Direct evidence for multiple endothelin receptors.

Competition binding experiments and peptide mapping techniques were employed in order to directly address the possible existence of endothelin (ET) receptor subtypes in the atria. Competition binding assays for 125I-labeled ET-1 or 125I-labeled ET-3 to bovine atrial membrane preparations suggest the existence of two ET receptor subtypes, one of which binds ET-1 and ET-3 with a similar affinity while the other shows preference for ET-3. However, cross-linking experiments of both peptides to this tissue resulted in the identification of a single 50-kDa protein. To identify directly the existence of multiple ET receptors, peptide mapping of cross-linked 125I-labeled ET-1 or 125I-labeled ET-3 receptors was conducted. Different peptide maps were obtained only under conditions that preferentially label one receptor subtype. These results indicate, for the first time, the existence of two ET receptor subtypes in the atria which differ from each other in both their binding characteristics and primary structure.     

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Venous smooth muscle contains vasoconstrictor ETB-like receptors.

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.     

Purification of an endothelin receptor from human placenta

We have identified an endothelin (ET) binding protein on the membranes of human placenta and purified it to homogeneity. It is a polypeptide with an apparent Mol. Wt. of 40,000 and is a major protein to be labeled by cross-linking with either 125I-ET-1, -2, or -3. Binding studies with Scatchard analysis indicated the presence of a single class, high-affinity binding site with Kds of 57 pM, 480 pM and 40 nM for 125I-labeled ET-1, ET-2 and ET-3, respectively. These results suggest that the 40K protein is a major ET receptor in placenta and, most likely, can bind differentially to ET-1, ET-2 and ET-3.     

Molecular Determinants for Ligand Selectivity of the Cell-Free Synthesized Human Endothelin B Receptor

Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms.     

Cadmium directly acts on endothelin receptor and inhibits endothelin binding activity.

The binding of endothelin (ET) to human placenta ET receptor was strongly inhibited by cadmium ions (Cd2+) (IC50 = 2 x 10(-5) M). Experiments with affinity cross-linking showed that the major 40 kDa receptor was inhibited to form a [125I]ET-1/receptor complex. The mode of inhibition was noncompetitive with respect to ET-1. The inhibitory effect of Cd2+ on solubilized ET receptor was partially reversed by the chelating agent, ethylenediaminetetraacetic acid (EDTA), whereas the effect was irreversible for the membrane-associated receptor. The rat aorta contractions by ET were prevented by pretreatment or addition of Cd2+.     

Non-isopeptide-selective endothelin receptors in human Girardi heart cells.

We characterized the endothelin (ET) receptor in Girardi heart (GH) cells derived from human atrium. The ET isopeptides ET-1, ET-2 and ET-3 induced the monotonous and long-lasting rise in cytosolic free Ca2+ concentration [( Ca2+]i) with almost the same potency in GH cells. Scatchard analysis of [125I]ET-1 and [125I]ET-3 binding revealed that GH cells have almost the same number of binding sites for either labeled ligand. All ET isopeptides displaced either [125I]ET-1 or [125I]ET-3 binding in GH cells almost equipotently. These results reveal that the functional ET receptors in GH cells are of the ETB-type. GH cells are the first cell line to be found to express the functional ETB-receptor.     

A potent and specific agonist, Suc-[Glu9,Ala11,15]-endothelin-1(8-21), IRL 1620, for the ETB receptor.

A series of C-terminal linear peptides of endothelin (ET)-1 and their N alpha-succinyl (Suc) analogs were synthesized and their binding affinities for the two subtypes of ET receptor, ETA and ETB, in porcine lung membranes were examined. Among the synthetic analogs, Suc-[Glu9,Ala11,15]-ET-1(8-21), IRL 1620, was the most potent and specific ligand for the ETB receptor (KiETA/KiETB approximately equal to 120,000) as judged by the Ki values for ETA (1.9 microM) and ETB (16 pM) receptors. IRL 1620 was 60 times more selective for the ETB receptor than ET-3 (KiETA/KiETB approximately equal to 1,900). IRL 1620 (10(-9)-10(-7) M) induced contractions of the guinea pig trachea with a comparable potency to those of ET-1 or ET-3, suggesting that IRL 1620 is a potent ETB receptor agonist.     

