CDB15:0000465 EDN3 — EDNRB

ABSTRACT: We report the cloning of human cDNA encoding an ETB (non-isopeptide-selective) subtype of the endothelin receptor. The predicted amino acid sequence of the human ETB endothelin receptor was 87.8% and 62.9% identical with the previously cloned rat ETB and ETA receptors, respectively. COS cells transiently transfected with the cloned cDNA expressed specific, high-affinity binding sites for endothelin isopeptides and responded to the peptides with a transient increase of [Ca2+]i; endothelin-1 and endothelin-3 exhibited approximately equal potencies both in displacing 125I-labeled endothelin-1 binding and in eliciting [Ca2+]i transients. The ETB receptor mRNAs were expressed in various human tissues and also in the intact porcine aortic intimal cells ex vivo.

ABSTRACT: Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.

ABSTRACT: The amount of endothelin receptors in human prostate tissue was measured by radioligand binding techniques using 125I-Endothelin -1 and -3 (125I-ET-1, -3). Specimens of the non-hypertrophy group were obtained from 6 patients who underwent total cystectomy under the diagnosis of bladder cancer and those of the hypertrophy group from 6 prostatic hypertrophy patients who underwent open prostatectomy. 125I-ET-1 bound to the prostate tissue with the KD value of 0.033 +/- 0.012 nM in the non-hypertrophy group and with the KD value of 0.035 +/- 0.012 nM in the hypertrophy group. 125I-ET-3 bound to the prostate tissue with the KD value of 0.023 +/- 0.011 nM in the non-hypertrophy group and with the KD value of 0.029 +/- 0.016 nM in the hypertrophy group. The KD values were not significantly different between the hypertrophy and non-hypertrophy groups. The KD values of 125I-ET-1 and 125I-ET-3 were similar. The Bmax values (fmol/mg protein) of 125I-ET-1 binding to the prostate tissue were 32.18 +/- 3.69 to the non-hypertrophy group and 85.66 +/- 20.65 to the hypertrophy group. The Bmax values (fmol/mg protein) of 125I-ET-3 binding to the prostate tissue were 27.48 +/- 5.25 to the non-hypertrophy group and 75.90 +/- 13.46 to the hypertrophy group. The Bmax values of both 125I-ET-1 and 125I-ET-3 were significantly higher in the hypertrophy group than in the non-hypertrophy group.(ABSTRACT TRUNCATED AT 250 WORDS)

ABSTRACT: Defects in the gene encoding the endothelin-B receptor produce aganglionic megacolon and pigmentary disorders in mice and humans. We report that a targeted disruption of the mouse endothelin-3 ligand (EDN3) gene produces a similar recessive phenotype of megacolon and coat color spotting. A natural recessive mutation that results in the same developmental defects in mice, lethal spotting (ls), failed to complement the targeted EDN3 allele. The ls mice carry a point mutation of the EDN3 gene, which replaces the Arg residue at the C-terminus of the inactive intermediate big EDN3 with a Trp residue. This mutation prevents the proteolytic activation of big EDN3 by ECE-1. These findings indicate that interaction of EDN3 with the endothelin-B receptor is essential in the development of neural crest-derived cell lineages. We postulate that defects in the human EDN3 gene may cause Hirschsprung's disease.

ABSTRACT: To investigate the endothelin-3 (ET-3) binding region of the endothelin-B (ETB) receptor, we have transiently produced various chimeric endothelin receptors in transfected Chinese hamster ovary cells. Using 125I-ET-1 as the radioactive ligand in the displacement experiment, the replacement of both the second and third extracellular regions including the flanking transmembranes of the ETB receptor with the corresponding domains of the endothelin-A (ETA) receptor, increased the apparent Ki value for ET-3 from 5 x 10(-11) M to 10(-8) M. The affinity of this chimeric receptor, ETB-BC, for ET-3 was about two orders lower than ETB yet one order higher than ETA. Previously we have reported the involvement of Lys-140 located in the C-terminus of the second transmembrane region of the ETA receptor for ET-1 binding (Eur. J. Biochem., 220, 37-43, 1994). To assess the importance of the corresponding Lys-161 of the ETB receptor in ET-3 binding, we have replaced it with Ile in the ETB receptor. The mutant receptor had a 5.6-fold reduction in its affinity for ET-3, but its affinity for ET-1 remained similar. These results demonstrate that Lys-161 of the receptor is important for high affinity binding with ET-3 which, in part, confers the non-selective binding characteristics of the ETB receptor for ET isopeptides.

