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, Robert; Alken, Martina; Rutz, Claudia; Krause, Gerd; Oksche, Alexander; Rosenthal, Walter; Schülein, Ralf

doi:10.1074/jbc.m111674200

Cited by 60 publications

(60 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells were grown on coated coverslips for 48 h and washed once with phosphate-buffered saline (PBS), and coverslips were transferred into a self-made humidified chamber. Cells were covered with 1 ml of PBS containing 0.05% trypan blue for plasma membrane staining (17) and immediately subjected to microscopic analysis on a Zeiss LSM510 inverted laser scanning microscope. Fluorescence signals were detected using the following configurations: GFP: exc ϭ 488 nm, BP em ϭ 496 -517 nm; YFP: exc ϭ 514 nm, BP em ϭ 517.7-646 nm.…”

Section: Methodsmentioning

confidence: 99%

The Golgi Protein RCAS1 Controls Cell Surface Expression of Tumor-associated O-Linked Glycan Antigens

Engelsberg

Hermosilla

Karsten

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Tumor immunology has received a large impetus from the identification of tumor-associated antigens. Among them, a monoclonal antibody, 22.1.1, was instrumental in defining a novel tumor-associated antigen that was termed "receptor binding cancer antigen expressed on SiSo cells" (RCAS1). RCAS1 was proposed to induce growth arrest and apoptosis on activated immune cells, mediated by a putative death receptor. Structurally, RCAS1 was predicted to exist as a type II transmembrane protein and in a soluble form. Here, we analyzed occurrence, membrane topology, and subcellular localization of the RCAS1-encoded gene product. RCAS1 was shown to be a ubiquitously expressed type III transmembrane protein with a Golgi-predominant localization. Monoclonal antibody 22.1.1 failed to recognize RCAS1, as demonstrated by confocal microscopy. Instead, we showed that the cognate 22.1.1 epitope is identical with the tumor-associated O-linked glycan Tn (N-acetyl-D-galactosamine, GalNAc). Overexpression of RCAS1 in cell lines that are negative for 22.1.1 surface staining led to the generation of Tn and the closely related TF (Thomsen-Friedenreich, Gal␤1-3GalNAc) antigen, thus providing a functional link to the generation of the 22.1.1 epitope. We suggest that RCAS1 modulates surface expression of tumor-associated, normally cryptic O-linked glycan structures and contributes indirectly to the antigenicity of tumor cells.

show abstract

Section: Methodsmentioning

confidence: 99%

The Golgi Protein RCAS1 Controls Cell Surface Expression of Tumor-associated O-Linked Glycan Antigens

Engelsberg

Hermosilla

Karsten

et al. 2003

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…We have previously shown that the GFP moiety fused to the C terminus of the ET B receptor does not alter the functional properties of the receptor (7,8,13). The fusion protein allowed visualization of the receptor in living cells and its detection in immunoblot experiments.…”

Section: Generation Of Hek293 Cell Clones Expressing Wild-type and Mumentioning

confidence: 99%

“…Thus, the tight binding of ET1 to the ET B receptor and the transport of the receptor/ligand complex into lysosomal compartments could provide a molecular basis for the efficient removal of ET1 from the circulation. The ET B receptor possesses a cleavable signal peptide (comprising 26 amino acids), which is removed in the ER lumen during receptor biosynthesis (6,12,13). In addition to the removal of the signal peptide, the N terminus of the mature protein undergoes proteolytical cleavage.…”

mentioning

confidence: 99%

The Extracellular N Terminus of the Endothelin B (ETB) Receptor Is Cleaved by a Metalloprotease in an Agonist-dependent Process

Grantcharova

Furkert

Reusch

et al. 2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Endothelins, which exist in three isoforms, exert their action via two endothelin receptor subtypes: (i) the endothelin A (ET A ) 1 and (ii) the endothelin B (ET B ) receptor (1, 2). Both receptors belong to the large family of GPCRs. The ET A receptor is predominantely expressed in smooth muscle cells, and its stimulation causes a long-lasting vasoconstriction (3). The ET B receptor is mainly expressed in endothelial cells, and its activation results in a transient vasodilatation (4).The ET B receptor displays several unique properties, which are not shared by the ET A receptor or other members of the GPCR family. For example, the ET B receptor binds the ligand ET1 almost irreversibly (5). The receptor-ligand complex is resistant to acid washes and 2% SDS and even survives SDSpolyacrylamide gel electrophoresis at low temperature (6). In living cells, internalized ET1-ET B receptor complexes remain stable for more than 2 h. This is remarkable, as the ligandreceptor complex is transported into late endosomal/lysosomal compartments within 30 min (7,8). The tight binding of ET1 to the ET B receptor is evolutionary conserved, since it was also observed for the ET B receptor of calf, dog, mouse, and guinea pig (9). Mutagenesis studies revealed that aspartate 75 and proline 93 within the extracellular N terminus of the human ET B receptor are important determinants for the formation of a stable ligand/receptor complex (9). The physiological significance of this tight receptor/ligand association is still not understood. Recently it was shown that the ET B receptor is crucially involved in the regulation of circulating ET1 plasma levels: blockade of the ET B receptor causes a significant increase in ET1 plasma levels (10, 11). Thus, the tight binding of ET1 to the ET B receptor and the transport of the receptor/ligand complex into lysosomal compartments could provide a molecular basis for the efficient removal of ET1 from the circulation. The ET B receptor possesses a cleavable signal peptide (comprising 26 amino acids), which is removed in the ER lumen during receptor biosynthesis (6,12,13). In addition to the removal of the signal peptide, the N terminus of the mature protein undergoes proteolytical cleavage. Protein analysis of purified ET B receptors from human placenta revealed two different isoforms: (i) a full-length receptor comprising 416 amino acids (after removal of the signal peptide) and starting with glutamate 27, and (ii) an N-terminal-truncated ET B receptor, comprising 378 amino acids and starting with serine 65 (6, 14). The existence of the two ET B receptor isoforms has been demonstrated for the dog, pig, and calf receptor, indicating that the proteolytical processing of the mature ET B receptor is evolutionary conserved (12,(15)(16)(17). The proteolytically released Nterminal peptide fragment harbors the only N-linked glycosylation. Thus, the N-terminal proteolysis yields unglycosylated ET B receptors.So far, the mechanism and physiological significance of the N-terminal proteolysis are unknown. In o...

show abstract

“…For GPCRs, signal peptides are usually rare, and only about 5-10 % of GPCRs are predicted to possess one. These are typically found in GPCRs which possess very long Ntermini involved in ligand binding, such as members of the glycoprotein hormone receptor family, as well as secretin and glutamate receptors [53,55,56]. Only very few class A GPCRs exhibit predicted signal peptides and their function is in most cases not well understood [53].…”

Section: Discussionmentioning

confidence: 99%

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

Knospe

Müller

Rosa

et al. 2013

Purinergic Signalling

View full text Add to dashboard Cite

The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110 3.24 47 , were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenineinduced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.

show abstract

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

Cited by 60 publications

References 32 publications

The Golgi Protein RCAS1 Controls Cell Surface Expression of Tumor-associated O-Linked Glycan Antigens

The Golgi Protein RCAS1 Controls Cell Surface Expression of Tumor-associated O-Linked Glycan Antigens

The Extracellular N Terminus of the Endothelin B (ETB) Receptor Is Cleaved by a Metalloprotease in an Agonist-dependent Process

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

Contact Info

Product

Resources

About