FUNCTIONAL DOMAINS OF HUMAN ENDOTHELIN RECEPTOR

The ligand binding site to the ET(A) receptor was investigated by subs titution of each 5-amino acid sequence located in the second extracell ular (B) region of the ET(A) receptor with the cognate sequences of th e beta2-adrenergic receptor. A 5-amino acid sequence (140-KLLAG-144) i n the B-loop region was implicated as the most important element requi red for ligand binding. In addition, both the third and the fourth ext racellular regions (C- and D-loops), including the flanking transmembr ane regions, were found to play an important role in ligand selection. As for the biological significance of the intracellular regions of th e ET(A) receptor, we have found that the C-terminal 8-amino acid resid ues 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 t hird loop and C-terminal domains seem to contribute to the three-dimen sional structure of the ligand binding site located in the extracellul ar domains. The same lines of experiment showed that the ET(A) recepto r requires >13 amino acid residues at the proximal cytoplasmic tail an d 10 amino acid residues in the C-terminal region of the third intrace llular loop to induce an ET-1-dependent increase in [Ca2+]i. Both regi ons are possibly involved in the interaction with G-protein.