title: The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process.
abstract: 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.
authors.affiliation: Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin Buch, Robert-Roessle-Strasse 10, 13125 Berlin, Federal Republic of Germany.
authors.forename: Evelina
authors.initials: E
authors.lastname: Grantcharova
authors.forename: Jens
authors.initials: J
authors.lastname: Furkert
authors.forename: H Peter
authors.initials: HP
authors.lastname: Reusch
authors.forename: Hans-Willi
authors.initials: HW
authors.lastname: Krell
authors.forename: Gisela
authors.initials: G
authors.lastname: Papsdorf
authors.forename: Michael
authors.initials: M
authors.lastname: Beyermann
authors.forename: Ralf
authors.initials: R
authors.lastname: Schulein
authors.forename: Walter
authors.initials: W
authors.lastname: Rosenthal
authors.forename: Alexander
authors.initials: A
authors.lastname: Oksche
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection
chemical_list: D007295:Inositol Phosphates; D008024:Ligands; D008164:Luminescent Proteins; D010455:Peptides; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D008666:Metalloendopeptidases