Lysosomal traffic of liganded endothelin B receptor

The endothelin B receptor (ETB) is an endothelial cell receptor found in caveolae. Studies with GFP-tagged ETB have suggested that the protein is constitutively endocytosed and targeted to lysosomes where it is rapidly degraded. We report that iodinated endothelin-1 ligand (ET-1) is taken up by cells transfected with ETB and remains undegraded for at least 17 h. Analysis of the intracellular traffic of endocytosed ET-1 on isotonic Ficoll gradients shows that it is rapidly internalised to lysosomes by a chloroquine sensitive and cholesterol dependent pathway. Low-temperature nonreducing SDS gels show that the ET-1 initially binds to full-length GFP-tagged ETB, which is rapidly clipped at the amino-terminus and is then stable for at least 6 h. Analysis of GFP tagged ETB on reducing SDS gels shows that it is proteolytically cleaved with a half time of approximately 3 h. However, nonreducing gels show that the receptor is virtually intact, suffering only a similar cleavage to the liganded receptor. We conclude that the ETB receptor shows remarkable stability in lysosomes, held together by disulfide bonds, and maintaining ligand binding for long periods of time.     

Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Self-regulation of the endothelin receptor system in choriocarcinoma cells

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.     

Effects of human peptide endothelin-1 and two of its sterically unrestrained C-terminal fragments on coronaryvascular smooth muscle

Clearance of human peptide endothelin-1 (ET-1) has been proposed to follow a receptor pathway involving a cascade of ET-1 receptor endocytosis and lysosomal degradation by a family of proteinases expressed constitutively by most cells. Genetically distinct endopeptidases produce ET-1 and degrade mature peptide. The ET-1 degradation products were considered to be inactive, however, recent evidence suggests that ET-1 fragments sustain most of the homeostatic response produced by parent peptides. The purpose of this study was to establish whether the overall structure of human ET-1 or the structure of its C-terminus is responsible for the subtype-selectivity, down-regulation and clearance of endothelin, and whether D-aminoacid substitution in the moiety of synthetic peptide is involved in effective ET-1 antagonism in coronary vascular smooth muscle. To characterize specific mechanism(s) leading to subtype-selective ET-receptor down-regulation and/or to ET-1 antagonism, ligand binding studies were accomplished with radioactive human (1-21)ET-1 and with C-terminal ET-1 fragments, both peptide agonists and antagonists, in adult male porcine coronary artery vascular smooth muscle (CVSM). The subcellular membranes of CVSM were isolated by isopycnic gradient centrifugation. Exposure of porcine coronary artery to exogenous ET-1 induced endothelin-ETB selective down-regulation. ETA-mediated subtype-ETB down-regulation was observed with distribution of ligand-ETB receptor complexes in light, endosomal, membranes. The ETA selective PD151242 significantly attenuated [3H]-thymidine incorporation, and the ETB selective antagonist BQ788 blocked down-regulation observed in porcine vascular fibroblasts (PF). Preincubation of coronary arteries with ETB selective BQ3020 was accompanied with a more intense down-regulation. CONCLUSION: our data are indicative of short-term ETB selective down-regulation of endothelin receptors in coronary vascular smooth muscle after exposure to ET-1. The presence in the carboxy-terminus of (Ala11,15) substitution in peptide fragments IRL1620 and BQ3020 determined the differential specificity of ETB-receptor coupling and was important for subtype-ETB-receptor down-regulation. The activation of the dominating ETA-receptor by ET-1 facilitated mitogenic responses to ET-1 in porcine vascular fibroblasts.     

Cross-linking reconsidered: binding and cross-linking fields and the cellular response.

