Functional domains of human endothelin receptor.

The ligand binding site to the ETA receptor was investigated by substitution of each 5-amino acid sequence located in the second extracellular (B) region of the ETA receptor with the cognate sequences of the beta 2-adrenergic receptor. A 5-amino acid sequence (140-KLLAG-144) in the B-loop region was implicated as the most important element required for ligand binding. In addition, both the third and the fourth extracellular regions (C- and D-loops), including the flanking transmembrane regions, were found to play an important role in ligand selection. As for the biological significance of the intracellular regions of the ETA receptor, we have found that the C-terminal 8-amino acid residues located in close proximity to the seventh transmembrane region and the C-terminal 16-amino acid residues in the third intracellular loop are important for the binding of ET-1. Therefore, the intracellular third loop and C-terminal domains seem to contribute to the three-dimensional structure of the ligand binding site located in the extracellular domains. The same lines of experiment showed that the ETA receptor requires > 13 amino acid residues at the proximal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce an ET-1-dependent increase in [Ca2+]i. Both regions are possibly involved in the interaction with G-protein.