Identification of a region of the human endothelin ETA receptor required for interaction with bosentan.
Bosentan (Ro 47-0203, 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2- methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzenesulfonamide) is a new non-peptidic mixed antagonist of endothelin receptors whose binding activity was two orders higher for the endothelin ETA receptor than that for the endothelin ETB receptor. To identify which region of the human endothelin ETA receptor interacts with bosentan, we created various chimeric endothelin receptors containing domains from the endothelin ETA and ETB receptors in Chinese hamster ovary cells and studied the effect of bosentan on the binding of endothelin-1 to the chimeric receptors. We found that the chimeric endothelin ETB receptor containing domains from the endothelin ETA receptor, the second extracellular region including the proximal transmembrane region (B-region) revealed an affinity toward bosentan which was similar to that of the endothelin ETA receptor. In contrast, the chimeric endothelin ETA receptor, containing the B-region of the endothelin ETB receptor, reduced the binding affinity to the level of the endothelin ETB receptor. Since bosentan competes with endothelin-1 for binding to the endothelin ETA receptor, this receptor antagonist seems to interact with the (140-144) KLLAG sequence located at the carboxylterminus of the second transmembrane region of the endothelin ETA receptor, required for the natural ligand binding.
Department of Molecular Genetics, Nippon Roche Research Center, Kamakura, Japan.
M
M
Adachi
Y
Y
Furuichi
C
C
Miyamoto
D000595:Amino Acid Sequence; D000818:Animals; D000077300:Bosentan; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D017466:Receptors, Endothelin / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects; D013449:Sulfonamides / Q000737:chemistry* / Q000494:pharmacology*
D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D017466:Receptors, Endothelin; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D000077300:Bosentan; D002118:Calcium