LIGAND-BINDING RESULTS IN DIVALENT-CATION DISPLACEMENT FROM THE ALPHA(2)BETA(1) INTEGRIN-I DOMAIN - EVIDENCE FROM TERBIUM LUMINESCENCE SPECTROSCOPY

The alpha(2)beta(1) integrin serves as a cell surface collagen or coll agen/laminin receptor. Binding of the integrin to its ligands is large ly mediated by the at subunit I domain and requires the presence of di valent cations. Terbium ion (Tb3+), a fluorescent trivalent cation tha t often binds divalent cation-binding sites on proteins, supported bin ding of the I domain to collagen with half-maximal binding occurring a t 5.2 +/- 1.7 mu M Tb3+. By fluorescence resonance energy transfer spe ctroscopy, Tb3+ showed specific and saturable binding to the recombina nt I domain with a K-d of 27 +/- 4 mu M. Although both Mg2+ and Mn2+ w ere capable of quenching Tb3+ fluorescence, Mn2+ was much more effecti ve than Mg2+. The alpha(2)beta(1) integrin also binds the pro-alpha 1( I) collagen carboxyl-terminal propeptide in a Mg2+-dependent manner vi a the I domain. Recombinant propeptide was used to examine the effect of ligand on the Tb3+ binding properties of the alpha(2) integrin I do main. As propeptide bound to the I domain, Tb3+ fluorescence progressi vely diminished suggesting that as ligand binds to the I domain, eithe r Tb3+ is displaced or its fluorescence is quenched. Consistent with t he former possibility, little dissociation of collagen-bound I domain occurred upon the addition of EDTA and subsequent incubation. These da ta support a model in which (1) the divalent cation is required for in itial ligand-binding activity of the I domain and (2) ligand binding r esults in subsequent metal ion displacement to generate a metal-free I domain-ligand complex.