superfamily of integral membrane proteins which have in their structure seven transmembrane alpha-helices, and for that reason, they are also known as 7 transmembrane receptors (7TMRs). 7TMRs constitute the largest class of cell surface receptors and they regulate a wide range of physiological and pathological processes, because of that, 7TMRs are currently among the most important pharmacological targets. Angiotensin II receptor type 1 (AT1), the main mediator of the renin-angiotensin system, is classically activated by the octapeptide angiotensin II (Ang II), but it can also be activated by biased agonists, so named for their capacity of activating a determined signaling pathway, such as the ones mediated by G proteins or ²-arrestins. While studying structure and function of the AT1 receptor, Prof. Claudio M. Costa-Neto's group constructed human and rat AT1 mutant receptors bearing alterations in proline residues from transmembrane regions. Among them, the mutant AT1-P207A (in which the proline at position 207 was substituted for alanine) was highlighted by demonstrating inability to activate G proteins (Gq, Gs, and Gi), inability to recruit ²-arrestins (1 and 2), and inability to transactivate EGFR, and yet, ability to induce ERK1/2 phosphorylation when stimulated with SII, a biased agonist towards the ²-arrestins pathway. The functional response analysis for this mutant strongly suggests the existence of a new signaling pathway which has not been described yet, and, therefore, the existence of additional molecular partners for the receptors. The purpose of this project is precisely to identify those partners, which will be accomplished through immuneprecipitation of signaling complexes (ligand/receptor/effectors), followed by mass spectrometry, and to validate the functional contribution and relevance for endogenous signal transduction, through the knockdown of identified targets. The project falls within the context of the Thematic Project number 2012/20148-0. (AU)