growth factor (IGF) binding to human fibroblast and glioblastoma cells: the
modulating effect of cell released IGF binding proteins (IGFBPs)
McCusker RH, Camacho-Hubner C, Bayne ML, Cascieri MA, Clemmons DR
Department of Medicine, University of North Carolina, Chapel Hill
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
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
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
PMID: 2166057 [PubMed - indexed for MEDLINE]