The amino-terminal sequence of pro-opiomelanocortin directs intracellular targeting to the regulated secretory pathway.

The molecular signal which targets the pro-opiomelanocortin (POMC) prohormone into the regulated secretory pathway was investigated. DNA sequences encoding the first 10, 26, 50, and 101 N-terminal amino acids of mouse POMC were fused in frame to the chloramphenicol acetyltransferase (CAT) gene and expressed in AtT-20 cells. Immunofluorescence microscopy using antibody directed against CAT indicated that fusion proteins carrying 26, 50 and 101 amino acids of N-POMC were directed to secretory granules. This finding was confirmed by secretion studies in which 1 microM forskolin stimulated the release of fusion proteins carrying 26 and 101 amino acids of N-POMC, whereas no regulated secretion was observed with the shortest fusion protein. Subcellular fractionation studies also indicated the presence of the fusion proteins with 26 and 101 amino acids of N-POMC in secretory granules. These results provide evidence that the signal directing POMC to secretory granules is contained within the N-terminus of the prohormone, with the first 26 amino acids being sufficient for targeting. Binding studies showed that N-POMC1-76 bound to secretory granule membranes specifically on the luminal side and in a pH-sensitive manner. Only N-POMC1-76 bound optimally to secretory granule membranes at pH 5 to 6.5, but not the ACTH1-39 (mid), corticotropin-like intermediate lobe peptide (CLIP) and beta-lipotropin (C-terminal) domains of POMC. Such binding may be involved in the mechanism of sorting POMC to the regulated secretory pathway.