Post-translational Modifications of Endothelin Receptor B from Bovine Lungs Analyzed by Mass Spectrometry

Roos, Martin; Šoškić, Vukic; Poznanović, Slobodan; Godovac‐Zimmermann, Jasminka

doi:10.1074/jbc.273.2.924

Cited by 48 publications

(48 citation statements)

References 46 publications

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“…The reason for this is unknown. Differential cell-type-specific posttranslational modification of the ET B receptor molecule may account for the lack of endothelial binding, because the epitope used for immunization lies in close proximity to a potential glycosylation site at amino acid residue 60 (Roos et al 1998). However, there are no studies addressing this issue in vascular smooth muscle and endothelial cells.…”

Section: Discussionmentioning

confidence: 99%

Muscular ET_BReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

Wendel

Kummer

Knels

et al. 2005

J Histochem Cytochem.

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S U M M A R YThe endothelin/endothelin-receptor system is a key player in the regulation of vascular tone in mammals. We raised and characterized an antiserum against rat ET B receptor and investigated the distribution of ET B receptors in different vascular beds during postnatal development (day 0 through day 28) and in the adult rat. We report the tissuespecific and age-dependent presence of vasoconstrictor ET B receptors. At the time of birth, vascular smooth muscle cells from all tissues examined did not exhibit ET B receptor immunoreactivity. The occurrence of ET B receptor immunoreactivity in the postnatal development was time dependent and started in small coronary and meningeal arteries at day 5, followed by small mesenteric arteries as well as brachial artery and vein at day 14. At day 21, ET B receptors were present in the media of muscular segments of pulmonary artery, large coronary arteries, and intracerebral arterioles. At day 28, ET B receptor immunoreactivity was evident in interlobular renal arteries, vas afferens, and efferens. Large renal arteries, mesenteric artery, and elastic segments of pulmonary arteries, as well as coronary and mesenteric veins, did not exhibit ET B receptor immunoreactivity. These data demonstrate the age-dependent and tissue-specific presence of ET B receptors, mainly on arterial smooth muscle cells in the vascular system of the rat. E ndothelin -1 (ET-1) is a potent endothelium-derived vasoconstrictor peptide (Yanagisawa et al. 1989). It takes part in the regulation of vascular tone after birth (for review see Perreault and Coceani 2003) and is implicated in the pathophysiology of cardiovascular, renal, and pulmonary diseases (Saito et al. 1990;Watanabe et al. 1990;Larriviere and Lebel 2003;Michel et al. 2003).ET-1 exerts its biological effects through two distinct ET-receptor subtypes, ET A and ET B . ET A receptors on smooth muscle cells exclusively mediate vasoconstriction, whereas ET B receptors can mediate both vasodilation as well as vasoconstriction, depending on their location on endothelial cells and vascular smooth muscle, respectively. While the role of the ET A receptor subtype in mediating vasoconstriction is beyond debate, the existence of ET B receptors on vascular smooth muscle cells in distinct vascular beds is still controversial, and systematic histological investigations on the distribution pattern of vascular ET receptors are missing. Recently, colocalization of ET A and ET B receptors on arterial smooth muscle cells in the coronary circulation (Wendel-Wellner et al. 2002) and small pulmonary arteries of adult rats were demonstrated (Soma et al. 1999).Pharmacological studies aimed at defining the role of vasoconstrictor ET B receptors are highly controversial, and studies from different groups lead to contradictory results even for identical organ systems (Takase et al.

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Section: Discussionmentioning

confidence: 99%

Muscular ET_BReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

Wendel

Kummer

Knels

et al. 2005

J Histochem Cytochem.

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“…Synthesis of Polyadenylated Met-Lys-Bradykinin-Met-Lys-bradykinin derivatized with EMCS at the ⑀-NH 2 group of Lys 2 was prepared by a synthesis similar to that used recently to prepare an analogous endothelin derivative (12). 1 mg (400 nmol) of Met-Lys-bradykinin was dissolved in 400 l of 35% acetonitrile, 0.05% trifluoroacetate, further diluted with 50 mM sodium borate and 0.015% Triton X-305, pH 8.2, and a 10-fold excess (4 mol) of the heterobifunctional cross-linker EMCS dissolved in 200 l of acetonitrile was added.…”

Section: Methodsmentioning

confidence: 99%

Correlations in Palmitoylation and Multiple Phosphorylation of Rat Bradykinin B2 Receptor in Chinese Hamster Ovary Cells

