Manipulation of neuropeptide biosynthesis through the expression of antisense RNA for peptidylglycine alpha-amidating monooxygenase.
Stable cell lines with significantly elevated or diminished levels of a key neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), were generated by transfection of a mouse pituitary cell line with expression vectors containing PAM cDNA in the sense or antisense orientation. By evaluating the ability of these cell lines to alpha-amidate endogenous neuropeptides, a rate-limiting role for PAM in neuropeptide alpha-amidation was demonstrated. Overexpression of either the full-length PAM precursor with its trans-membrane domain or a soluble protein containing only the monooxygenase domain of PAM led to increased alpha-amidation of endogenous neuropeptides. Overexpression of the full-length PAM led to an unexpected decrease in the endoproteolytic processing of endogenous prohormone; conversely, underexpression of PAM led to significantly enhanced endoproteolytic processing of endogenous prohormone. These data suggest that PAM may have additional functions in peptide processing.
Mains
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Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Bloomquist
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D000324:Adrenocorticotropic Hormone / Q000096:biosynthesis; D000818:Animals; D015153:Blotting, Western; D003594:Cytoplasmic Granules / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004723:Endorphins / Q000096:biosynthesis; D004792:Enzyme Precursors / Q000235:genetics; D015870:Gene Expression; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics*; D009097:Multienzyme Complexes; D009479:Neuropeptides / Q000096:biosynthesis* / Q000235:genetics; D011498:Protein Precursors / Q000096:biosynthesis; D016372:RNA, Antisense / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004723:Endorphins; D004792:Enzyme Precursors; D009097:Multienzyme Complexes; D009479:Neuropeptides; D011498:Protein Precursors; D016372:RNA, Antisense; D000324:Adrenocorticotropic Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase