Sites Within the 39-kDa Protein Important for Regulating Ligand Binding to the Low-Density Lipoprotein Receptor-Related Protein

A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/α₂-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione 5-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1 - 114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (K_d ~ 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1 - 114 inhibits both tissue-type plasminogen activator (t-PA) and α₂-macroglobulin-methylamine (α₂M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and α₂M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1 - 114, alanine 103 and leucine 104 are required for inhibition of t-PA and α₂M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and α₂M* binding as well as for the direct binding of amino-terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of α₂M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.