Characterization of Critical Residues in the Extracellular and Transmembrane Domains of the Endothelin Type B Receptor for Propagation of the Endothelin-1 Signal

We have previously reported the crystal structures of endothelin-1 (ET-1)-bound, ligand-free, and antagonist bosentan-bound forms of the thermostabilized ET type B receptor (ETB). Although other agonist-bound structures of ETB have been determined, the interactions for high-affinity binding and ETB receptor activation, as well as the roles of rearrangement of the hydrogen-bond network surrounding the ligand in G protein activation, remain elusive. ET-1, a 21-amino acid residue peptide, plays fundamental roles in basal vascular tone, sodium balance, cell proliferation, and stress-responsive regulation. We studied the interactions between the ET-1(8-21) peptide and ETB in the ligand binding and activation of ETB using a series of Ala-substituted ET-1(8-21) analogues and the mutated ETB. We found that while D8, L17, D18, I20, and W21 were responsible for high-affinity binding and potent G protein activation, Y13 and F14 in the helical region of ET-1 are prerequisites for the full activation of ETB via interactions near the extracellular side. Furthermore, we introduced the mutation into the residues around the ET-1 binding pocket of ETB. The results showed that while S184(3.35), W336(6.48), N378(7.45), and S379(7.46) in a conserved polar network are required for full activation, N119(1.50), D147(2.50), and N382(7.49) are essential for G protein activation via direct interactions after rearrangement upon ET-1 binding. These results demonstrate that both interactions near the extracellular side and within the transmembrane helices with ET-1 play crucial roles in the full activation of the ETB receptor.