Šoškić¹,

Nyakatura²,

Roos³

et al. 1999

Journal of Biological Chemistry

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Rat bradykinin B 2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B 2 receptor was isolated on oligo(dT)-cellulose using N-(⑀-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA) 30 -mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isolated rat bradykinin B 2 receptor showed phosphorylation at Bradykinin, a member of the kinin family (1), is a nonapeptide with diverse biological activities ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, renal homeostasis, and pain generation (2, 3). Bradykinin mediates its physiological effects by binding to and activation of the bradykinin B 2 receptor. Molecular cloning has revealed the primary structure of the B 2 receptor (4) and classified it as a member of the G protein-coupled receptor superfamily. The consensus bradykinin receptor topology predicts four extracellular domains (ED 1 1-4) and intracellular domains (ID1-4), each separated by seven transmembrane helical regions (TM1-7) spanning the lipid bilayer. B 2 receptors are post-translationally modified by glycosylation (5), phosphorylation (6), and presumably by palmitoylation of the cytoplasmic surface.Based on homology with other G protein-coupled receptors there have been indications regarding possible structural features probed by agonists, antagonists, and anti-idiotypic antibodies (5, 6), functional regions (7), and sites of post-transitional modifications for the B 2 receptor (8). Site-directed mutagenesis indicated the importance of Tyr residues and of ID4 for the signaling and the uptake of the B 2 receptor in receptor in rat-1 cells transfected with wild and mutant receptor cDNAs (9). However, as yet there is still rather little direct evidence at the protein level for attributes such as the precise sites and the roles of glycosylation, palmitoylation, and phosphorylation of bradykinin B 2 or other G protein-coupled receptors. Indeed phosphorylation sites for few G protein-coupled receptors have been mapped to date (10 -12), and most of these sites reflect the in vitro phosphorylation of the isolated receptor.We report here on the isolation of rat B 2 receptor from transfected Chinese hamster ovary (CHO) cells using oligo(dA) covalently linked to bradykinin via a specially developed bifunctional cross-linker. Affinity chromatography has been carried out under very mild conditions using oligo(dT) columns analogous to methods used for isolation of eukaryotic mRNA. In-gel digestion of electrophoretically separated receptor, subsequent peptide mass fingerprinting by matrix-assisted laser desorption...

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“…45 Other studies, where the putative N-glycosylation site of ETB was mutated, did not reveal any modification of the expression of ETB at cell surface, binding to ET1 or coupling to signaling pathways. 46,47 Moreover, Roos et al 48 found, by mass spectrometry approach, that ETB, extracted from bovine lung, was not glycosylated in its N-terminal domain. These results indicate that the glycosylation of ETB does not influence the structure and function of the receptor, and has very few chances to play a role in the discriminating properties of rendomab-B4 toward CHO-ETB and UACC-257 cells.…”

Section: (C and D) Living Uacc-257 Cells Were Incubated For 2 H At 4 mentioning

confidence: 99%

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

Borrull

Allard

Wijkhuisen

et al. 2016

mAbs

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Metastatic melanoma is an aggressive cancer with a poor prognostic, and the design of new targeted drugs to treat melanoma is a therapeutic challenge. A promising approach is to produce monoclonal antibodies (mAbs) against the endothelin B receptor (ETB), which is known to be overexpressed in melanoma and to contribute to proliferation, migration and vasculogenic mimicry associated with invasiveness of this cancer. We previously described rendomab-B1, a mAb produced by DNA immunization. It is endowed with remarkable characteristics in term of affinity, specificity and antagonist properties against human ETB expressed by the endothelial cells, but, surprisingly, had poor affinity for ETB expressed by melanoma cells. This characteristic strongly suggested the existence of a tumor-specific ETB form. In the study reported here, we identified a new mAb, rendomab-B4, which, in contrast to rendomab-B1, binds ETB expressed on UACC-257, WM-266-4 and SLM8 melanoma cells. Moreover, after binding to UACC-257 cells, rendomab-B4 is internalized and colocalizes with the endosomal protein EEA-1. Interestingly, rendomab-B4, despite its inability to compete with endothelin binding, is able to inhibit phospholipase C pathway and migration induced by endothelin. By contrast, rendomab-B4 fails to decrease ERK1/2 phosphorylation induced by endothelin, suggesting a biased effect on ETB. These particular properties make rendomab-B4 an interesting tool to analyze ETB-structure/function and a promising starting point for the development of new immunological tools in the field of melanoma therapeutics.

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Post-translational Modifications of Endothelin Receptor B from Bovine Lungs Analyzed by Mass Spectrometry

Cited by 48 publications

References 46 publications

Muscular ET_BReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

Muscular ET_BReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

Correlations in Palmitoylation and Multiple Phosphorylation of Rat Bradykinin B2 Receptor in Chinese Hamster Ovary Cells

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

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Post-translational Modifications of Endothelin Receptor B from Bovine Lungs Analyzed by Mass Spectrometry

Cited by 48 publications

References 46 publications

Muscular ETBReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

Muscular ETBReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

Correlations in Palmitoylation and Multiple Phosphorylation of Rat Bradykinin B2 Receptor in Chinese Hamster Ovary Cells

Rendomab B4, a monoclonal antibody that discriminates the human endothelin B receptor of melanoma cells and inhibits their migration

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Product

Resources

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Muscular ET_BReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature

Muscular ET_BReceptors Develop Postnatally and Are Differentially Distributed in Specific Segments of the Rat Vasculature