Ligand Binding Domain of the Human Endothelin-B Subtype Receptor

Wada, Kenji; Hashido, Kento; Terashima, Hiroyuki; Adachi, Miki; Fujii, Yuki; Hiraoka, Osamu; Furuichi, Yasuhiro; Miyamoto, Chikara

doi:10.1006/prep.1995.1029

Cited by 10 publications

(10 citation statements)

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“…6). This is in agreement with a previous report, which demonstrated that even the sequence up to residue Arg-64 is not essential for a functional ligand binding pocket (32). The ET B receptor may, however, contain another folded domain within its N tail.…”

Section: Signal Peptides Are Present Preferentially In Those Gpcrs Thsupporting

confidence: 93%

The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane

Köchl

Alken

Rutz

et al. 2002

Journal of Biological Chemistry

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Section: Signal Peptides Are Present Preferentially In Those Gpcrs Thsupporting

confidence: 93%

The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane

Köchl

Alken

Rutz

et al. 2002

Journal of Biological Chemistry

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“…Through proteolytic truncation experiments, it has been shown that ET-1 binds to a 29-amino-acid sequence in the NH 2 -terminal region of the ET B receptor near the first transmembranespanning domain (18,31). Thus we can speculate that ET-3 binding in the sl/sl inner medulla must be to an ET-like receptor that contains an NH 2 -terminal region of 29 amino acids near the first transmembrane segment.…”

Section: Discussionmentioning

confidence: 92%

Unique endothelin receptor binding in kidneys of ET_Breceptor deficient rats

Taylor¹,

Gariepy

Pollock

et al. 2003

American Journal of Physiology-Regulatory, Integrative and Comp

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.) developed rescued spottinglethal rats that carry a naturally occurring deletion of the endothelin (ET) type B receptor gene resulting in a lack of functional renal ETB receptor expression. It has been shown that rats homozygous (sl/sl) for the deletion have elevated plasma ET-1 levels; thus, the purpose of this study was to determine whether this deletion would result in a downregulation of ETA receptors in renal tissue. ET-1 and ET-3 binding experiments were performed with cortex, outer medullary, and inner medullary membranes of heterozygous (sl/ϩ) and sl/sl ETB receptor-deficient rats.125 I-labeled ET-1 binding in sl/sl cortex and outer medulla was significantly lower than cortex and outer medulla from sl/ϩ rats. In contrast to sl/ϩ rats, [125 I]ET-3 binding was not detected in the cortex and outer medulla of sl/sl rats, indicating a lack of ETB receptor expression. The inner medulla of sl/ϩ rats also demonstrated an abundance of ETB receptors. Surprisingly, however, we also observed significant [ 125 I]ET-3 binding in the sl/sl inner medulla. Furthermore, ET-3 binding in the inner medulla could be blocked with an ETA receptor antagonist in sl/sl rats but not in tissue from sl/ϩ rats. These studies indicate that rats deficient in ETB receptors have decreased renal cortical and outer medullary ETA receptor number, most likely in response to elevated plasma ET-1 levels. In addition, homozygous ETB-deficient rats express a novel inner medullary ET-3 binding site.endothelin B receptor; kidney; receptor binding; spottinglethal rats IN 1988, YANAGISAWA ET AL. (33) isolated a potent vasoconstrictor produced by endothelial cells that was named endothelin (ET). Inoue and colleagues (14) determined through DNA analysis that there are three distinct genes encoding three ET isopeptides (ET-1, ET-2, and ET-3). G protein-coupled receptors were discovered and then characterized by their selective affinity to each of the ET isopeptides, location, and their biological function (18). Endothelin A (ET A ) receptors bind ET-1 with higher affinity than ET-2 or ET-3, are localized to vascular smooth muscle cells, and cause vasoconstriction. Endothelin B (ET B ) receptors are found on endothelial cells and have been shown to produce vasodilation and bind all three ET isopeptides with equal affinity. Additionally, there is evidence that ET B receptors in pulmonary endothelial cells "clear" ET-1 from the circulation (7). However, this classification of ET receptors may be oversimplified, with increasing evidence suggesting additional receptor subtypes. It is now known that there exist two pharmacologically distinct subtypes of the ET B receptor, ET B1 and ET B2 , whose biological activity is a function of their localization. ET B1 are located on the vascular endothelium and produce vasodilation upon agonist binding, whereas ET B2 are found on vascular smooth muscle cells and mediate non-ET A vasoconstriction.Within the kidney, both ET receptor subtypes have been identified in various regions differing both in number and function. ...

