Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs).
The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.
McCusker
R H
RH
Department of Medicine, University of North Carolina, Chapel Hill 27599.
Camacho-Hubner
C
C
Bayne
M L
ML
Cascieri
M A
MA
Clemmons
D R
DR
D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005910:Glioma; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins / Q000378:metabolism; D013002:Somatomedins / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II