Expression of porcine cholecystokinin cDNA in a murine neuroendocrine cell line. Proteolytic processing, sulfation, and regulated secretion of cholecystokinin peptides.
The cDNA for porcine preprocholecystokinin (pre-pro-CCK) was engineered for expression in mammalian cells under the control of the Rous sarcoma virus-long terminal repeat promoter. This expression construct was transfected into the murine anterior pituitary cell line, AtT-20. A stable cell line (AtT-20/CCK) was derived that expresses CCK mRNA indistinguishable from the CCK mRNA found in pig brain or gut. The AtT-20/CCK cells carry out proteolytic processing and sulfation reactions to generate authentic sulfated CCK8 from pro-CCK. The cells also store and secrete CCK-immunoreactive peptides. This secretion can be stimulated with corticotropin releasing factor, the natural secretagogue for anterior pituitary cells. In contrast, monkey kidney epithelial cells (COS cells), which are transiently transfected to express CCK, predominantly secrete nonsulfated pro-CCK into the medium. These studies show that a murine neuroendocrine cell line contains the complete processing machinery required to generate authentic porcine CCK8. The processing events include simultaneous proteolytic processing at one and two basic amino acid sites and sulfation of tyrosine residues. The cell line thus duplicates exactly the processing patterns found to occur in pig brain cortex.
Lapps
W
W
Department of Molecular Genetics, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Eng
J
J
Stern
A S
AS
Gubler
U
U
D000595:Amino Acid Sequence; D000818:Animals; D002766:Cholecystokinin / Q000032:analysis / Q000235:genetics*; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D005786:Gene Expression Regulation; D011498:Protein Precursors / Q000032:analysis; D012333:RNA, Messenger / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013431:Sulfates / Q000378:metabolism; D013552:Swine
D011498:Protein Precursors; D012333:RNA, Messenger; D013431:Sulfates; C042082:preprocholecystokinin; D004247:DNA; D002766:Cholecystokinin; D003346:Corticotropin-Releasing Hormone; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases