MULTIVALENT CATIONS DEPRESS LIGAND-BINDING TO CELL-ASSOCIATED INSULIN-LIKE-GROWTH-FACTOR BINDING PROTEIN-5 ON HUMAN GLIOBLASTOMA CELLS

The current studies quantified the effect of the multivalent cations z inc, cadmium, lanthanum, chromium, and gold (Zn2+, Cd2+, La3+, Cr3+, a nd Au3+) on [I-125]-insulin-like growth factor ([I-125]-IGF) binding t o T98G human glioblastoma cells. The major binding site for the IGFs o n T98G cells is IGF binding protein-5 (IGFBP-5), as determined by affi nity labeling. Competitive binding studies, using either [I-125]-IGF-I or [I-125]-IGF-II, indicated that La3+ and Cr3+ did not affect [I-125 ]-IGF-I or [I-125]-IGF-II binding to cell-associated IGFBP-5. Zn2+, Au 3+, and Cd2+ depressed binding of both [I-125]-IGF-I and [I-125]-IGF-I I. [I-125]-IGF-I and [I-125]-IGF-II binding resulted in nonlinear conc ave-down Scatchard plots, indicating the presence of high- and low-aff inity equilibrium constant of association (K-a) sites. Assuming a pree xisting asymmetric model with independent high (K-aHi) and low (K-aLo) sites; Zn2+, Cd2+, and Au3+ eliminated K-aHi and Zn2+, and Au3+ lower ed K-aLo, compared with control values. The same results were found, i ndependent of whether [I-125]-IGF-I or [I-125]-IGF-II was used. Simila rly, assuming a ligand-induced model of negative cooperativity, all th ree cations eliminated the initial affinity for the high affinity site s (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 approximat ely 20 mu M, 14-60 mu M, and 50-65 mu M, respectively. Zn2+, Au3+, and Cd2+ bind to similar sites on proteins (a zinc-binding motif), indica ting similar mechanisms of action. A zinc-binding motif is present wit hin the IGFBPs but not the IGFs. We demonstrate, for the first time. t hat multivalent cations have the potential to modulate IGF activity by decreasing the amount of IGF bound to cell-associated IGFBP-5.