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Multivalent Cations Depress Ligand Binding to Cell-Associated Insulin-Like Growth Factor Binding Protein-5 on Human Glioblastoma Cells*

The Department of Animal Sciences, Laboratory of Developmental Endocrinology, The University of Illinois, Urbana, Illinois 61801

Address all correspondence and requests for reprints to: Robert H. McCusker, Ph.D., 210 Meat Science Laboratory, 1503 South Maryland Drive, Urbana, Illinois 61801-4737. E-mail: rmccuske{at}staff.uiuc.edu

The current studies quantified the effect of the multivalent cations zinc, cadmium, lanthanum, chromium, and gold (Zn²⁺, Cd²⁺, La³⁺, Cr³⁺, and Au³⁺) on [¹²⁵I]-insulin-like growth factor ([¹²⁵I]-IGF) binding to T98G human glioblastoma cells. The major binding site for the IGFs on T98G cells is IGF binding protein-5 (IGFBP-5), as determined by affinity labeling. Competitive binding studies, using either [¹²⁵I]-IGF-I or [¹²⁵I]-IGF-II, indicated that La³⁺ and Cr³⁺ did not affect [¹²⁵I]-IGF-I or [¹²⁵I]-IGF-II binding to cell-associated IGFBP-5. Zn²⁺, Au³⁺, and Cd²⁺ depressed binding of both [¹²⁵I]-IGF-I and [¹²⁵I]-IGF-II. [¹²⁵I]-IGF-I and [¹²⁵I]-IGF-II binding resulted in nonlinear concave-down Scatchard plots, indicating the presence of high- and low-affinity equilibrium constant of association (K_a) sites. Assuming a preexisting asymmetric model with independent high (K_aHi) and low (K_aLo) sites; Zn²⁺, Cd²⁺, and Au³⁺ eliminated K_aHi and Zn²⁺, and Au³⁺ lowered K_aLo, compared with control values. The same results were found, independent of whether [¹²⁵I]-IGF-I or [¹²⁵I]-IGF-II was used. Similarly, assuming a ligand-induced model of negative cooperativity, all three cations eliminated the initial affinity for the high affinity sites (K_e), whereas Zn²⁺ and Au²⁺ reduced the final affinity for the low affinity sites (K_f). Dose-response studies indicated that Zn²⁺, Au³⁺, and Cd²⁺ depressed binding with half-maximal activities of approximately 20 µM, 14–60 µM, and 50–65 µM, respectively. Zn²⁺, Au³⁺, and Cd²⁺ bind to similar sites on proteins (a zinc-binding motif), indicating similar mechanisms of action. A zinc-binding motif is present within the IGFBPs but not the IGFs. We demonstrate, for the first time, that multivalent cations have the potential to modulate IGF activity by decreasing the amount of IGF bound to cell-associated IGFBP-5.