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“…In addition, the observation that ET-1 partly inhibits antibody binding might shed some light on the location of its binding domain. In fact, the precise delineation of the ET-1-binding site on ET B R remains a subject of controversy, despite the huge number of publications devoted to the question: numerous receptor domains have been implicated in ET-1 binding, including several transmembrane helices, TM1 and TM2 (Wada et al, 1995), TM3 (Lee et al, 1994), or TM5 (Boivin et al, 2004). To our knowledge, however, the two receptor regions that we identified here in the N-terminal domain have never been reported to play a direct role in endogenous ligand binding.…”

Section: Discussionmentioning

confidence: 99%

“…Since the major linear epitopes of our antibodies (at least those present in the two selected B3 sera) were delineated, we wondered whether these antibodies could be used to give any new insight into the localization of the ligand-binding site in hET B R, which is still a subject of great controversy (Lee et al, 1994;Wada et al, 1995;Boivin et al, 2004;Aubin et al, 2008;Lä ttig et al, 2009). For this purpose, we investigated whether endothelin-1 could compete with these antibodies for receptor binding.…”

Section: Et-1 Competes With Antibodies For Et B Receptor Bindingmentioning

confidence: 98%

Electroporation-Aided DNA Immunization Generates Polyclonal Antibodies Against the Native Conformation of Human Endothelin B Receptor

Allard

Priam

Deshayes

et al. 2011

DNA and Cell Biology

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Endothelin B receptor (ET(B)R) is a G protein-coupled receptor (GPCR) specific for endothelin peptides (including endothelin-1, ET1), which mediates a variety of key physiological functions in normal tissues, such as modulation of vasomotor tone, tissue differentiation, or cell proliferation. Moreover, ET(B)R, overexpressed in various cancer cells including melanoma, has been implicated in the growth and progression of tumors, as well as in controlling T cell homing to tumors. To gather information on receptor structure and function, antibodies are generally considered choice molecular probes, but generation of such reagents against the native conformation of GPCRs is a real technical challenge. Here, we show that electroporation-aided genetic immunization, coupled to cardiotoxin pretreatment, is a simple and very efficient method to raise large amounts of polyclonal antibodies highly specific for native human ET(B)R (hET(B)R), as assessed by both flow cytometry analysis of different stably transfected cell lines and a new and rapid cell-based enzyme-linked immunosorbent assay that we also describe. The antibodies recognized two major epitopes on hET(B)R, mapped within the N-terminal extracellular domain. They were used to reveal hET(B)R on membranes of three different human melanoma cell lines, by flow cytometry and confocal microscopy, a method that we show is more relevant than mRNA polymerase chain reaction in assessing receptor expression. In addition, ET-1 partially competed with antibodies for receptor binding. The strategy described here, thus, efficiently generated new immunological tools to further analyze the role of ET(B)R under both normal and pathological conditions, including cancers. Above all, it can now be used to raise monoclonal antibodies against hET(B)R and, more generally, against GPCRs that constitute, by far, the largest reservoir of potential pharmacological targets.

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Ligand Binding Domain of the Human Endothelin-B Subtype Receptor

Cited by 10 publications

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The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane

The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane

Unique endothelin receptor binding in kidneys of ET_Breceptor deficient rats

Electroporation-Aided DNA Immunization Generates Polyclonal Antibodies Against the Native Conformation of Human Endothelin B Receptor

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Ligand Binding Domain of the Human Endothelin-B Subtype Receptor

Cited by 10 publications

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The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane

The Signal Peptide of the G Protein-coupled Human Endothelin B Receptor Is Necessary for Translocation of the N-terminal Tail across the Endoplasmic Reticulum Membrane

Unique endothelin receptor binding in kidneys of ETBreceptor deficient rats

Electroporation-Aided DNA Immunization Generates Polyclonal Antibodies Against the Native Conformation of Human Endothelin B Receptor

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Unique endothelin receptor binding in kidneys of ET_Breceptor deficient rats