ABSTRACT: 1. We determined competition binding characteristics of endothelin ETB receptor selective ligands in human left ventricle and compared these values to those obtained with rat left ventricle. Sarafotoxin S6c, ET-3, BQ788 and IRL2500 competed against [125I]-PD151242 (ETA selective radioligand) with low affinity in human left ventricle, confirming the ETB selectivity of these compounds. 2. ET-3 competed with moderate selectivity for ETB over ETA receptors in human left ventricle and with slightly higher selectivity in rat left ventricle (460 and 1,400 fold, respectively). There was a small difference in the affinity of ETA receptors for ET-3 (KD ETA in human left ventricle = 0.07 +/- 0.02 microM; KD ETA in rat left ventricle = 0.27 +/- 0.08 microM; P = 0.05) but no difference in the affinity of ETB receptors for this ligand (KD ETB in human left ventricle = 0.15 +/- 0.06 nM; KD ETB in rat left ventricle = 0.19 +/- 0.03 nM). 3. The selectivity of sarafotoxin S6c for ETB over ETA receptors in human left ventricle was 5,900 fold compared with 59,400 fold in rat left ventricle. The affinity of ETA receptors for sarafotoxin S6c was higher in human than in rat left ventricle (KD ETA = 2.00 +/- 0.20 microM and 3.50 +/- 0.26 microM, respectively; P = 0.03), while the affinity of ETB receptors for this ligand was higher in rat left ventricle (KD ETB = 0.06 +/- 0.02 nM) than in human left ventricle (KD ETB = 0.34 +/- 0.13 nM) (P = 0.02). The affinity of ETB receptors for sarafotoxin S6c in rat left ventricle determined in the absence or presence of GTP was the same indicating that differing affinity states of ETB receptors in human and rat left ventricle do not account for the variation observed between species. 4. There was no difference in the affinity of ETA receptors for BQ788 (KD ETA = 1.01 +/- 0.20 microM and KD ETA = 1.39 +/- 0.35 microM) or for the novel ETB selective antagonist. IRL2500 (KD ETA = 30.0 +/- 20.8 microM and KD ETA = 55.6 +/- 9.93 microM) in human and rat left ventricle, respectively. ETB receptors had a significantly higher affinity for BQ788 (KD ETB = 9.8 +/- 1.3 nM and KD ETB = 31.0 +/- 5.4 nM; P = 0.02) and IRL2500 (KD ETB = 78.2 +/- 9.7 nM and KD ETB = 300.0 +/- 75.1 nM; P = 0.03) in human and rat left ventricle, respectively. The synthetically synthesized ETB selective antagonist RES-701-1 (0.1 -3 microM) failed to inhibit [125I]-ET-1 binding in either tissue. 5. In conclusion, we have compared equilibrium dissociation constants for a number of ETB selective compounds in human and rat heart. The affinity of ETB receptors for sarafotoxin S6c, BQ788 and IRL2500 differed in human and rat left ventricle. No difference in affinity was detected for ET-3 binding at ETB receptors. Sarafotoxin S6c binding was unaffected by GTP indicating that the different receptor affinities in human and rat heart cannot be explained by differing ETB receptor affinity states. This study highlights the need to consider differences in binding characteristics that may arise from the use of tissues obtained from different species.