Yao, Zemin,Kohen Avramoglu, Rita.2009-03-232009-03-2320012001Source: Dissertation Abstracts International, Volume: 63-05, Section: B, page: 2370.9780612679641http://hdl.handle.net/10393/9094http://dx.doi.org/10.20381/ruor-7637The 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.146 p.Biology, Molecular.Thesis