The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process
| Autor: | H. Peter Reusch, Walter Rosenthal, Jens Furkert, Alexander Oksche, Gisela Papsdorf, Hans-Willi Krell, Ralf Schülein, Michael Beyermann, Evelina Grantcharova |
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| Rok vydání: | 2002 |
| Předmět: |
Time Factors
Proteolysis media_common.quotation_subject Inositol Phosphates Recombinant Fusion Proteins Green Fluorescent Proteins Immunoblotting Molecular Sequence Data Cleavage (embryo) Ligands Transfection Biochemistry Cell Line Cell membrane chemistry.chemical_compound medicine Extracellular Cyclic AMP Humans Phenylarsine oxide Amino Acid Sequence Internalization Receptor Molecular Biology media_common medicine.diagnostic_test Dose-Response Relationship Drug Chemistry Receptors Endothelin Cell Membrane Temperature Metalloendopeptidases Cell Biology Molecular biology Endothelin 1 Receptor Endothelin B Protein Structure Tertiary Kinetics Luminescent Proteins medicine.anatomical_structure Mutation Electrophoresis Polyacrylamide Gel Peptides Protein Binding |
| Zdroj: | The Journal of biological chemistry. 277(46) |
| ISSN: | 0021-9258 |
| Popis: | The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure. |
| Databáze: | OpenAIRE |
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