title: Molecular analysis of ligand binding to the second cluster of complement-type repeats of the low density lipoprotein receptor-related protein. Evidence for an allosteric component in receptor-associated protein-mediated inhibition of ligand binding.
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 receptor-associated 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).PAI-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, Ca2+-dependent ligand binding sites in the cluster II domain (Cl-II) of LRP. t-PA.PAI-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 Ca2+-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-PA.PAI-1 complex and Fab A8 binding to LRP by RAP (IC50 congruent with 0.3 nM), whereas in the reverse experiment, competition of t-PA. PAI-1 complexes and Fab A8 for RAP binding to LRP could only be shown at high concentrations of competitors (>/=1 microM). 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.
authors.affiliation: Department of Biochemistry, Academic Medical Center, University of Amsterdam, 1105 AZ, Amsterdam, The Netherlands.
authors.forename: I R
authors.initials: IR
authors.lastname: Horn
authors.forename: B M
authors.initials: BM
authors.lastname: van den Berg
authors.forename: P Z
authors.initials: PZ
authors.lastname: van der Meijden
authors.forename: H
authors.initials: H
authors.lastname: Pannekoek
authors.forename: A J
authors.initials: AJ
authors.lastname: van Zonneveld
mesh_terms: D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D018832:Molecular Chaperones / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism*; D011973:Receptors, LDL / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments; D026563:LDL-Receptor Related Protein-Associated Protein; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D018832:Molecular Chaperones; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator