Investigation of the role of AT1 receptor biased agonists at formation of signaling complex ligand-receptor-Beta-arrestin-kinase

• Grant number: 13/15495-5
• Support type: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): December 01, 2013
• Effective date (End): November 30, 2015
• Field of knowledge: Biological Sciences - Pharmacology - Biochemical and Molecular Pharmacology
• Principal Investigator: Claudio Miguel da Costa Neto
• Grantee: Vanessa Lima
• 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

Abstract

In the last few years has been reported that agonists of G-protein coupled receptors (GPCRs) are capable of promoting a preferential activation profile or biased to a particular intracellular signaling pathway. This phenomenon has received different names in the literature, such as collateral efficacy, pluridimensional efficacy, functional selectivity or biased agonism. In general, this preference of activation has been best studied for canonical signaling (via activation of G protein) and non-canonical signaling (via B-arrestins) that occurs after the formation and internalization of a complex that includes ligand, receptor, B-arrestin (a scaffold protein), and kinases. Various drugs have been discovered as biased agonists to one of these signaling pathways. Despite many studies showing the existence and relevance of biased agonists at different receptor systems, so far there are no studies in the literature evaluating whether a group of biased agonists for the same pathway (e.g. for B-arrestins) may present differences in signaling downstream the recruitment of B-arrestins. Therefore, the aim of this project is to investigate whether activation of the AT1 receptor by AngII and different biased agonists lead to the formation of different signaling complexes. After stimulation and internalization it will be evaluated the interaction of B-arrestins 1 and 2 with complex Raf-1/MEK1/ERK1/2, Ask1/MKK4/JNK3. B-arrestins will be immunoprecipitated and followed by Western blotting with specific antibodies for each protein of the signaling pathways. Furthermore, the activation profile of ERK1/2 and JNK3 will be evaluated by AlphaScreen SureFire (TGR BioSciences / PerkinElmer) and the "biased" activation of the signaling pathway (ERK1/2 or JNK3) for each condition will be calculated by determining the reasons of "coupling efficiencies" tau.