Characterization of the molecular mechanisms involved in the binding and signaling of angiotensin II in the AT1 receptor

G protein-coupled receptors (GPCRs) superfamily mediates most of the physiological processes and is target for about 40% of the drugs currently available on the market. Despite recent advances in the study of the binding process of small and rigid molecules in GPCRs, the process of binding of complex molecules such as peptides and proteins remains poorly understood. In the first part of this work we used a series of angiotensin II (AngII) analogs with reduced conformational flexibility to study the mechanisms involved in AngII binding in the angiotensin II type 1 receptor (AT1). Using biochemical and pharmacological assays we have demonstrated that AngII seems to bind in the AT1 receptor by a stepwise mechanism involving an initial step of ligand recognition at extracellular sites of the receptor, then its accommodation in an intermediate site until its stabilization in the orthosteric site of the receptor. In addition, based on the structure of one of the analogs studied, our results suggest a conformation of AngII when stabilized in the orthosteric site, which stabilizes the receptor in its active state (s). In the second part of this work, using other analogues we address the molecular mechanisms involved in the AT1 receptor tachyphylaxis, which are not known yet, even tachyphylaxis being therapeutically important. Our results revealed that the studied analogs induce a dynamic of signaling, internalization and recycling of the receptors different to AngII. In addition, our results showed that the non-tachyphylactic analogs dissociate more quickly from the AT1 receptor and that the slow dissociation of AngII is a key factor for the occurrence of tachyphylaxis in this receptor. (AU)