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/alpha2-macroglobulin receptor (LRP/alpha2MR) binds and internalizes several plasma proteins including tissue-type plasminogen activator (t-PA) and alpha2-macroglobulin-protease complexes (alpha2M*). A 39-kDa protein that copurifies with LRP/alpha2MR inhibits the binding and uptake of ligands by LRP/alpha2MR, including t-PA and alpha2M*. To define domains on the 39-kDa protein which are essential for inhibition of t-PA and alpha2M* binding to LRP/alpha2MR, 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 alpha2M* binding to LRP/alpha2MR 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 alpha2M* binding to LRP/alpha2MR is also mediated by residues 200-225 and 311-319. The same 39-kDa protein constructs that inhibit alpha2M* and t-PA binding to MH1C1 cells are able to bind directly to purified LRP/alpha2MR 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 alpha2M* binding to LRP/alpha2MR.