br Conflict of interest br Introduction Endothelin receptors
Conflict of interest
Introduction Endothelin receptors are members of the superfamily of G-protein-coupled receptors (GPCRs) and central regulators of vascular tension and other physiological functions in higher eukaryotes . The human endothelin system comprises the highly homologous endothelin-A (ETA) and endothelin-B (ETB) receptors, as well as the three related natural 21-amino-acid peptide agonists endothelin (ET)-1, ET-2 and ET-3. The two receptors recognize the agonists ET-1 and ET-2 with similar high affinity, but they have a marked differential selectivity for ET-3 and numerous pharmaceutically relevant artificial agonists. This variation in their ligand portfolio in combination with complex AP1903 and patterns of the receptors and ligands in various tissues is supposed to be a major determinant for their sometimes divergent effects on human physiology , . Unbalanced ET-1 levels affect vasoconstriction and cause severe diseases such as pulmonary arterial hypertension . Activating ETB as vasodilator by ETB-selective agonists  or the blockade of vasoconstriction caused by ETA  are currently discussed as future strategies for medical applications. ETB displays selectivity for the ET-1 analog ET-3 for the related sarafotoxin 6c from the venom of the snake black mamba and for artificial agonists such as the linear peptides [Ala1,3,11,15]-endothelin-1 (4Ala-ET-1) and Suc-[Glu9,Ala11,15]-endothelin-18–21 (IRL1620) (Fig. 1a). In contrast, ETA is rather selective to small chemical compounds such as bosentan and sitaxentan . A multiple molecular interaction network of the ETB/ET-1 complex was recently revealed by crystallization , and a conformational dynamics has been observed in ligand-bound and ligand-free forms , . Similar to other GPCRs such as opioid receptors or the neurotensin receptor, ET-1 docks into the ETB orthosteric pocket mainly formed by transmembrane domains and covered by a conserved signature β-hairpin motif in the extracellular loop 2 (ECL2) of peptide binding GPCRs . However, the molecular mechanisms of ligand discrimination between ETB and ETA are unknown. Despite diverse physiological functions and ligand portfolios, GPCRs have common mechanisms to trigger downstream signaling by G-protein activation . These mechanisms are determined by the more conserved membrane-integrated seven-transmembrane helix (TM1–7) bundle and the intracellular domains (Fig. 1b) , . The initial ligand recognition is modulated by the extracellular domains, and understanding the molecular details of the ETB ligand selectivity could provide valuable inputs for directed drug design . The ETA and ETB receptors share an overall 57% sequence identity and variations in between the N-terminal domain and the extracellular loops ECL1–3 are most likely relevant for ligand discrimination (Fig. S1). We have constructed chimeric ETB receptors by swapping structural units of the N-terminal domain and of the ECL1–3 loops of ETA with corresponding units of ETB (Fig. S2). The chimeras were analyzed for their affinity to the common non-selective agonist ET-1 as well as to a selection of ETB-selective agonists. All GPCR derivatives were cell-free synthesized in the presence of preformed empty nanodiscs (NDs) assembled with the lipid 1,2-dielaidoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DEPG) that has previously been shown to provide a suitable environment for the membrane integration and folding of ETB , . The strategy of co-translational insertion of the GPCRs into defined membranes ensured that GPCR folding and ligand interaction is not affected by detergent contacts, as all studies could be performed in native-like membrane environments . Previous modeling studies as well as more recent crystal structures of ETB proposed several models of ETB/ligand interaction , , . Our studies indicate the second β-strand and a short linker region in ECL2 as well as the N-terminus of ETB (Fig. S1) as essential selectivity filters for several agonists such as IRL1620, sarafotoxin 6c and 4Ala-ET-1. Interactions of the ligands with the N-terminal domain of ETB further support selectivity at different extents and are most important for IRL1620 and 4Ala-ET-1. In contrast, the tight binding of ET-3 requires synergic interactions with both ECL2-B2 and the N-terminal domain, thus indicating a more complex recognition and interaction network for this ETB-selective agonist. We further present evidence that the overall topology of the common agonist ET-1 and constraints within its N-terminal domain are further discrimination parameters for ETB and ETA. Our data agree with other selectivity models of peptide binding GPCRs, including the human δ-opioid receptor , the human orexin receptors  and the neuropeptide Y receptors , . Several engineered ETB derivatives were generated that have lost the binding of ETB specific agonists, while high affinity to the agonist ET-1 common to ETB and ETA remained conserved. The documented strategy could therefore open alternative options to address ligand selectivity of GPCRs and to analyze GPCR/drug interactions on the molecular level.