Inducible overexpression and secretion of int-1 protein.
The int-1 proto-oncogene is a target for insertional activation of transcription by mouse mammary tumor virus in many murine mammary tumors. Whereas no expression of int-1 is seen in normal mammary tissue, int-1 RNA can be detected in normal mice in the neural tubes of midgestation embryos and in postmeiotic spermatocytes from adult testes. I report here the results of a study in which several different antibodies against synthetic peptides were produced and used to characterize the processing and secretion of int-1 protein. CHO cells were transfected with an inducible int-1 expression vector that was subsequently amplified to generate cell lines expressing very high levels of int-1 protein. Immunoprecipitation of [35S]cysteine-labeled cell lysates from these CHO cells yielded large amounts of four immature forms of int-1 glycoprotein (molecular weights of 36,000, 38,000, 40,000, and 42,000). A significant fraction of these int-1 species formed disulfide-linked multimers. Pulse-chase and glycosidase digestion studies demonstrated that some of the immature species of int-1 protein move through the secretory pathway and are processed to a mature heterogeneous glycoprotein with a molecular weight of about 44,000. Suramin treatment of the CHO cells during pulse-chase experiments increased the amount of 44,000-molecular-weight int-1 protein in the culture medium.
Papkoff
J
J
Syntex Research, Palo Alto, California 94304.
D000818:Animals; D002460:Cell Line; D004220:Disulfides / Q000032:analysis; D006031:Glycosylation; D006358:Hot Temperature; D010446:Peptide Fragments / Q000032:analysis; D010957:Plasmids; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D014162:Transfection; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029961:Zebrafish Proteins
D004220:Disulfides; D010446:Peptide Fragments; D011498:Protein Precursors; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029961:Zebrafish Proteins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase