The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each ≈ 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca2+ cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. α2-macroglobulin (α2M)–proteinase complexes, lipoprotein-containing particles and serine proteinase–inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA–PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3–CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA–PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp958,CR5, Asp999,CR6, Trp953,CR5 and Trp994,CR6), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP)–the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA–PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA–PAI-1 as well as for the binding of RAP.
Abbreviations used: LDLR, low-density-lipoprotein receptor; LRP, α2-macroglobulin (α2M) receptor/LDLR-related protein; RAP, receptor-associated protein; RAPd3, RAP residues 216–323; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; uPAR, uPA receptor; CR, complement-type repeat from LRP; VLDLR, very-low-density-lipoprotein receptor; apoER2, apolipoprotein E receptor 2; SPR, surface plasmon resonance; α2M∗, transformed α2M; EGF, epidermal growth factor; LB, complement-type repeat from LDLR; wt, wild-type; W994S, a mutant in which Trp994 has been mutated to Ser (and similarly for other mutants); serpin, serine-proteinase-inhibitor.
These authors contributed equally to this work.