Previous modeling studies as well as more recent crystal str

Previous modeling studies as well as more recent crystal structures of ETB proposed several models of ETB/ligand interaction [7], [9], [18]. 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 Pirfenidone [19], the human orexin receptors [20] and the neuropeptide Y receptors [21], [22]. 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.
Results
Discussion
Materials and Methods
Acknowledgment This work was funded by the Collaborative Research Centre (SFB) 807 of the German Research Foundation (DFG) and supported by the state of Hessen (Center for Biomolecular Magnetic Resonance) and the German Research Foundation (DO545/11, SFB 807). We thank Birgit Schäfer for technical assistance. Author Contributions: F.D., R.B.R. and F.B. conceptualized the project. F.D. and R.B.R. conducted experiments. F.D., S.K., V.D. and F.B. wrote the manuscript. Declaration of Interests: The authors declare no competing financial interests. The article is the authors\' original work, has not received prior publication and is not under consideration for publication elsewhere.
Introduction Preeclampsia (PE) affects 2–8% of all pregnancies worldwide [1], and it is a leading factor of maternal mortality, maternal morbidity, fetal growth restriction (FGR), and preterm birth. Although its pathogenesis remains unclear, a number of hallmarks are central. One of these is the development of a pro-inflammatory state [2], [3] including the B-cell production of functionally-active autoantibodies directed against the Angiotensin II, type 1 receptor (AT1-AA) [4], [5] as well as the endothelin-1 ETA receptor (ETA-AA) [6]. In concordance with endothelin 1 [7], [8], [9], these two autoantibodies increase vasoconstriction activation [10]. Animal studies have demonstrated that AT1-AAs are involved in pathways leading to preeclampsia. Thus, administration of human AT1-AA to pregnant mice induces hypertension, proteinuria, and glomerular endotheliosis, the effects of which can be prevented by the AT1 receptor antagonist losartan [11]. Furthermore, the RUPP rat model of preeclampsia, in which placental ischemia is surgically induced, displays increased expression of TNF-a, IL-6 and AT1-AA [12], [13]. Also the involvement of ETA-AA is rendered probably, as administration of a specific Endothelin 1 receptor A antagonist to RUPP rats and to the rodent sFlt-1 model normalized their blood pressure [14], [15].