Karne S et al. (1993), Cloning and characterization of an endothelin-3...

XB-ART-22221

J Biol Chem 1993 Sep 05;26825:19126-33.

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Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores.

Karne S , Jayawickreme CK , Lerner MR .

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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.

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Species referenced: Xenopus laevis
Genes referenced: edn3 ednra ednrb gcg tbx2

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	FIG. 1. Pigment dispersion of melanophores in response to ETs. Wild type melanophores plated at a concentration of 15,000 cells/well in a 96-well tissue culture dish were treated with 1 nM melatonin for 1h followed by treatment with 10 pM of ET-1 (0)E, T- 2 )(,. or ET-3 (0)T.h e pigment dispersion was measured with a microplate reader at the indicated time points. Each point in the graph is the mean from triplicate samples with error bars representing the corresponding standard errors of means.
	FIG.2 . Pigment dispersionin duced by ET-3. A, three-dimensional representation of pigment dispersion in response to ET-3. Wild type melanophores were pretreated with 1 nM melatonin for 1 h followed by treatment with varying concentrations of ET-3 ranging from 0.1 to 100 nM. Pigment dispersion was measured with a microplate reader. The resulting data are analyzed with respect to time and concentration of ET-3 applied using the distance weighted least squares algorithm (Statistica I11 Program, Statsoft, Inc.). B, twodimensional representation of the data from A. Pigment dispersion response to the seven concentrations of ET-3 was measured following pretreatment with 1 nM melatonin for 1 h 100 (O), 50 (0);2 5 (O), 12.5 (m), 6.25 (A), 3.125 (A),1 .0 (01,an d 0 (e)n M. Each point represents the mean of three samples with error bars representing the corresponding standard errors of the mean.
	FIG. 3. Dose-response curves of wild type melanophores stimulated for 30 min with ET-1 (O), ET-2 (O), and ET-3 (m. The data were derived as outlined in Fig. 1 and as described under "Experimental Procedures."
	FIG. 4. Expression of the PCR-generated fragment in frog melanophores (M) and fibroblasts (F). 1 pg of poly(A') RNA was electrophoresed on 1.5% agarose/formaldehyde gel, transferred onto nylon, and hybridized at 42 'C in 50% formamide solution for 12 h with a random-hexamer primer labeled PCR-generated fragment. The blot was washed with 0.1 X SSPE with 0.1% SDS solution at 65 "C for 20 min and exposed to x-ray film with intensifying screen at -80 'C for 10 h. The quality of the RNA in both lanes was confirmed by staining the blot with 0.2% methylene blue and was found to be same in both lanes. MW, RNA ladder in kb
	FIG. 5. The predicted amino acid sequence, along with the 6'- and 3'-untranslated nucleotide sequence of pETcR. The lines under the amino acid sequence display hydrophobic segmeanst ds etermined byK yte-Doolittle analysis using MacVect3o.r5 (IBI, an Eastman Kodak Co.). The first hydrophobic region consisting of the initial 20 amino acids is a putative signal sequence and the remaining Beven indicate the predicted TM domains. Also indicated are the consensus N-linked glycosylation sites (f). The + designate boundaries of the PCR-generated fragment isolated and useidn screening
	FIG. 6. Amino acid sequence alignment of X. laevis ETcR, bovine ETAR (Arai et d., l990), and rat ETsR (Sakurai et d., 1990). The residues which are common to all three receptor subtypes are displayed with a (-), while gaps between receptors are indicated by (.). The sequence alignment was generated using the PILEUP program of the GCG Sequence Analysis software.
	FIG. 7. Comparison of the amino acid sequence between the different domains of (A) ETA and ETB receptors, (B) ETA and ETc, and (C)E TB and ETc. The domains designated by Arai et al. (1990) and Sakurai et al. (1990) for ET, and ETB receptors, respectively, were used in comparing the sequences. In cases where the lengths of the regions differed between the subtype of ET receptors, the extra amino acids were not used to calculating the percent of identity. The identity between the receptors were divided into three ), 30-60 (O), and 530% (m). ID, intracellular domain; ED, extracellular domain. The numbers in boxes designate transmembrane domains.
	FIG. 8. Competition binding experiments on (A) wild type melnnophorea and (B) Hehi cells transiently transfected with PET&. A, displacement of ['"I]ET-3 from wild type melanophores by unlabeled ET-1 (0) and ET-3 (0). Competition binding experiments were performed in 24-well tissue culture plates at 4 "C with 20 PM ['"I]ET-3 exactly as detailed under "Experimental Procedures." Each point is the mean of three samples from a single experiment, with error bars corresponding to the standard error of mean. Ki values were determined from three experimenta and are given as the average of three experimenta. Nonspecific binding was determined in the presence of 1 p~ ET-3 and WBB found to be less than 5%. B, displacement of ['161]ET-3 from HeLa cells transiently transfected with PET& by unlabeled ET-1 (0) and ET-3 (0). HeLa cells, which had been infected with vaccinia virus for 30 min, were transfected with PET& in pBlueacript byth e lipofection methoda s described by Blakely et al. (1991). Competition binding experiments, as outlined in Fig. 7A, were performed 12-16 h after HeLa cells had been transiently transfected with pETcRN. o specificb inding was detected in wild type HeLa cells, in HeLa cells which had been infected with vaccinia virus, or transfected witht he vector, pBluescript, alon(dea ta not shown). BIB,, B, bound; Bo, maximal bound.