Expression and posttranslational processing of preprodynorphin complementary DNA in the mouse anterior pituitary cell line AtT-20.
A recombinant plasmid containing the rat prodynorphin cDNA was introduced into the mouse anterior pituitary corticotroph cell line AtT-20. These cells normally express and posttranslationally process proopiomelanocortin, but not prodynorphin. Stable transformants were isolated and analyzed for the expression and processing of prodynorphin. The stably transformed AtT-20 cells that expressed a 1.3-kilobase prodynorphin mRNA also expressed prodynorphin protein and processed it to dynorphin peptides. The peptides included leucine-enkephalin, beta-neoendorphin, dynorphin-A8, and dynorphin-B, as identified by gel filtration and reverse phase HPLC followed by RIA using peptide-specific antisera. These results demonstrate that AtT-20 cells efficiently and accurately process prodynorphin at both dibasic sites and monobasic cleavage sites, indicating that the AtT-20 cells contain enzymes capable of cleaving the precursor not only at dibasic residues but also at monobasic residues. The release of prodynorphin-derived peptides paralleled secretion of endogenous proopiomelanocortin-derived peptides when stimulated by CRF, a natural secretagogue for ACTH.
Devi
L
L
Department of Pharmacology, New York University Medical Center, New York 10016.
Gupta
P
P
Douglass
J
J
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000235:genetics; D004399:Dynorphins / Q000096:biosynthesis / Q000235:genetics*; D004723:Endorphins / Q000096:biosynthesis / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior / Q000473:pathology; D010911:Pituitary Neoplasms / Q000235:genetics* / Q000473:pathology; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014407:Tumor Cells, Cultured / Q000378:metabolism
D004723:Endorphins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C036448:pre-prodynorphin; D004399:Dynorphins; D004247:DNA