Truncation of N-terminal extracellular or C-terminal intracellular domains of human ETA receptor abrogated the binding activity to ET-1.
We have investigated the function of N-terminal and C-terminal domains of the human ETA receptor by expressing truncated mutants in COS-7 cells. Three kinds of ETA receptors truncated in the N-terminal extracellular or C-terminal intracellular domains were produced. Deletion of the entire extracellular N-terminal or intracellular C-terminal domain completely inactivated the ET-1 binding activity. However, the deletion of one half of the N-terminal extracellular domain of the ETA receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ETA receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ETA receptor was heterogeneous due to differential glycosylation and distributed in 48K, 45K and 42K dalton bands in Western blot analysis. These results demonstrated that a part of the N-terminal domain in close proximity to the first transmembrane region is required for the ligand binding activity of the ETA receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.
Hashido
K
K
Department of Molecular Genetics, Nippon Roche Research Center, Kamakura, Japan.
Gamou
T
T
Adachi
M
M
Tabuchi
H
H
Watanabe
T
T
Furuichi
Y
Y
Miyamoto
C
C
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D016232:Endothelins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017466:Receptors, Endothelin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D013329:Structure-Activity Relationship
D016232:Endothelins; D017466:Receptors, Endothelin