Identification of domains on the 39-kDa protein that inhibit the binding of ligands to the low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein/α₂-macroglobulin receptor (LRP/α₂MR) binds and internalizes several plasma proteins including tissue-type plasminogen activator (t-PA) and α₂-macroglobulin- protease complexes (α₂M*). A 39-kDa protein that copurifies with LRP/α₂MR inhibits the binding and uptake of ligands by LRP/α₂MR, including t-PA and α₂M*. To define domains on the 39-kDa protein which are essential for inhibition of t-PA and α₂M* binding to LRP/α₂MR, we have generated bacterial expression constructs encoding discrete regions of the 39-kDa protein as fusion proteins with glutathione S-transferase. Inhibition of t-PA and α₂M* binding to LRP/α₂MR on rat hepatoma MH₁C₁ cells are shown to require amino acid residues 18-24 and 100-107 on the 39-kDa protein. Inhibition of t-PA but not α₂M* binding to LRP/α₂MR is also mediated by residues 200-225 and 311-319. The same 39-kDa protein constructs that inhibit α₂M* and t-PA binding to MH₁C₁ cells are able to bind directly to purified LRP/α₂MR immobilized on nitrocellulose. Thus, our studies demonstrate several specific regions on the 39-kDa protein which are required for the inhibition of t-PA and α₂M* binding to LRP/α₂MR.