Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores.
We report here the presence of a receptor specific for endothelin-3 (termed ETc receptor or ETcR) on Xenopus laevis dermal melanophores. Activation of ETcR causes the dispersion of the pigment granules within the melanophores. The EC50 for ET-3 to induce the pigment dispersion is 24 +/- 7 nM, compared to greater than 10 microM for both ET-1 and -2. This effect desensitizes in a manner that is dependent on both time and the concentration of ET-3 used to stimulate the cells. A cDNA encoding for ETcR 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 library. The cloned cDNA consists of 2,240 nucleotides, with an open reading frame coding for 444 amino acids containing an initial 20-amino acid signal sequence. The predicted mature peptide consists of 424 amino acids with a heptahelical structure common to the G-protein-coupled receptor surperfamily. Its deduced amino acid sequence is 47 and 52% identical to ETA and ETB receptors, respectively, while ETA and ETB are 48% identical to each other. Expression of cDNA in HeLa cells, which do not contain endothelin receptors, enables the cells to specifically bind [125I]ET-3. Competition binding experiments performed on HeLa cells transiently expressing pETc show that the apparent Ki values for ET-3 and ET-1 to displace [125I]ET-3 are 45.5 +/- 16 and 114 +/- 22 nM, respectively.
Karne
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Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, Connecticut 06510.
Jayawickreme
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CK
Lerner
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MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016232:Endothelins / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D008547:Melanophores / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010860:Pigments, Biological / Q000378:metabolism; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D017466:Receptors, Endothelin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014982:Xenopus laevis
D016232:Endothelins; D007457:Iodine Radioisotopes; D010860:Pigments, Biological; D021382:Protein Sorting Signals; D017466:Receptors, Endothelin; D004247:DNA