Conformational study of the Angiotensin II AT1 receptor under shear stress by means of molecular dynamics simulation and site-directed mutagenesis

• Grant number: 14/13940-4
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Effective date (Start): October 01, 2014
• Effective date (End): August 08, 2016
• Field of knowledge: Health Sciences - Medicine
• Acordo de Cooperação: Coordination of Improvement of Higher Education Personnel (CAPES)
• Principal Investigator: José Eduardo Krieger
• Grantee: Matheus Malta de Sá
• Host Institution: Instituto do Coração Professor Euryclides de Jesus Zerbini (INCOR). Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

AT1 receptor is part of the G protein-coupled receptors family. The angiotensin II binding leads to the activation of the heterodimeric G protein and, consequently, the activation of phospholipase C and protein kinase calcium dependent (PKC). Next, the receptor is phosphorylated by ²-arrestin and internalized. Studies have shown that ²-arrestin also generates second messengers which can activate other signaling pathways. Moreover, it is believed that this activation can occur independently of G protein by molecules known as biased agonists. This AT1 selective activation can lower blood pressure by not activating G protein and, at the same time, retain the beneficial effects of ²-arrestin (cell survival and renewal). The AT1 receptor can also be activated by mechanical forces, such as strain and shear. It has been recently shown that shear stress can activate the G protein dependent and independent pathways, without any ligand. As a protein activity is dependent on its conformation, AT1 ability to produce various pharmacological responses brings the existence of several conformational states into question. In this work we propose to study the conformational changes and amino acid residues that are responsible for AT1 responsiveness to shear stress by means of molecular dynamics simulation and experimental validation by site-directed mutagenesis. The computational approach makes possible to study conformational changes at atomic level at low cost when compared to experimental techniques to solve protein structures. Site mutagenesis will be produced by the Stratagene QuickChange® Site-Directed Mutagenesis kit and the AT1 activity will be detected by measuring the interaction of ²-arrestin with the activated AT1 receptor via a chemiluminescence reaction using a complementary fragment of the enzyme ²-gal. Alternatively, we intend to study the AT1 conformational changes in presence of biased agonists. We expect to obtain details about the conformational states of AT1 that help to elucidate the biased mechanism of action of this receptor. (AU)