We analyze a model for the reversible cross-linking of cell surface receptors by a collection of bivalent ligands with different affinities for the receptor as would be found in a polyclonal anti-receptor serum. We assume that the amount of cross-linking determines, via a monotonic function, the rate at which cells become activated and divide. In addition to the density of receptors on the cell surface, two quantities, the binding field and the cross-linking field, are needed to characterize the cross-linking curve, i.e., the equilibrium concentration of cross-linked receptors plotted as a function of the total ligand site concentration. The binding field is the sum of all ligand site concentrations weighted by their respective binding affinities, and the cross-linking field is the sum of all ligand site concentrations weighted by the product of their respective binding and cross-linking affinity and the total receptor density. Assuming that the cross-linking affinity decreases if the binding affinity decreases, we find that the height of the cross-linking curve decreases, its width narrows, and its center shifts to higher ligand site concentrations as the affinities decrease. Moreover, when we consider cross-linking-induced proliferation, we find that there is a minimum cross-linking affinity that must be surpassed before a clone can expand. We also show that under many circumstances a polyclonal antiserum would be more likely than a monoclonal antibody to lead to cross-linking-induced proliferation.     

Absence of endothelin receptors and receptor mRNA in mammalian fibroblasts transformed with SV40 or ras oncogene

Endothelin-1 (ET-1), a peptide isolated from the culture medium of endothelial cells, mediates a variety of physiological and pathological responses including mitogenesis. We have compared the expression of ET receptors in untransformed versus ras-transformed NIH-3T3 murine fibroblasts and in untransformed versus SV40-transformed Wl38 (VA13) human fibroblasts by ligand binding and Northern analysis. NIH-3T3 and Wl38 cells displayed high affinity (200 and 220 pM) and high density (23,000 sites/cell and 14,000 sites/cell for NIH-3T3 and Wl38 cells, respectively) ET receptors. Competition binding experiments using subtype-selective ligands identified these receptors as the ETA subtype. Addition of ET-1 to the cells produced a concentration-dependent increase in intracellular calcium release. Both ras-transformed NIH-3T3 cells and SV40-transformed Wl38 cells (VA13) completely lacked [125I]ET-1 binding and failed to release calcium when exposed to ET-1. Northern analysis of the polyadenylat ed RNA (polyA RNA) isolated from untransformed and transformed cells revealed that the steady-state level of ETA receptor RNA was 90-95% less in transformed cells compared to untransformed cells. Thus, the loss of ET receptors as well as the receptor-mediated responses in transformed cells can be explained by down-regulation of ET receptor mRNA.     

Endothelin and structurally related analogs distinguish between endothelin receptor subtypes.

The endothelin (ET) analog ET-1[1,3,11,15-Ala] was compared with ET-1, ET-2, ET-3 and sarafotoxins (SRTX) S6b and S6c for receptor binding and function. All the peptides exhibited high affinity binding and contracted rabbit pulmonary artery with near equal potency. In rat aorta both ET-3 and ET-1 [1,3,11,15-Ala] bound with much lower affinity than ET-1 while ET-3 displayed weak contractile potency and ET-1 [1,3,11,15-Ala] and SRTX-c were inactive. In rat left atria, ET-1 [1,3,11,15-Ala] and SRTX-c were weak inhibitors of binding and were also functionally inactive, whereas ET-1, ET-2, ET-3, and SRTX-b were equipotent in producing contractile responses. The data support the idea of there being a predominance of ETA receptors in rat aorta and ETB receptors in rabbit pulmonary artery. In rat left atria, the ET receptor could not be readily classified into ETA or ETB and suggests the existence of a new receptor subtype.     

