Effects of the N-terminal cysteine mutation on prolactin expression and secretion in transfected cells .
We developed an experimental cell model to look for motif(s) of rat PRL sequence encoding a sorting signal to secretory granules. An efficient expression vector (pCMV-rPRL) was used to transfect several eukaryotic cell lines in culture, i.e., one neuronal cell line (C6) and three glandular pituitary derived cell lines (AtT20, GC, GH3CDL). Despite the ubiquitous transcription of pCMV-rPRL, the synthesis and secretion of rPRL were detected primarily in GH3CDL cells that derived from lactotrophs, suggesting a cell-specific post-transcriptional control of rPRL expression. During transient expression, exogenous native PRL was transported through intracellular compartments of the secretory pathway and underwent regulated release. Abolition by mutagenesis (C4S) of the N-terminal disulfide bond increased by 50% the PRL secretion rate (medium to cell ratio) and multiplied by 5 the specific activity of medium PRL from pulse-labeled cells. These results support the hypothesis that N-terminal disulfide bond plays a role in the control of PRL intracellular transit and storage.
Morin
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Collège de France, Groupe de Neuroendocrinologie et Neurobiologie Cellulaires et Moléculaires, URA 1115 CNRS, Paris, France.
Picart
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R
Tixier-Vidal
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D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011233:Precipitin Tests; D011388:Prolactin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011388:Prolactin; D003545:Cysteine