Nickel is a specific inhibitor for the binding of activated alpha(2)-macroglobulin to the low density lipoprotein receptor-related protein alpha(2)-macroglobulin receptor

The low density receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)-MR) binds to several ligands involved in lipoprotein and protease clearance. The receptor-associated protein (RAP) inhibits the binding of all known ligands. We studied the inhibition by Ni2+ of the binding of different ligands to cells and to the purified LRP/alpha(2)-MR. Ni2+ inhibited all of the specific binding of radiolabeled methylamine-activated alpha(2)-macroglobulin (I-125-alpha(2)-M*) to rabbit aortic smooth muscle cells (SMC), rat hepatoma Fu5AH, and mouse fibroblast L cells. Ni2+ also inhibited the binding of trypsin-activated alpha(2)-macroglobulin to SMC but did not affect the binding of RAP, Pseudomonas exotoxin A, or low density lipoproteins. The inhibition of alpha(2)-M* binding by Ni2+ was not due to its interaction with alpha 2-M*. Preincubation of SMC with Ni2+ followed by ligand binding suggested that Ni2+ binds to cell-surface molecules and inhibits the binding of alpha(2)-M* but does not affect RAP binding. Most of the binding of alpha(2)-M* to SMC was due to its binding to the LRP/alpha(2)-MR, as opposed to the recently described signaling receptor, as demonstrated by the inhibition of this binding by the RAP. Moreover, the inhibition of alpha(2)-M* binding to the LRP/alpha(2)-MR by Ni2+ was demonstrated using purified receptor immobilized on microtiter plates. Two to three molecules of Ni-63(2+) bound to the immobilized receptor with equal affinity but not to alpha(2)-M*. The specific binding of alpha(2)-M* to the immobilized receptor was inhibited in the presence of nickel. Furthermore, preincubation of the immobilized LRP/alpha(2)-MR with Ni2+ inhibited the binding of alpha(2)-M* but did not inhibit RAP or exotoxin A binding. These data suggest that Ni2+ is a site-specific inhibitor for the alpha 2-M* binding site present on the LRP/alpha(2)-MR. Nickel may be a useful tool for investigating different ligand binding domains of the LRP/alpha(2)-MR.