Characterization of Endothelin Receptors ETA arid ETB Expressed in COS Cells

Takasuka, Tsuyoshi; Adachi, Miki; Miyamoto, Chikara; Furuichi, Yasuhiro; Watanabe, T.

doi:10.1093/oxfordjournals.jbchem.a123911

Cited by 40 publications

(22 citation statements)

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“…3,6 125 I-ET-1 and the ET B ⅐GFP receptor form tight, sodium dodecyl sulfate-stable complexes, which can be detected by autoradiography without cross-linking. 19 Therefore, VSMCs expressing ET B ⅐GFP receptor were incubated with 100 pmol/L 125 I-ET-1 at 4°C (to prevent receptor internalization) for 30 minutes and then either lysed or incubated for another 3 hours at 37°C. Lysates were then separated by lowtemperature polyacrylamide gel electrophoresis at 4°C.…”

Section: Agonist-dependent and Agonist-independent Cleavage Of The Etmentioning

confidence: 99%

N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

Grantcharova

Reusch

Großmann

et al. 2006

ATVB

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Objective-The extracellular N terminus of the endothelin B (ET B ) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ET B receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs). Methods and Results-VSMCs expressing either the full-length ET B receptor or an N-terminally truncated ET B receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ET B receptor, IRL1620 (an ET B -selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22␣, and ␣-actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of G i proteins. In contrast, in VSMCs expressing N-terminally truncated ET B receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression. Key Words: transactivation Ⅲ EGF receptor Ⅲ ERK1/2 Ⅲ differentiation E ndothelin-1 (ET-1), one of the most potent vasoactive peptides, acts on 2 G protein-coupled receptors (GPCRs): the ET A receptor mainly expressed in vascular smooth muscle cells (VSMCs) and the endothelin B (ET B ) receptor predominantly expressed in endothelial cells. 1,2 Whereas VSMCs normally express only low levels of ET B receptors, a dramatic upregulation occurs during atherosclerosis, hypercholesterolemia, and in models of focal ischemia. [3][4][5][6] In atherosclerotic plaques, VSMCs close to foam cells show a predominant expression of ET B receptors and an enhanced immunoreactivity of ET-1. It was therefore suggested that ET-1 and ET B receptors may play a role in atherogenesis. 4 A protective role for the ET B receptor has been reported for the monocrotalin-induced pulmonary hypertension in mice. 7 Similarly, inhibition of the ET B receptor deteriorated remodeling after vascular injury in mice, 8 pointing to a protective role of the ET B receptor in atherosclerosis. Conclusions-ThisHowever, the precise role of ET B receptors in VSMCs remains elusive.VSMCs in native vessels are characterized by a slow proliferation rate and an abundant expression of contractile proteins, such as smooth muscle (SM) myosin and SM ␣-actin. On vascular injury, VSMCs show a loss of contractile protein expression and increased proliferative and migratory responses to mitogens or cytokines. 9,10 However, within 3 to 6 months after vascular injury, VSMCs may regain a contractile phenotype through yet unknown mechanisms. 11 It is notable that thrombin via the protease-activated receptor 1 (PAR1) 12,13 and rapamycin, a blocker of the mammalian target of rapamycin, induce differentiation of VSMCs b...

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Section: Agonist-dependent and Agonist-independent Cleavage Of The Etmentioning

confidence: 99%

N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

Grantcharova

Reusch

Großmann

et al. 2006

ATVB

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“…Conversely, the ET, subtype has similar affinities for the three ET isopeptides, as seen after transient and stable heterologous expression [2,[12][13][14]. The newly described ET, subtype which was identified in frog melanophores exhibits an ET-3-selective profile when expressed transiently in HeLa cells [3].…”

mentioning

confidence: 99%

Mutational analysis of human endothelin receptors ET_A and ET_B

Becker¹,

Haendler²,

Hechler³

et al. 1994

European Journal of Biochemistry

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Two endothelin(ET)-receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ETA displaying an ET-1-selective profile and ET, a nonselective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS-7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand-binding analysis. COS-7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3-'251]iodotyrosyl ET-1 (*"I-ET-l) binding, with affinities comparable to those of the wild-type receptors (apparent K, = 1-6 X lo-").An average of 10'-106 binding sitedcell was calculated for the wild-type and mutant forms. In competition experiments using 'Z'I-ET-l and unlabeled ET-3, an ET,-selective agonist, we detected a clear switch from an ET-1-selective profile to a non-isopeptide-selective profile in ETA chimeras where the second extracellular loop and the flanking transmembrane domains IV and V, or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ET,. The opposite effect, namely a switch from a non-isopeptide-selective to an ET-1-selective binding, was observed for the mirror ET, chimeras where the symmetrical exchange had been operated. Using 1251-ET-l and the ETA-specific antagonist cyclo-(D-Trp-D-Asp-Pro-D-val-Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the Nterminal moiety of this receptor. Therefore, the ability for the interaction with ET-3 or BQ123 is governed by two different regions of the ET receptors.Cloning and sequencing of the three endothelin(ET)-receptor subtypes ETA, ET, and ET, [l -31 revealed remarkable structural similarities between them and the members of the guanine-nucleotide-binding-regulatory-protein(G-pr0-tein)-coupled, seven-transmembrane-domain receptor superfamily [4, 51. The hydrophobicity profile of the ET receptors is similar to that of bacteriorhodopsin [6], suggesting a common topological organisation. In this model seven hydrophobic domains span the lipid bilayer of the cell plasma membrane; the N-terminal part and three loops linking domains I1 and 111, IV and V, and VI and VII are located on the extracellular side whereas the three loops joining domains I and 11, 111 and IV, and V and VI, as well as the C-terminal tail are located on the cytoplasmic side (Fig. 1). The overall conservation across species is similarly high (= 90% amino acid identity) at least for the ETA and ET, subtypes, where data are available from human, rat and bovine cDNA clones [l, 2, 7-11]. In contrast, only 60% amino acid identity was calculated between ETA and ET, with the best conserved parts being the membrane-spanning domains and the cytoplasmic loops (Table 1).Bi...

