Rat PC12 pheochromocytoma cells synthesize insulin-like growth factor-binding protein-6.
The PC12 cell line established from a rat pheochromocytoma has been extensively studied as a model of neuronal differentiation. Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic for PC12 cells under serum-starved conditions. IGF activity is modulated by a family of six IGF-binding proteins (IGFBPs). It recently was reported that PC12 cells produced an IGFBP that had a marked preferential binding affinity for IGF-II over IGF-I. We now show that the main IGFBP produced by PC12 cells is rat IGFBP-6 and compare its properties with those of human IGFBP-6. The predominant IGFBP in medium conditioned by undifferentiated and differentiated PC12 cells migrated on sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis with an apparent molecular mass of 22.5-25 kilodaltons and was recognized by polyclonal antiserum to rat IGFBP-6 by immunoblotting. Rat IGFBP-6 mRNA (1.4 kilobases) was detected by Northern hybridization of total RNA extracted from PC12 cells using a rat IGFBP-6 cDNA probe. Rat IGFBP-6, like human IGFBP-6, is O-glycosylated; incubation with neuraminidase, fucosidase, and O-glycanase reduced its apparent molecular mass to 21 kilodaltons. Competitive binding studies of rat and human IGFBP-6 with [125I]IGF-II and unlabeled IGF-II or IGF-I demonstrated that both IGFBPs bound IGF-II with similar affinities (Ka, 1.5-1.8 x 10(11) M-1) and bound IGF-I with approximately 25- to 35-fold lower affinity than IGF-II. Thus, differences in amino acid sequence, such as deletion of nine amino-terminal residues (including two conserved cysteine residues) in rat IGFBP-6 compared with human IGFBP-6, do not alter its binding characteristics. PC12 cells should provide a useful system to define the regulation of IGFBP-6 expression and the role of IGFBP-6 in modulating IGF action.
Bach
L A
LA
Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Tseng
L Y
LY
Swartz
J E
JE
Rechler
M M
MM
D000818:Animals; D001667:Binding, Competitive; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002850:Chromatography, Gel; D017077:Culture Media, Conditioned; D006031:Glycosylation; D006801:Humans; D018976:Insulin-Like Growth Factor Binding Protein 6; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008970:Molecular Weight; D016716:PC12 Cells / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D018976:Insulin-Like Growth Factor Binding Protein 6; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II