Folding and maturation of lipoprotein receptors

Low density lipoprotein receptor (LDLR) Related Proteins (LRPs) are a large family of transmembrane receptors with a broad set of roles in eukaryotic development and homeostasis, including cholesterol uptake, organogenesis, and bone deposition. The ectodomains of receptors of this family are made up of the following characteristic protein domains: LDL-A repeats, YWTD propellers, and EGF domains. In each receptor, these domains occur in different numbers and arrangements, and carry out different functions. Within each ectodomain, individual domains are arranged to generate complex interdomain interfaces, which are critical to biological function. On a microscopic level, how does the amino acid sequence of an individual protein domain encode a unique three-dimensional shape? From a biophysical standpoint, LRPs offer a unique opportunity to investigate the principles of protein folding and maturation. LDL-A repeats are small 40 residue domains with six cysteines and an acidic C-terminal patch of residues and folding requires both disulfide bond formation and coordination of a calcium ion. In Chapter 2, I show that the coupling of C-terminal disulfide bond formation and calcium coordination drives folding of the C-terminus, while formation of the N-terminal disulfide bond appears to order the N-terminus of the domain. On a macroscopic level, how does the cell ensure the proper folding of multiple protein domains, and their arrangement into natively packed interdomain interfaces? The second part of this thesis characterizes the presumed folding chaperone MESD, which is required for the cell-surface expression of LRP6. We show using computational and biophysical methods that MESD is likely to be largely unfolded. Enforced expression of full-length human MESD promotes the secretion of soluble minireceptors derived from LRP6 that contain either one or two β-propeller-EGF domain pairs, but not secretion of a control minireceptor with a point mutation (G158V) that renders it completely incompetent for secretion in the presence of added MESD. Conversely, siRNA mediated knockdown of human MESD expression blocks secretion of native LRP6 minireceptors, and dramatically reduces the level of cell-surface expression of full-length LRP6. Cell-surface expression is only rescued by simultaneous delivery of siRNA-resistant forms of mouse MESD that contain most or all of the unstructured N- and C-termini, implicating the flexible parts of MESD in its function of promoting LRP maturation. These studies reinforce the conclusion that MESD is required for the maturation of nascent LRPs from the ER, to the cell surface.