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/alpha(2)-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by t:his receptor. We recen tly utilized glutathione S-transferase (GST)-39-kDa fusion protein con structs to demonstrate that constructs encoding amino-terminal residue s 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein in dependently bind to purified LRP and to LRP on hepatoma cells with sim ilar affinities as the full-length GST-39-kDa protein (Kd similar to 8 -10 nM). These regions, however, inhibit ligand binding to LRP differe ntly: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha(2)-macroglobulin-methylamine (alpha(2)M) binding whereas G ST/1 15 -3 19 only potently inhibits t-PA binding. Four domains, conta ining residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are r equired for inhibition of ligand binding. In the present study, we gen erated additional 39-kDa protein constructs to precisely define residu es within each domain required for inhibition of t-PA and alpha(2)M b inding 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 leucin e 104 are required for inhibition of t-PA and alpha(2)M binding. Thes e residues, however, are not required for binding either to purified L RP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is re quired for inhibition of t-PA and alpha(2)M binding as well as for th e direct binding of amino-terminal constructs to LRP. Within carboxy-t erminal domains 200-225 and 311-319, leucine 222 and leucine 319 are b oth required for inhibition of t-PA binding. Deletion of leucine 319 c hanges the ligand specificity from inhibition of t-PA binding, to inhi bition of alpha(2)M binding. Thus, leucine 319 is not required for di rect binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.