The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process

The extracellular N terminus of the endothelin B (ETB) receptor is susceptible to limited proteolysis (cleavage at R64 down arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ETB receptor or an ETB-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degreesC, only the full-length ETB receptor was detected at the cell surface. However, when cells were incubated at 37 degreesC, 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 ETB receptor at the cell surface. Functional analysis of a mutant ETB receptor lacking the first 64 amino acids ([Delta2-64]ETB 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 ETB 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.