IDENTIFICATION OF SPECIFIC INTRACELLULAR DOMAINS OF THE HUMAN ET(A) RECEPTOR REQUIRED FOR LIGAND-BINDING AND SIGNAL-TRANSDUCTION

We have investigated the function of the C-terminal and the third intracellular domains of the ET(A) receptor by expressing truncated and mutated ET(A) receptors in COS-7 and CHO cells. All the C-terminal truncated ET(A) receptors were produced at a similar expression level and were detected in the cell membrane using indirect immunostaining. The sizes of the truncated ET(A) receptors were decreased in proportion to the molecular mass of the truncated amino acid sequence. When the ligand binding activities were determined for various truncated ET(A) receptors, it was found that more than eight amino acid residues at the proximal cytoplasmic tail of the ET(A) receptor were required for ET-1 binding. In addition, the deletion of 16 C-terminal amino acid residues from the third intracellular loop severely decreased the ligand binding activity. It seems that deletion of these cytoplasmic domains of the ET(A) receptor influences the three-dimensional structure of the ligand binding site located in the extracellular domains. The ET(A) receptor required more than 13 amino acid residues in the proximity of C-terminal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce the ET-1 dependent increase in intracellular calcium concentration. Both regions are possibly coupled with G-protein to transmit the ET-1 signal.