Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines.
A previous report from our laboratory described an approximately 30 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) that inhibited the binding of insulin-like growth factor I (IGF-I) by its receptor and was secreted by a subline of the B104 rat neuronal cell line. To better understand the biology of this IGFBP, it was purified from media conditioned by these B104 cells, and the chemical and biological properties of the protein were examined. The IGFBP existed as a 24 kDa form and a 28 kDa form when the conditioned media were analyzed by ligand blot. Deglycosylation studies indicated the 28 kDa species was the N-linked glycosylated form of the 24 kDa IGFBP. Multiple forms at both mol wts were found using two-dimensional electrophoresis, suggesting that there were posttranslational modifications in addition to glycosylation. The amino acid sequence of the 12 amino-terminal residues was identical to that of rat IGFBP-4. Increased synthesis of IGFBP-4 by the subline contrasted with neglible production by other B104 cells. Blot hybridization with rat IGFBP-4 complementary DNA showed differential expression of a 2.6 kilobase transcript among B104 cell lines that correlated with quantities of IGFBP-4 secreted in media. The difference persisted even when the cells were xenografted into athymic nude mice. Purified IGFBP-4 inhibited the binding of [125I]IGF-I by its receptor and blunted stimulation of [3H]thymidine incorporation by IGF-I. These findings suggest a role for IGFBP-4 in neural cell function and indicate the B104 cell lines may be a useful model for further examination of IGFBP-4 biology.
Cheung
P T
PT
Department of Pediatrics, Children's Hospital Medical Center, Cincinnati, Ohio 45229.
Smith
E P
EP
Shimasaki
S
S
Ling
N
N
Chernausek
S D
SD
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D018974:Insulin-Like Growth Factor Binding Protein 4; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D002352:Carrier Proteins; D018974:Insulin-Like Growth Factor Binding Protein 4; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; D004247:DNA