SCHMIDT, KE; DESAI, P; SAK, KM; BALOGH, LM; KELLEY, KM: Membrane Binding of an Insulin-like Growth Factor (IGF)-binding Protein (IGFBP) in a Reptilian Heart Cell: Impact on IGF-I Action

A heart cell line (IgH2) derived from Iguana iguana secretes a 30-kDa IGFBP that also localizes to the cell membranes. Competition binding assays on soluble iguana IGFBP demonstrate that it binds ¹²⁵I-IGF-I and -II in a highly specific manner, with 50% inhibition of ¹²⁵I-IGF-I binding between 0.1-0.5 ng/ml of added unlabeled IGF-I. Affinity crosslinking of ¹²⁵I-IGF to cell monolayers indicates that the IGFBP is bound to the cell membranes at levels rivaling that of the cellular type-I IGF receptor. Addition of excess unlabeled IGF-I (>10 ng/ml) results in removal of membrane-bound IGFBP, upon which a corresponding increase in its levels in the medium are observed. This “membrane-removal” effect occurs rapidly, detectable within 5 min of IGF addition. When cells are precultured for 15 min in medium containing 100 ng/ml IGF-I, followed by replacement of fresh identical medium (i.e., IGFBP is removed from membrane & the medium containing released IGFBP discarded), the mitogenic effect of subsequent IGF-I addition is enhanced, as compared with cells in which the membrane IGFBP remains. This suggests that the membrane-bound IGFBP serves to inhibit IGF action in the iguana IgH2 cell through a unique mechanism by which IGF binding to abundant cell surface-associated IGFBP is followed by release of the IGF/IGFBP complex from proximity to the cellular type-I IGF receptor. Recent studies also show that [Ala31][Leu60]-IGF-I, an analog with greatly reduced binding affinity to IGF receptors but normal IGFBP binding, has a similar effect as wild-type IGF-I, suggesting that the membrane-releasing effect is a result of ligand binding to the IGFBP. [Support in part by NSF grants IBN-9600783 & -0115975]