A novel 31-amino-acid-length endothelin, ET-1(1-31), can act as a biologically active peptide for vascular smooth muscle cells

Human chymase produces a novel endothelin-1 with 31 amino-acid length ?ET-1(1-31)?, which is longer than conventional ET-1, ?ET-1(1-21)?. The aim of our study was to investigate the role of ET-1(1-31) on porcine coronary vascular smooth muscle cell (VSMC). Although the increase in [Ca(2+)](i) by ET-1(1-31) was 10 times weaker than that of ET-1(1-21), ET-1(1-31) showed equivalent potency in VSMC proliferation, c-fos/c-myc mRNA expression and cell cycle analysis with ET-1(1-21). ET-1(1-31) significantly induced expression of cyclin D1 but not those of cyclin D2 or D3. These effects were specifically inhibited by BQ485, an ET(A) receptor antagonist, although that of ET-1(1-21) was not specific to BQ485, suggesting different receptor specificity from ET-1(1-21). These results indicate that ET-1(1-31) also can involve a VSMC proliferation process such as atherosclerosis, although it has weaker vasoconstricting potency and different receptor subtypes on VSMC from those of ET-1(1-21).     

Characteristic defects in neural crest cell-specific Galphaq/Galpha11- and Galpha12/Galpha13-deficient mice

The endothelin/endothelin receptor system plays a critical role in the differentiation and terminal migration of particular neural crest cell subpopulations. Targeted deletion of the G-protein-coupled endothelin receptors ET(A) and ET(B) was shown to result in characteristic developmental defects of derivatives of cephalic and cardiac neural crest and of neural crest-derived melanocytes and enteric neurons, respectively. Since both endothelin receptors are coupled to G-proteins of the G(q)/G(11)- and G(12)/G(13)-families, we generated mouse lines lacking Galpha(q)/Galpha(11) or Galpha(12)/Galpha(13) in neural crest cells to study their roles in neural crest development. Mice lacking Galpha(q)/Galpha(11) in a neural crest cell-specific manner had craniofacial defects similar to those observed in mice lacking the ET(A) receptor or endothelin-1 (ET-1). However, in contrast to ET-1/ET(A) mutant animals, cardiac outflow tract morphology was intact. Surprisingly, neither Galpha(q)/Galpha(11)- nor Galpha(12)/Galpha(13)-deficient mice showed developmental defects seen in animals lacking either the ET(B) receptor or its ligand endothelin-3 (ET-3). Interestingly, Galpha(12)/Galpha(13) deficiency in neural crest cell-derived cardiac cells resulted in characteristic cardiac malformations. Our data show that G(q)/G(11)- but not G(12)/G(13)-mediated signaling processes mediate ET-1/ET(A)-dependent development of the cephalic neural crest. In contrast, ET-3/ET(B)-mediated development of neural crest-derived melanocytes and enteric neurons appears to involve G-proteins different from G(q)/G(11)/G(12)/G(13).     

Endothelin stimulates glucose uptake and GLUT4 translocation via activation of endothelin ETA receptor in 3T3-L1 adipocytes

Endothelin-1 (ET-1) is a 21-amino acid peptide that binds to G-protein-coupled receptors to evoke biological responses. This report studies the effect of ET-1 on regulating glucose transport in 3T3-L1 adipocytes. ET-1, but not angiotensin II, stimulated glucose uptake in a dose-dependent manner with an EC50 value of 0.29 nM and a 2.47-fold stimulation at 100 nM. ET-1 stimulated glucose uptake in differentiated 3T3-L1 cells but had no effect in undifferentiated cells, although ET-1 stimulated phosphatidylinositol hydrolysis to a similar degree in both. The 3T3-L1 cells expressed approximately 560,000 sites/cell of ETA receptor, which was not altered during differentiation. Western blot analysis and immunofluorescence staining show that ET-1 stimulated the translocation of insulin-responsive aminopeptidase and GLUT4 to the plasma membrane. The effect of ET-1 on glucose uptake was blocked by A-216546, an antagonist selective for the ETA receptor. ET-1 treatment did not induce phosphorylation of insulin receptor beta-subunit, insulin receptor substrate-1, or Akt but stimulated the tyrosyl phosphorylation of a 75-kDa protein. Genistein (100 microM), an inhibitor of tyrosine kinases, inhibited ET-1-stimulated glucose uptake. Our results show that ET-1 stimulates GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes via activation of ETA receptor.