Dissection of the Domain Architecture of the α 2 macroglobulin‐Receptor‐Associated Protein

136

150

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic cell-surface receptor that binds and internalizes a diverse array of ligands. The receptor contains four putative ligand-binding domains, generally referred to as clusters I, II, III, and IV. In this study, soluble recombinant receptor fragments, representing each of the four individual clusters, were used to map the binding sites of a set of structurally and functionally distinct ligands. Using surface plasmon resonance, we studied the binding of these fragments to methylamine-activated ␣ 2 -macroglobulin, pro-urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1, t-PA⅐plasminogen activator inhibitor-1 complexes, lipoprotein lipase, apolipoprotein E, tissue factor pathway inhibitor, lactoferrin, the light chain of blood coagulation factor VIII, and the intracellular chaperone receptor-associated protein (RAP). No binding of the cluster I fragment to any of the tested ligands was observed. The cluster III fragment only bound to the anti-LRP monoclonal antibody ␣ 2 MR␣3 and weakly to RAP. Except for t-PA, we found that each of the ligands tested binds both to cluster II and to cluster IV. The affinity rate constants of ligand binding to clusters II and IV and to LRP were measured, showing that clusters II and IV display only minor differences in ligandbinding kinetics. Furthermore, we demonstrate that the subdomains C3-C7 of cluster II are essential for binding of ligands and that this segment partially overlaps with a RAP-binding site on cluster II. Finally, we show that one RAP molecule can bind to different clusters simultaneously, supporting a model in which RAP binding to LRP induces a conformational change in the receptor that is incompatible with ligand binding.The low density lipoprotein receptor-related protein (LRP) 1 is a 600-kDa membrane glycoprotein that is a member of the low density lipoprotein (LDL) receptor family of endocytic receptors (reviewed in Refs. 1 and 2). LRP can bind and internalize a diverse spectrum of structurally unrelated ligands in a calcium-dependent manner including apolipoproteins, lipases, proteinases, proteinase-inhibitor complexes, Kunitz-type inhibitors, matrix proteins, and other proteins such as lactoferrin, Pseudomonas exotoxin A, and malaria circumsporozoite protein (1, 2). In addition, the blood coagulation factor VIII was recently identified as a ligand of LRP (3). The broad range of ligands suggests a role for the receptor in distinct physiological and pathophysiological processes, ranging from lipoprotein metabolism, cell growth and cell migration, fibrinolysis, and thrombosis to atherosclerosis and Alzheimer's disease.LRP is synthesized as a single polypeptide chain and is cleaved in the trans-Golgi network by the endopeptidase furin into two subunits, resulting in a 515-kDa fragment that contains the ligand binding domains and an 85-kDa fragment comprising the transmembrane and cytoplasmic domains. The subunits rema...

Section: Kinetics Of Ligand Binding To Lrp Cluster II and Cluster Imentioning

confidence: 99%

Section: Kinetics Of Ligand Binding To Lrp Cluster II and Cluster Imentioning

confidence: 99%

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

Neels¹,

Berg²,

Lóokene³

et al. 1999

136

150

“…The RAP domains were expressed as fusion proteins. The constructs (carrying the first 34 N-terminal residues of the phage CII repressor, a hexahistidine affinity tag, and a factor Xa recognition site at the N terminus of the RAP sequences) were constructed, expressed, and purified as described (37 RAP were labeled with 125 I as described (48). Rat PN-1 and human PAI-1 were prepared as described (49,50).…”

Section: Methodsmentioning

confidence: 99%

“…Mature human RAP consists of 323 amino acids (35). Due to internal sequence homology, a three-domain structure has been proposed (36,37). The three-dimensional structure of the N-terminal domain was determined by NMR spectroscopy (38).…”

mentioning

confidence: 99%

Ligand Binding Properties of the Very Low Density Lipoprotein Receptor

Rettenberger

Oka

Ellgaard

et al. 1999

Self Cite

The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the M r 40,000 receptor-associated protein (RAP) and a variety of serine proteinaseserpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA⅐PAI-1, and uPA⅐PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed ϳ4-fold lower binding of RAP than VLDLR-I and ϳ10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA⅐PAI-1 and uPA⅐PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA⅐PN-1, but not uPA⅐PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.

“…Polymerase chain reaction products were cleaved using BamHI and HindIII and subcloned into the vector pT7H6UB (33). To construct the plasmid encoding Plg kringle 1, the following primers were used, 5Ј-CGT CCT GGA TCC ATC GAG GGT AGG TCA GAG TGC AAG ACT GGG AAT GG-3Ј and 5Ј-CGA CCG AAG CTT ATT CAC ACT CAA GAA TGT CGC-3Ј.…”

Section: Construction Of Expression Plasmids and Site-directed Mutagementioning

confidence: 99%

Mutational Analysis of Affinity and Selectivity of Kringle-Tetranectin Interaction

Graversen¹,

Jacobsen²,

Sigurskjold³

et al. 2000

Self Cite

C-type lectin-like domains are found in many proteins, where they mediate binding to a wide diversity of compounds, including carbohydrates, lipids, and proteins. The binding of a C-type lectin-like domain to a ligand is often influenced by calcium. Recently, we have identified a site in the C-type lectin-like domain of tetranectin, involving Lys-148, Glu-150, and Asp-165, which mediates calcium-sensitive binding to plasminogen kringle 4. Here, we investigate the effect of conservative substitutions of these and a neighboring amino acid residue. Substitution of Thr-149 in tetranectin with a tyrosine residue considerably increases the affinity for plasminogen kringle 4, and, in addition, confers affinity for plasminogen kringle 2. As shown by isothermal titration calorimetry analysis, this new interaction is stronger than the binding of wild-type tetranectin to plasminogen kringle 4. This study provides further insight into molecular determinants of importance for binding selectivity and affinity of C-type lectin kringle interactions.