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Abstract. Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-U, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF-I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors.

Insulin-like growth factor (IGF)-binding protein 1 (IGFBP-1) contains an Arg-Gly-Asp (RGD) integrin recognition sequence. In vitro mutagenesis was used to alter this RGD sequence to Trp-Gly-Asp (WGD). Migration of Chinese hamster ovary (CHO) cells expressing the wild-type protein was more than 3-fold greater in 48 hr compared with ceUs expressing the WGD mutant form of IGFBP-1. Similarly, wild-type IGFBP-1 added to the media of control CHO cells stimulated migration 2-fold compared with the WGD protein.A synthetic RGD-containing peptide, when added to the medium with wild-type IGFBP-1, blocked the effect of IGFBP-1 on cell migration. The addition of IGF-I to the culture medium had no effect on the migration of cells expressing IGFBP-1 or vector alone. Affmity chromatography of 12I-labeled CHO cel membrane proteins, using IGFBP-1 coupled to agarose, iden- These studies demonstrate that IGFBP-1 stimulates CHO cell nmgration and binds to the a4s31 integrin receptor, both by an RGD-dependent mechanism. The effect of IGFBP-1 on migration is independent of IGF-I and is probably mediated through the a43.B integrin.The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of homologous but distinct proteins that specifically bind IGF-I and IGF-II with high affinity and are ubiquitous in biologic fluids, tissues, and the extracellular matrix of most cell types (1). Currently six IGFBPs have been purified, cloned, and sequenced (2,3). The roles of these proteins in intercellular transport, extracellular localization, and the modulation of the actions of IGF-I and IGF-II are areas of active study. The major focus of studies published to date has been to examine the effects of the IGFBPs on modifying IGF physiology. However, few direct non-IGFmediated effects of a purified form of IGFBP on cellular functions have been reported. IGFBP-1 is a phosphorylated protein of approximately 25 kDa that is expressed in greatest amounts during fetal development. It has been reported to potentiate (4) or inhibit (5) IGF actions, depending upon the experimental conditions and degree of IGFBP-1 phosphorylation (6). IGFBP-1 contains an Arg-Gly-Asp (RGD) integrin recognition sequence (7,8) and has been shown to bind to cell surfaces (9). The specific mechanism by which IGFBP-1 or any of the other IGFBPs bind to cell surfaces has not been previously described.We undertook these studies after observing that transfected Chinese hamster ovary (CHO) cells expressing humanThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.IGFBP-1 appeared to spread and migrate on tissue culture plates more rapidly than CHO cells not expressing IGFBP-1. Cell migration is known in many cell types to involve cellular integrin receptor binding to RGD sequences in extracellular matrix proteins (10). Since IGFBP-1 binds to cell surfaces and contains an RGD sequence, we sought to determine ...

The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). We and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted us to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells (transfected with a human IGFBP-l cDNA construct) and human hepatoma (HepG2) cells secrete 32P-labeled IGFBP-1 following incubation with [32P orthophosphate. Phospho amino acid analysis of 32P-labeled IGFBP-l revealed only phosphoserine residues. A method was developed that could separate nonphosphorylated IGFBP-l from four or five phosphorylated isoforms. Using this technique we demonstrated that human amniotic fluid and human fetal serum contain a large proportion of nonphosphorylated IGFBP-1, as well as phosphorylated forms. In contrast, HepG2 cells and human decidual cells secrete predominantly the phosphorylated isoforms. These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo. Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation. We conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification.

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 1251 and immunoprecipitated with specific anti-integrin antibodies. Integrins containing aV (vitronectin receptor), a5 (fibronectin receptor), and a3 (collagen/ laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in a5-integrin subunit protein and a 25% increase in aV subunit. More importantly, ligand binding of aVf33 was increased by 2.4-fold. We therefore examined whether the function of the aVf83 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to aV.83 and not to a5j31 or other abundant integrins. The related disintegrin echistatin specifically inhibited 1251-labeled kistrin binding to cVI83, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the aiV integrin. Furthermore, an aVf83 blocking antibody inhibited SMC migration by 80%o. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and aVj33 integrin antagonists appear to be impor-