The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

Abstract

Cardiovascular diseases (CVDs) like hypertension are a major cause for death worldwide. In the cardiovascular tissue, the endothelin system—consisting of the receptor subtypes A (ETAR) and B (ETBR) and the mixed agonist endothelin 1 (ET‐1)—is a major key player in the regulation of vascular tone and blood pressure. Tight control of this system is required to maintain homeostasis; otherwise, the endothelin system can cause severe CVDs like pulmonary artery hypertension. The high sequence homology between both receptor subtypes limits the development of novel and selective ligands. Identification of small differences in receptor–ligand interactions and determination of selectivity constraints are crucial to fine‐tune ligand properties and subsequent signaling events. Here, we report on novel ET‐1 analogs and their detailed pharmacological characterization. We generated simplified ET‐1‐derived monocyclic peptides to provide an accessible synthesis route. By detailed in vitro characterization, we demonstrated that both G protein signaling and the subsequent arrestin recruitment of activated ETBR remain intact, whereas activation of the ETAR depends on the intramolecular ring size. Increasing of the intramolecular ring structure reduces activity at the ETAR and shifts the peptide toward ETBR selectivity. All ET‐1 analogs displayed efficient ETBR‐mediated signaling by G protein activation and arrestin 3 recruitment. Our study provides in‐depth characterization of the ET‐1/ETAR and ET‐1/ETBR interactions, which has the potential for future development of endothelin‐based drugs for CVD treatment. By identification of Lys9 for selective labeling, novel analogs for peptide‐mediated shuttling by ET‐1 are proposed.

Structural basis of peptide recognition and activation of endothelin receptors

“…This sequence difference elicits a hypothesis that ET B R has a larger pocket space to accommodate the bulkier N-terminal region of ET-3, which is supported by a smaller pocket size (1976 Å 3 , calculated by POVME 31 ) for ET A R relative to ET B R (2059 Å 3 ). This conclusion is in agreement with a previous finding that increasing intramolecular ring size decreased peptide activity at ET A R and shifted the peptide towards ET B R selectivity 32 .…”

Orthogonal Peptide‐Templated Labeling Elucidates Lateral ETAR/ETBR Proximity and Reveals Altered Downstream Signaling

“…For activation of the ET B R the recently described monocyclic ET-1 analog [4Ala 1,3,11,15 , Nle 7 ]-ET-1 was used due to its missing ET A R activation properties. [69] As we demonstrated the reduction of ET B R signaling also for ET A R occupied by the competitive antagonist sitaxentan, ligand scavenging by ET A R can be excluded as reason for the reduced ET B R activation. Further studies demonstrated that ET A R expression does not alter the signaling profile of other G q -coupled GPCRs as demonstrated for the AT 1 R. Therefore, it is likely that ET A R selectively impairs ET B R signaling due to close proximity in the cell membrane e. g. by masking intracellular interaction interfaces and, thus, delaying the initial effector coupling, sequestering endothelin receptor signaling.…”

Role of G-protein coupled receptors in cardiovascular diseases

“…The endothelin system includes two GPCRs: endothelin receptor A (ET A R) and B (ET B R). Endothelin 1 (ET-1) can promote vasoconstriction by activating ET A R or promote vasodilation by activating ET B R ( 59 ). Recent studies have shown that the binding of the orphan receptor GPR75 to 20-hydroxyeicosatetraenoic acid (20-HETE) activates the Gα q/11 protein, which causes vasoconstriction ( 69 ).…”