Alterations in the Synthesis of a Fibroblast Surface Associated 35 K Protein Modulates the Binding of Somatomedin-C/lnsulin-Like Growth Factor I*

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Abstract
Human fibroblasts, a cell type that is used extensively to determine the pleiotypic effects of the insulin-like growth factors, have been shown to secrete a 35K protein that binds somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) but not insulin. This 35 K protein is associated with the fibroblast surface and following transfer to the surface of cell types that do not have this protein on their surfaces, it alters the binding of radiolabeled Sm-C/IGF-I. In this study human fibroblast monolayers that were incubated with cycloheximide (50 .mu.g/ml) for 14 h at 37 C had no detectable 35 K protein on their cell surface, but type I Sm-C/IGF-I receptors were still present. Loss of the 35 K protein was associated with 60-70% increase in binding of Sm-C/IGF-I to type I receptors. The relative affinity of the type I receptor for Sm-C/IGF-I was apparently increased because unlabeled Sm-C/IGF-I (12 ng/ml) competitively displaced 63% of radiolabeled Sm-C/IGF-I after cycloheximide exposure, whereas in cultures not exposed to cycloheximide [125I]Sm-0C/IGF-I binding was increased by 11%. Coincubation of fibroblast conditioned media containing the 35 K protein with cycloheximide-treated fibroblast monolayers resulted in restoration of the paradoxical increase in Sm-C/IGF-I binding and loss of sensitivity to competition by unlabeled Sm-C/IGF-I. Exposure of suspended fibroblasts, which do not have 35 K on their cell surface, to media conditioned by fibroblast monolayers also induced both of these changes. Extensive washing the incubation of the monolayers in serum-free media, a condition that reduces protein synthesis, did not result in a complete removal of the 35 K protein from the cell surface and effected only a partial restoration of sensitivity to competition by unlabeled Sm-C/IGF-I. Cross-linking of 125I-35 K protein to the cell surface did not reveal a specific protein receptor for the binding protein. Furthermore, immunoprecipitation studies showed no structural homology between the type I receptor and the 35 Kilodalton binding protein. It appears that cultured fibroblast monolayers synthesize and secrete a 35 K protein that alters Sm-C/IGF-I binding to the cell surface. This protein may be an important modulator of the cellular response to Sm-C/IGF-I.

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