[PDF] Characterization Of Ligand Binding To The Low Density Lipoprotein Receptor Related Protein Lrp eBook

Author : Rita Kohen Avramoglu
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Page : 0 pages
File Size : 44,5 MB
Release : 2001
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The low density lipoprotein receptor-related protein (LRP) is a 600 kDa endocytic plasma membrane receptor. LRP is involved in the uptake of numerous structurally and functionally unrelated ligands, including alpha2-macroglobulin:protease complexes (alpha2M*), apolipoprotein E-enriched remnant lipoproteins, Pseudomonas exotoxin A (PEA), and receptor associated protein (RAP). To date, analysis of LRP ligand-binding regions has proven challenging due to its large size and repeating modular structure. Using a recombinant DNA approach, full-length somatic chicken LRP, as well as several deletion mutants, were stably expressed in a mutant CHO-derived cell line deficient in endogenous LRP expression. LRP100, LRP67 and LRP25 encode 100%, 67% and 25% of the protein respectively, with LRP67 and LRP25 encoding large internal deletions but retaining an N-terminal portion, transmembrane region, and cytoplasmic tail. These three mutants were found to possess varying degrees of ligand-binding activity toward alpha 2M*, RAP, and PEA. Generation of this expression system has allowed the mapping of several LRP ligands to the N-terminal portion of the receptor and should allow further study of domains within LRP responsible for its multifunctionality. It was recently postulated that eight spacer regions within LRP containing repeating YWTD motifs adopt beta-propeller conformations. While developing cell, lines expressing refined mutants encoding deletions of putative ligand-binding regions, disruption of the naturally occurring, ordered arrangement of beta-propeller domains was found to have an effect on intracellular trafficking and plasma membrane presentation of mutant receptors. Although highly expressed, several mutants possessing crippled beta-propellers were not detected at the plasma membrane by biotin labeling. These mutants also exhibited delayed or no resistance to endoglycosidase H (endo H) suggesting ER exit was delayed or impaired. Restoration of integral beta-propellers and flanking EGF modules restored ER exit and plasma membrane presentation to a transport incompetent mutant. The beta-propeller domains may play an important role in conferring structural stability to LRP. Caution should therefore be exercised in the mutagenesis of this enormous receptor.

Author : Miklos Guttman
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Page : 184 pages
File Size : 18,25 MB
Release : 2009
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The LDLR family of receptors mediates the uptake of lipoprotein particles, and is essential for cholesterol homeostasis. The LDL receptor-related protein 1 (LRP-1) mediates internalization of a large number of diverse ligands and is widely implicated in Alzheimer's disease. Clusters of complement-type ligand binding repeats (CRs) in the LDL receptor family are thought to mediate the interactions between these receptors and their various ligands. Apolipoprotein E, a key ligand for cholesterol homeostasis, has been shown to interact with LDLR, LRP and VLDLR, through these clusters. LDLR and VLDLR each contain a single ligand-binding repeat cluster, whereas LRP contains three large clusters of ligand binding repeats, each with ligand binding functions. In order to study smaller units of these ligand binding clusters we have engineered a new approach to express and refold complement repeat (CR) domains in E. coli. This successfully produced high yields of refolded protein with the benefit of inexpensive isotope labeling for NMR studies. We have expressed a subdomain of sLRP3 (CR16-18) that has previously been shown to recapitulate ligand binding to the isolated receptor binding portion of ApoE (residues 130-149). Binding experiments with the ApoE recognition region of LDLR (LA3-5) and CR16-18 showed that each CR could interact with ApoE(130-149) and that a conserved W25/D30 pair within each repeat appears critical for high affinity. The triple repeat LA3-5 showed the expected interaction with the lipid complexed ApoE(1-191)*DMPC, but surprisingly CR16-18 did not interact with this form of ApoE. To understand these differences in ApoE binding affinity, we introduced mutations of conserved residues from LA5 into CR18, and produced a CR16-18 variant capable of binding ApoE(1-191)*DMPC. This change cannot fully be accounted for by the interaction with ApoE's proposed receptor binding region, therefore we speculate that LA5 is recognizing a distinct epitope on ApoE that may only exist in the lipid bound form. The combination of avidity effects with this distinct recognition process likely governs the ApoE-LDL receptor interaction. Since even the strongest interaction between ApoE(130-149) and a single repeat (CR17) was relatively weak, we constructed a CR17-ApoE(130-149) fusion protein to stabilize the interface for structural studies. The structure revealed a motif seen previously in all ligand CR interactions, in which lysine residues of the ligand interact with the calcium binding site of the CR. Like many ligands of CRs ApoE(130-149) binds as a helix, but with an unexpected turn at H140. These studies also revealed that little structural rearrangement occurs within CR17 upon binding. In addition, dynamics measurements of the free and bound CR17 reveal that certain regions become more ordered, while others become less ordered upon binding. The cytoplasmic tail of LRP, containing two NPXY motifs, has been implicated in the onset of Alzheimer's disease. To examine the intracellular interactions of LRP, as well as to separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY450-- (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY44--3 (membrane proximal) bound many fewer proteins and only to the phosphorylated form.