Molecular Analysis of Ligand Binding to the Second Cluster of Complement-type Repeats of the Low Density Lipoprotein Receptor-related Protein

Abstract

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor gene family, mediates the cellular uptake of a diversity of ligands. A folding chaperone, the 39-kDa receptorassociated protein (RAP) that resides in the early compartments of the secretory pathway inhibits the binding of all ligands to the receptor and may serve to prevent premature binding of ligands to the receptor during the trafficking to the cell surface. To elucidate the molecular interactions that underlie the interplay between the receptor, RAP, and the ligands, we have analyzed and delineated the binding sites of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (t-PA)⅐PAI-1 complexes, RAP, and the anti-LRP Fab fragment Fab A8. To that end, we have generated a series of soluble recombinant fragments spanning the second cluster of complement-type repeats (C3-C10) and the amino-terminal flanking epidermal growth factor repeat (E4) of LRP (E4-C10; amino acids 787-1165). All fragments were expressed by stably transfected baby hamster kidney cells and purified by affinity chromatography. A detailed study of ligand binding to the fragments using surface plasmon resonance revealed the presence of three distinct, Ca 2؉ -dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA⅐PAI-1 complexes as well as PAI-1 bind to a domain located in the amino-terminal portion of Cl-II, spanning repeats E4-C3-C7. Adjacent to this site and partially overlapping is a high affinity RAP-binding site located on repeats C5-C7. Fab A8, a pseudo-ligand of the receptor, binds to a third Ca 2؉ -dependent binding site on repeats C8-C10 at the carboxyl-terminal end of Cl-II. Next, we studied the RAP-mediated inhibition of ligand binding to LRP and to Cl-II. As expected, we observed a strong inhibition of t-PA⅐PAI-1 complex and Fab A8 binding to LRP by RAP (IC 50 Х 0.3 nM), whereas in the reverse experiment, competition of t-PA⅐PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors ( 1 M).Interestingly, even though the equilibrium dissociation constants for the binding of RAP to LRP and to Cl-II are similar, the binding of the ligands to Cl-II is only prevented by RAP at concentrations that are at least 2 orders of magnitude higher than those required for inhibition of ligand binding to LRP. Our results favor models that propose RAP-induced allosteric inhibition of ligand binding to LRP that may require LRP moieties that are located outside Cl-II of the receptor.The low density lipoprotein receptor-related protein (LRP), 1 a 600-kDa glycoprotein, is a multifunctional endocytic receptor (1-3). LRP binds and internalizes a broad spectrum of structurally unrelated ligands including two important components of the fibrinolytic system, tissue-type plasminogen activator (t-PA) and its specific inhibitor plasminogen activator inhibitor type-1 (PAI-1) (4, 5). In rodents, LRP was shown to be one of the in vivo receptor systems involved in th...

The Second and Fourth Cluster of Class A Cysteine-rich Repeats of the Low Density Lipoprotein Receptor-related Protein Share Ligand-binding Properties

“…It is as yet unclear how RAP is capable of antagonizing all ligand binding to LRP. In a previous study we have shown that, even though the affinity of RAP for cluster II and LRP is similar, RAP is a poor inhibitor of ligand binding to the isolated cluster II compared with its inhibition of ligand binding to the intact LRP (9). This suggests that RAP does not inhibit ligand binding solely in a competitive or sterical manner.…”

“…Procedures to purify active PAI-1 were essentially as described (25,26). t-PA⅐PAI-1 complexes were prepared as described (9). Human tissue factor pathway inhibitor was a generous gift from Dr. L. Aarden (Central Laboratory of Blood Transfusion, Amsterdam, The Netherlands).…”

“…Receptor Fragments-The construction of plasmids, encoding recombinant LRP cluster II fragments, was described previously (9). One of these plasmids, pZEM229R-II, was modified to facilitate the construction of plasmids, encoding recombinant LRP cluster I, III, and IV fragments.…”

“…Specifically, it has been demonstrated that ␣ 2 -macroglobulin-light chain, complexes between urokinase-type plasminogen activator (u-PA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1), lactoferrin, an anti-LRP Fab fragment (denoted Fab A8), and RAP bind to cluster II. In addition, complexes between tissue-type plasminogen activator (t-PA) and PAI-1 were shown to bind to cluster II, while no binding of t-PA⅐PAI-1 complex to cluster IV could be detected (7,9). The present study was performed to systematically examine the binding of a large number of structurally and functionally distinct ligands to each of the clusters I to IV to obtain more insight into the molecular elements that contribute to the remarkable ligand binding capacity of LRP.…”

“…RAP inhibits the binding of all ligands to the receptor and is thought to prevent premature binding of ligands to the receptor during the trafficking to the cell surface (15)(16)(17). Some ligands for LRP occupy distinct binding sites (9,18) while other ligands compete with each other for binding to LRP (19 -22). Although RAP competes with all known ligands, most ligands are not able to compete for RAP binding to LRP.…”