Characterization of Angiotensin II Molecular Determinants Involved in AT₁ Receptor Functional Selectivity

Abstract

The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT 1 ), a G protein-coupled receptor. The AT 1 receptor engages and activates several signaling pathways, including heterotrimeric G proteins G q and G 12 , as well as the extracellular signal-regulated kinases (ERK) 1/2 pathway. Additionally, following stimulation, barrestin is recruited to the AT 1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favor the activation of some signaling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of six different signaling modalities of the AT 1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4, and 8, we sought to better characterize the molecular determinants of AngII that underlie functional selectivity of the AT 1 receptor in human embryonic kidney 293 cells. The results reveal that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over G q signaling, and position 8 confers a bias toward barrestin recruitment over ERK activation and G q signaling. Interestingly, the analogs modified in position 8 were also partial agonists of the protein kinase C (PKC)-dependent ERK pathway via atypical PKC isoforms PKCz and PKCi.

Angiotensin II Signal Transduction: An Update on Mechanisms of Physiology and Pathophysiology

“…Position 8 mutations also reduce G 12 activation. Position 8 is also important to partially mediate EGFR-dependent as well as PKC-dependent ERK activation (239). As mentioned above, SII also activates Akt leading to mTOR-dependent protein synthesis in HEK293 cells (482).…”

Angiotensin II cyclic analogs as tools to investigate AT1R biased signaling mechanisms

“…Therefore, upon treatment with PD168393, the ERK response was PKC-dependent with a maximal response obtained after This is the postprint version of the following article: St-Pierre D, et al (2018), Biochem Pharmacol. doi: 10.1016/j.bcp.2018.04.021, which has been accepted and published in final form at https://www.sciencedirect.com/science/article/pii/S0006295218301643 a maximal response obtained after 5 min [19]. Where specified, Go6983 (1 uM) or PD168393 (250 nM) were added 30 min before stimulation.…”

Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the β-Arrestin Pathway Using Molecular Dynamics Simulations

“…The ability to insert in the hydrophobic core and elicit the conformational change could be unique to the phenyl moiety, as mutation of this residue to a tyrosine or diphenylalanine are the only tolerated changes that do not lead to an AngII analog with an antagonist profile on the G q/11 pathway. Indeed, substitution of Phe 8 for other hydrophobic residues, such as alanine, leucine, or isoleucine, turns the ligand into an antagonist on the G q/11 pathway (15,(63)(64)(65). However, a larger side chain such as (pentabromo)Phe-8 or a change of orientation from L-Phe-8 to D-Phe-8 also results in conferring an antagonistic nature on the G q/11 pathway (66,67).…”

“…To verify this hypothesis, we used microsecond time scale MD simulations of the WT-AT 1 receptor, N111G-AT 1 receptor, and D74N-AT 1 receptor to explore their conformational landscape by looking at specific structural determinants. Furthermore, based on a previously developed model of the AT 1 receptor in complex with AngII (14), we looked at how the presence of AngII in the binding pocket modified the conformational landscapes of the WT-AT 1 , D74N-AT 1 , and N111G-AT 1 receptors and also how the ␤-arrestin-biased agonist [Sar 1 ,Ile 8 ]AngII (SI8) (15,16) modified the conformational landscapes of the WT-AT 1 receptor. The simulations suggest that the N111G mutation destabilizes the ground state of the receptor.…”