Transfected human neuropeptide Y cDNA expression in mouse pituitary cells. Inducible high expression, peptide characterization, and secretion.
An expression vector was constructed that placed the cDNA for human neuropeptide Y (NPY) under the control of the mouse metallothionein promoter and was used to transfect the AtT-20 mouse anterior pituitary corticotrope cell line. AtT-20 cells normally process the pro-ACTH/endorphin precursor but do not produce detectable levels of NPY. The resulting AtT-20/NPY cell line (Mt.NPY1a) was used to study the ability of the corticotrope cells to synthesize, process, and secrete the foreign proNPY-related peptide products. The stable cell line created contains approximately 40 copies of proNPY cDNA per cell. NPY mRNA levels and proNPY synthesis were increased at least 35-fold when maximally induced with cadmium; proNPY synthesis was also induced by glucocorticoids. Upon induction the NPY secretion rate was equimolar to that of the endogenous peptides. ProNPY, NPY, and the COOH-terminal peptide produced by this cell line had molecular weight and amino acid-labeling pattern predicted from cDNA sequence data and from previous isolation of NPY-related molecules from NPY-producing cells. The structures of secreted proNPY, NPY, and COOH-terminal peptide, as well as determination of the site of proteolytic cleavage between NPY and the COOH-terminal peptide, were determined by tryptic mapping and Edman degradation of secreted biosynthetically labeled peptide products. The proNPY molecule appears to be processed in the same pathway responsible for cleavage of the endogenous pro-ACTH/endorphin precursor. Secretion of proNPY-derived peptides paralleled secretion of endogenous pro-ACTH/endorphin-derived products, under both basal and stimulated conditions. With induction proNPY expression there is a dose-dependent inhibition of both proNPY and pro-ACTH/endorphin proteolytic processing.
Dickerson
I M
IM
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Dixon
J E
JE
Mains
R E
RE
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009478:Neuropeptide Y / Q000235:genetics*; D010446:Peptide Fragments / Q000032:analysis; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D010957:Plasmids; D011863:Radioimmunoassay
D009478:Neuropeptide Y; D010446:Peptide Fragments; D004247:DNA