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.
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Section on Cellular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda 20892.
Andreasson
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D004247:DNA / Q000032:analysis / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010911:Pituitary Neoplasms / Q000737:chemistry / Q000473:pathology / Q000503:physiopathology; D011333:Pro-Opiomelanocortin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013347:Subcellular Fractions; D014162:Transfection; D014407:Tumor Cells, Cultured
D005576:Colforsin; D011333:Pro-Opiomelanocortin; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase