High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation

Abstract

The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three-domain protein that contains the following two high affinity binding sites for LRP1: one is located within domains 1 and 2, and one is located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here, we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2-LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors, which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor.The LDL receptor-related protein 1 (LRP1) is a member of the LDL receptor family and is a highly efficient endocytic and signal-transducing receptor that plays an important role in vascular development, lipoprotein metabolism, and inflammation (1-3). Originally identified as the hepatic receptor responsible for the removal of ␣ 2 -macroglobulin (␣ 2 M) 3 -protease complexes (4), we now know that LRP1 recognizes numerous ligands, including lipoproteins, matrix proteins, growth factors (1, 2, 5, 6), and extracellular proteases (7-11). Deletion of the Lrp1 gene in mice results in early embryonic lethality at E13.5 (12, 13) due to extensive hemorrhaging resulting from a failure to recruit and maintain vascular smooth muscle cells and pericytes in the vessels. Selective deletion of LRP1 in vascular smooth muscle cells (smLRP1 Ϫ/Ϫ mice) reveals that LRP1 protects against the development of atherosclerosis by attenuating PDGF receptor activation (14, 15) and prevents formation of aneurysms (11, 16) in part by regulating the levels of proteases that are known to degrade matrix components (11).The efficient delivery of LRP1 and certain other members of the LDL receptor family to the cell surface require their association with a 39-kDa protein termed the receptor-associated protein (RAP). RAP was initially identified when it co-purified with LRP1 (17,18). Subsequent work demonstrated that RAP binds tightly to LRP1 and prevents ligands from associating with this receptor (19,20). In the endoplasmic reticulum (ER), RAP functions a...

Evidence That Factor VIII Forms a Bivalent Complex with the Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1)

“…This apparent discrepancy can be explained by the possibility that several lysine residues may compensate for one another. This also seems to be the case for the binding of D1D2 to LRP1 where we noted that although mutation of Lys-60 had a significant impact on binding of mutant D1D2 to LRP1 mutation of Lys-191 had a minimal impact on binding unless Lys-60 was also mutated (28).…”

“…These experiments were performed in duplicate, and replicates are plotted. only when both lysine 60 and lysine 191 were mutated was binding of D1D2 to LRP1 ablated (28).…”

“…This has been clearly demonstrated for RAP, which contains two binding sites for LRP1. The first binding site is located within domains 1 and 2 of RAP (D1D2), and recent studies reveal that lysine 60 in D1 and lysine 191 in D2 interact with distinct sites on LRP1 to form a bivalent D1D2⅐LRP1 complex (28). A second LRP1 binding site is located in domain 3 of RAP, and here lysines 256 and 270 are key residues that are necessary for high affinity binding of this domain to LRP1 (29,30).…”

Identification of Receptor Ligands in Apo B100 Reveals Potential Functional Domains

“…All three domains in RAP (RAP1d, RAP2d, and RAP3d) were shown to have high binding affinity for LRP (50). Lysine residues 0060 in motifs W K KLK and 0191 in L K EKL of RAP Domains 1 and 2, respectively, were shown experimentally to bind LDLR related protein 1 (LRP-1) with high affinity (51). In RAP3d, residues Lys 0253 , Lys 0257 , Tyr 0260 , Lys 0270 , Arg 0285 , and Arg 0296 were shown to interact with ligand adhesion modules LA3–4 of the LDLR and complement-like repeat CR5–6 of the LRP-1 (51).…”

“…Lysine residues 0060 in motifs W K KLK and 0191 in L K EKL of RAP Domains 1 and 2, respectively, were shown experimentally to bind LDLR related protein 1 (LRP-1) with high affinity (51). In RAP3d, residues Lys 0253 , Lys 0257 , Tyr 0260 , Lys 0270 , Arg 0285 , and Arg 0296 were shown to interact with ligand adhesion modules LA3–4 of the LDLR and complement-like repeat CR5–6 of the LRP-1 (51). The following ligand motifs are established in RAP: E K L, at positions 0024 in RAP1d, positions 0119 and 0193 in RAP2d, and at 0270 in RAP3d; E K V at 0148, L K E at 0191 and D K L at 0137 in RAP2d; and, E K H at 0256 in RAP3d; these motifs are involved in binding to Ligand Adhesion Modules, LA3–4 of the LDLR and CR5–6 of LRP-1 (50–52).…”