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 (ET_B). Although other agonist-bound structures of ET_B have been determined, the interactions for high-affinity binding and ET_B 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 ET_B in the ligand binding and activation of ET_B using a series of Ala-substituted ET-1(8-21) analogues and the mutated ET_B. 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 ET_B via interactions near the extracellular side. Furthermore, we introduced the mutation into the residues around the ET-1 binding pocket of ET_B. 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 ET_B receptor.

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