NMR Solution Structure of Complement-like Repeat CR3 from the Low Density Lipoprotein Receptor-related Protein

Background: Low density lipoprotein receptor (LDLR) mediates clearance of blood coagulation factor VIII (FVIII). Results:The region of complement-type repeats 2-5 in LDLR was identified as the binding site for FVIII and for ␣-2-macroglobulin receptor-associated protein (RAP). Conclusion: Binding sites of LDLR for FVIII, and also for RAP, were characterized. Significance: This provides new data on LDLR structure and function and on FVIII catabolism.

Section: Discussionsupporting

confidence: 68%

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Kurasawa

Shestopal

Karnaukhova

et al. 2013

“…LRP contains multiple copies of cysteine-rich repeats known as LDLR class A repeats (complement-type repeats) arranged in 4 clusters, forming ligand-binding sites (45)(46)(47)(48)(49). The binding site for many LRP ligands has been sublocalized (50), and NMR solution structure of the complement-like repeat CR3 from LRP has recently been determined (51). It will be of interest to sublocalize the binding site for CTGF within the extracellular domain of LRP.…”

Section: Discussionmentioning

confidence: 99%

The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Is a Receptor for Connective Tissue Growth Factor

Segarini¹,

Nesbitt²,

Li³

et al. 2001

189

138

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-␤ (TGF-␤) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of ϳ620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/␣ 2 -macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.

“…We have previously shown that the first complement-like repeat in the second cluster of LRP, CR3, binds RBD weakly, although with sufficient affinity (K d ϳ 140 M) to suggest that it forms part of the RBD binding site on LRP (19). We therefore expressed first CR3-4 and then CR3-4-5 to determine whether the full affinity of RBD for LRP could be reproduced by adding contiguous domains and to enable evaluation of the contributions of individual domains to the overall binding energy.…”

Section: Characterization Of Cr3-4 Cr3-4-5 and Cr5-6-7-mentioning

confidence: 99%

“…Given the importance both of LRP as an essential endocytosis and signaling receptor and of ␣ 2 M-proteinase complexes as an LRP ligand, an understanding of the specificity of their interaction is a very worthwhile goal. An excellent starting point is the known importance of RBD in binding, the identification of two lysine residues within RBD that are required for tight binding to LRP (18), the localization of high affinity ␣ 2 M binding to CR cluster 2 of LRP (17), and our earlier observation that RBD can bind to CR3, the first CR domain of cluster 2, with modest affinity (19). In the present study we have built on this and attempted to identify the minimum additional CR domains that together with CR3, might constitute the full binding site for RBD and, hence, ␣ 2 M on LRP.…”

mentioning

confidence: 99%

Three Complement-like Repeats Compose the Complete α2-Macroglobulin Binding Site in the Second Ligand Binding Cluster of the Low Density Lipoprotein Receptor-related Protein

Dolmer

Gettins

2006

Self Cite

Given the importance of the low density lipoprotein receptorrelated protein (LRP) as an essential endocytosis and signaling receptor for many protein ligands, and of ␣ 2 -macroglobulin (␣ 2 M)-proteinase complexes as one such set of ligands, an understanding of the specificity of their interaction with LRP is an important goal. A starting point is the known role of the 138-residue receptor binding domain (RBD) in binding to LRP. Previous studies have localized high affinity ␣ 2 M binding to the eight complement repeat (CR)-containing cluster 2 of LRP. In the present study we have identified the minimum CR domains that constitute the full binding site for RBD and, hence, for ␣ 2 M on LRP. We report on the ability of the triple construct of CR3-4-5 to bind RBD with an affinity (K d ‫؍‬ 130 nM) the same as for isolated RBD to intact LRP. This K d is 30-fold smaller than for RBD to CR5-6-7, demonstrating the specificity of the interaction with CR3-4-5. Binding requires previously identified critical lysine residues but is almost pH-independent within the range of pH values encountered between extracellular and internal compartments, consistent with an earlier proposed model of intracellular ligand displacement by intramolecular YWTD domains. The present findings suggest a model to explain the ability of LRP to bind a wide range of structurally unrelated ligands in which a nonspecific ligand interaction with the acidic region present in most CR domains is augmented by interactions with other CR surface residues that are unique to a particular CR cluster.The low density lipoprotein receptor-related protein (LRP) 2 is an essential member (1) of the low density lipoprotein (LDL) family of mosaic receptor proteins that are responsible for binding and internalization of a large number of protein ligands (2). Each of these receptors contains one or more clusters of ligand binding domains, known both as complement-like repeats (CR) and LDL receptor type A modules (3). Whereas LDLR contains only one such cluster of 7 CR domains, LRP has four clusters containing 2, 8, 10, and 11 repeats, counting from the N terminus (4 -6). Commonly used nomenclature, thus, describes cluster 2 as being composed of domains CR3 through CR10.Each CR domain contains three conserved disulfide bridges and a functionally required calcium binding site within 40 -42 residues. These domains are linked by variable length, flexible linkers that are likely to afford considerable orientational freedom to each CR domain with respect to others in the cluster (7). Although the cysteines and calcium-coordinating residues are highly conserved, most of the remaining ϳ30 residues are variable (8). This potentially gives LRP a wide variety of ligand recognition sites within its four clusters of 31 CR domains and may in part explain the very much broader range of ligands that can bind and be internalized by LRP compared with the much simpler LDLR. Thus, LRP binds ligands as diverse as ϳ760-kDa ␣ 2 M-protease complexes, various ϳ100-kDa serpin-proteinase complexes s...