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 intr acellular domains of the ET(A) receptor by expressing truncated and mu tated ET(A) receptors in COS-7 and CHO cells. All the C-terminal trunc ated ET(A) receptors were produced at a similar expression level and w ere detected in the cell membrane using indirect immunostaining. The s izes of the truncated ET(A) receptors were decreased in proportion to the molecular mass of the truncated amino acid sequence. When the liga nd binding activities were determined for various truncated ET(A) rece ptors, it was found that more than eight amino acid residues at the pr oximal cytoplasmic tail of the ET(A) receptor were required for ET-1 b inding. In addition, the deletion of 16 C-terminal amino acid residues from the third intracellular loop severely decreased the ligand bindi ng activity. It seems that deletion of these cytoplasmic domains of th e ET(A) receptor influences the three-dimensional structure of the lig and binding site located in the extracellular domains. The ET(A) recep tor required more than 13 amino acid residues in the proximity of C-te rminal cytoplasmic tail and 10 amino acid residues in the C-terminal r egion of the third intracellular loop to induce the ET-1 dependent inc rease in intracellular calcium concentration. Both regions are possibl y coupled with G-protein to transmit the ET-1 signal.