Patricia A. Young

High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation: CRITICAL ROLES OF LYSINES 60 AND 191.

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.

Evidence That Factor VIII Forms a Bivalent Complex with the Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1): IDENTIFICATION OF CLUSTER IV ON LRP1 AS THE MAJOR BINDING SITE.

Hemophilia A is a bleeding disorder caused by a deficiency in coagulation factor VIII (fVIII) that affects 1 in 5,000 males. Current prophylactic replacement therapy, although effective, is difficult to maintain due to the cost and frequency of injections. Hepatic clearance of fVIII is mediated by the LDL receptor-related protein 1 (LRP1), a member of the LDL receptor family. Although it is well established that fVIII binds LRP1, the molecular details of this interaction are unclear as most of the studies have been performed using fragments of fVIII and LRP1. In the current investigation, we examine the binding of intact fVIII to full-length LRP1 to gain insight into the molecular interaction. Chemical modification studies confirm the requirement for lysine residues in the interaction of fVIII with LRP1. Examination of the ionic strength dependence of the interaction of fVIII with LRP1 resulted in a Debye-Hückel plot with a slope of 1.8 ± 0.5, suggesting the involvement of two critical charged residues in the interaction of fVIII with LRP1. Kinetic studies utilizing surface plasmon resonance techniques reveal that the high affinity of fVIII for LRP1 results from avidity effects mediated by the interactions of two sites in fVIII with complementary sites on LRP1 to form a bivalent fVIII·LRP1 complex. Furthermore, although fVIII bound avidly to soluble forms of clusters II and IV from LRP1, only soluble cluster IV competed with the binding of fVIII to full-length LRP1, revealing that cluster IV represents the major fVIII binding site in LRP1.