An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocortin is packaged into secretory granules in AtT20 cells before its cleavage

We have raised a rabbit antiserum against a synthetic peptide corresponding to the cleavage site between beta-lipotropic hormone and the ACTH moieties of murine pro-opiomelanocortin (POMC). After affinity purification, the anti-cleavage site antibody immunoprecipitates POMC from extracts of AtT20 cells but it does not immunoprecipitate the ACTH in such extracts or any of the other products of cleavage of POMC. By contrast, an antiserum raised against pure swine ACTH immunoprecipitates both POMC and ACTH from AtT20 cell extracts. Using the anti-cleavage site antibody we have shown that all the POMC synthesized during a 15-min pulse-labeling with [35S]methionine is cleaved at this site within 1 h. By immunoelectron microscopy we show that approximately 25-30% of peripheral secretory granules in AtT20 cells can be labeled with the anti-cleavage site antibody while anti- ACTH antiserum labels all these granules. This establishes that at least some POMC is packaged into secretory granules before its proteolytic cleavage.