Truncation of N-terminal extracellular or C-terminal intracellular domains of human ET A receptor abrogated the binding activity to ET-1

Abstract

We have investigated the function of N-terminal and C-terminal domains of the human ET A receptor by expressing truncated mutants in COS-7 cells. Three kinds of ET A 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 ET A receptor, missing one of two N-linked glycosylation sites, maintained complete binding activity. Specific monoclonal antibodies detected all the truncated ET A receptors in the cell membrane fraction of transfected COS-7 cells. The size of the ET A 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 ET A receptor, and the C-terminal domain is perhaps necessary as an anchor for maintenance of the binding site.