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“…gene in the pCDM8 vector [33] was modified such that the BamHI and NotI sites were placed at the 5' and 3' ends of the structural gene, respectively, and a NheI site was introduced into the codons of Ser65 -Ah67 with a silent mutation of the Leu66 codon, TTG to CTA, by PCR. The modified ET,R gene fragment, about 1 kbp, was inserted into the BamHI/NorT sites of the transfer vector, pVL1393.…”

Section: Construction Of Mutant Etr Genes the Human Etrmentioning

confidence: 99%

Characterization of Human Endothelin B Receptor and Mutant Receptors Expressed in Insect Cells

Doi

Hiroaki

Arimoto

et al. 1997

European Journal of Biochemistry

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Endothelin type-B receptor (ET,R) forms a stable complex with its ligand, endothelin-I. To facilitate biochemical and biophysical studies of human ET,R, several ET,R mutants carrying a hexahistidine tag sequence at the N or C terminus were expressed in Sf9 cells and were purified by a combination of biotinylated endothelin-1 -1igand-affinity and nickel-affinity chromatographies. The ligand-free receptor was purified by dissociating the ligand . receptor complex with 2 M NaSCN, whereas the ligand-bound ET,R was purified by the use of thiol-sensitive biotinylated endothelin-I. While the wild-type ET,R was expressed at about 100 pmol '251-endothelin-l -binding activity/mg membrane protein, the deletion of 36 residues from the N-terminus reduced the expressed activity to about 30%. On the other hand, the lack of glycosylation and the replacement of 2-9 residues in the N-terminal tail resulted in a 20-40% reduction in the expressed activity. Among the mutant proteins, [H57-H62, G63 -G65]ETBR, carrying six His residues in the N-terminal tail, was studied extensively because it was purified most effectively. Ligand-free [H57 -H62, G63 -G65]ETBR, purified in digitonin, retained full ligand-binding activity, while other detergents led to partial denaturation of the receptor after solubilization or after elution with NaSCN. On the other hand, ligand-bound [H57-H62, G63 -G65]ETBR could be purified in various detergents, such as n-octyl-P-D-glucopyranoside or n-decyl-/b-maltopyranoside. Ligand-free [H57 -H62, G63 -G65]ETBR reconstituted in phospholipid vesicles stimulated the binding of guanosine 5'-3-0-(thi0)triphosphate by G, in the presence of endothelin-I. Ligand-bound [H57 -H62, G63 -G65]ETBR showed similar catalytic activity in nucleotide exchange by G,. These results indicate that the ligand . receptor complex in a detergent-micellar solution retained the biologically active structure, and that the presence of ligand, endothelin-1, in the receptor molecule reinforces the stable assembly of a helical bundle and therefore the active structure.Keywords: endothelin ; endothelin B receptor; guanine-nucleotide-binding regulatory protein ; reconstitution.Guanine-nucleotide-binding regulatory protein (G protein)-coupled receptors are cell-surface membrane proteins that mediate various signals through G proteins [l, 21. They are considered to have a common structural motif of a membrane-spanning heptahelical bundle [3], and cytoplasmic and extracellular domains. Many studies have shown that the interaction sites for ligands are in the transmembrane domain, and that G proteins bind to the cytoplasmic domain, In particular, the binding pockets for small ligands, such as the catecholamines for the adrenergic receptor and acetylcholine for the muscarinic acetylcholine receptor, are thought to be in the transmembrane helical cluster [4], similar to the binding of retinal in rhodopsin [3, 5, 61 and bacteriorhodopsin [7]. However, the binding pockets for many ligands, such as peptide and glycoprotein hormones, appear to Correspond...

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Characterization of Endothelin Receptors ETA arid ETB Expressed in COS Cells

Cited by 40 publications

References 0 publications

N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

Mutational analysis of human endothelin receptors ET_A and ET_B

Characterization of Human Endothelin B Receptor and Mutant Receptors Expressed in Insect Cells

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Characterization of Endothelin Receptors ETA arid ETB Expressed in COS Cells

Cited by 40 publications

References 0 publications

N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

N-Terminal Proteolysis of the Endothelin B Receptor Abolishes Its Ability to Induce EGF Receptor Transactivation and Contractile Protein Expression in Vascular Smooth Muscle Cells

Mutational analysis of human endothelin receptors ETA and ETB

Characterization of Human Endothelin B Receptor and Mutant Receptors Expressed in Insect Cells

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Mutational analysis of human endothelin receptors ET_A and ET_B