Expression of mouse proopiomelanocortin in an insulinoma cell line. Requirements for beta-endorphin processing.
Proopiomelanocortin (POMC) is a neuroendocrine precursor protein which is processed at paired basic amino acids in a tissue-specific manner. To study this phenomenon, a vaccinia virus recombinant, which directs the synthesis of mouse POMC (VV:mPOMC) was constructed and used to infect epithelial (BSC-40) and endocrine (Rin m5F) cell lines. Bona fide mPOMC was produced in both cell types and beta-endorphin immunoreactivity was secreted in a nonregulated manner from BSC-40 cells and in a regulated manner from Rin m5F cells. Although the precursor was not cleaved to smaller beta-MSH or beta-endorphin immunoreactive peptides in BSC-40 cell extracts, Rin m5F cells produced primarily authentic gamma-lipotropin and des-acetyl beta-endorphin. Furthermore, production of these peptides was restricted to the regulated secretory pathway in Rin m5F cells. Site-directed mutagenesis was then used to change the inefficiently recognized Lys-Lys potential cleavage site near the carboxyl terminus of beta-endorphin to Lys-Arg. Expression of the mutant precursor in Rin m5F cells resulted in the synthesis of both des-acetyl beta-endorphin and beta-endorphin.
Thorne
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BA
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Caton
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LW
Thomas
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D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D007340:Insulinoma / Q000378:metabolism*; D009074:Melanocyte-Stimulating Hormones / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics; D001615:beta-Endorphin / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000378:metabolism; D008083:beta-Lipotropin / Q000096:biosynthesis
D004274:DNA, Recombinant; C058549:desacetyl beta-endorphin 1-27; D001615:beta-Endorphin; D011333:Pro-Opiomelanocortin; D009074:Melanocyte-Stimulating Hormones; D008083:beta-Lipotropin