CLONING AND CHARACTERIZATION OF AN ENDOTHELIN-3 SPECIFIC RECEPTOR (ET(C) RECEPTOR) FROM XENOPUS-LAEVIS DERMAL MELANOPHORES

We report here the presence of a receptor specific for endothelin-3 (t ermed ET(C) receptor or ET(C)R) on Xenopus laevis dermal melanophores. Activation of ET(C)R causes the dispersion of the pigment granules wi thin the melanophores. The EC50 for ET-3 to induce the pigment dispers ion is 24 +/- 7 nm, compared to greater than 10 muM for both ET-1 and -2. This effect desensitizes in a manner that is dependent on both tim e and the concentration of ET-3 used to stimulate the cells. A cDNA en coding for ET(C)R was isolated by a polymerase chain reaction-mediated DNA amplification strategy using degenerate oligonucleotides prepared based on conserved regions of other known G-protein-coupled receptor sequences and by the subsequent screening of a frog melanophore cDNA l ibrary. The cloned cDNA consists of 2,240 nucleotides, with an open re ading frame coding for 444 amino acids containing an initial 20-amino acid signal sequence. The predicted mature peptide consists of 424 ami no acids with a heptahelical structure common to the G-protein-coupled receptor surperfamily. Its deduced amino acid sequence is 47 and 52% identical to ET(A) and ET(B) receptors, respectively, while ET(A) and ET(B) are 48% identical to each other. Expression of cDNA in HeLa cell s, which do not contain endothelin receptors, enables the cells to spe cifically bind [I-125]ET-3. Competition binding experiments performed on HeLa cells transiently expressing pET(C) show that the apparent K(i ) values for ET-3 and ET-1 to displace [I-125]ET-3 are 45.5 +/- 16 and 114 +/- 22 nM, respectively.