Angiotensin II type 1 receptor (AT1R) is a G-protein coupled receptor (GPCR) and an important regulator of blood pressure. It is a target for drug development, because abnormalities in its function are linked to hypertension, cardiac hypertrophy and heart failure. AT1R is composed of seven transmembrane helices connected by three extracellular loops and three intracellular loops. The extracellular loop 2 (ECL2) of AT1R directly interacts with the ligands. This loop is targeted by autoantibodies that activate AT1R in several pathologies such as preeclampsia, malignant hypertension and vascular allograft rejection. Therefore, we proposed that the conformation of ECL2 in AT1R is differentially regulated upon binding to agonists and antagonists.
We determined the conformation of the ECL2 of AT1R by reporter-cysteine accessibility mapping in different receptor states (i.e., empty, agonist-bound and antagonist-bound). We introduced cysteines at each position of ECL2 of a receptor surrogate lacking all non-essential cysteines and measured reaction of these with a cysteine-reactive biotin probe. The ability of biotinylated mutant receptors to react with a streptavidin-HRP-conjugated antibody was used as the basis for examining differences in accessibility.
Two segments of ECL2 were accessible in the empty receptor, indicating an open conformation of ECL2. These segments were inaccessible in the ligand-bound states of the receptor. Using the accessibility constraint, we performed molecular dynamics simulation to predict ECL2 conformation in different states of the receptor. Analysis suggested that a ‘lid’ conformation of ECL2 was induced upon binding to both agonists and antagonists, but exposing different accessible segments delimited by a highly conserved disulfide bond between ECL2 and TMIII. We propose that the ligand-induced ECL2-lid is coupled to movements of transmembrane helices through the conserved disulfide bond to achieve the transition to the active state of the receptor.
Our study reveals the ability of ECL2 to interact with diffusible ligands and adopt a ligand-specific ‘lid’ conformation. Distinct conformations induced by the agonist and the antagonist around the conserved disulfide bond suggest an important role for the disulfide bond in producing different functional states of the receptor. Dynamics of ECL2 may regulate the fundamental process of receptor activation and pathophysiological processes involving GPCRs.