The low-density lipoprotein (LDL) receptor (LDL-R) family consists of cell-surface receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. The LDL-R is the prototype of this family, which also contains very-low-density lipoprotein receptors (VLDL-R), apolipoprotein E receptor 2, LRP, and megalin. The family members contain four major structural modules: the cysteine-rich complement-type repeats, epidermal growth factor precursor-like repeats, a transmembrane domain, and a cytoplasmic domain. Each structural module serves distinct and important functions. These receptors bind several structurally dissimilar ligands. It is proposed that instead of a primary sequence, positive electrostatic potential in different ligands constitutes a receptor binding domain. This family of receptors plays crucial roles in various physiologic functions. LDL-R plays an important role in cholesterol homeostasis. Mutations cause familial hypercholesterolemia and premature coronary artery disease. LDL-R-related protein plays an important role in the clearance of plasma-activated alpha 2-macroglobulin and apolipoprotein E-enriched lipoproteins. It is essential for fetal development and has been associated with Alzheimer's disease. Megalin is the major receptor in absorptive epithelial cells of the proximal tubules and an antigenic determinant for Heymann nephritis in rats. Mutations in a chicken homolog of VLDL-R cause female sterility and premature atherosclerosis. This receptor is not expressed in liver tissue; however, transgenic expression of VLDL-R in liver corrects hypercholesterolemia in experiment animals, which suggests that it can be a candidate for gene therapy for various hyperlipidemias. The functional importance of individual receptors may lie in their differential tissue expression. The regulation of expression of these receptors occurs at the transcriptional level. Expression of the LDL-R is regulated by intracellular sterol levels involving novel membrane-bound transcription factors. Other members of the family are not regulated by sterols. All the members are, however, regulated by hormones and growth factors, but the mechanisms of regulation by hormones have not been elucidated. Studies of these receptors have provided important insights into receptor structure-function and mechanisms of ligand removal and catabolism. It is anticipated that increased knowledge about the LDL-R family members will open new avenues for the treatment of many disorders.

Lipoprotein biosynthesis is defective in abetalipoproteinemia due to mutations in the gene for microsomal triglyceride transfer protein (MTP) 1 whereas, in hypobetalipoproteinemia, plasma lipoprotein levels are low due to mutations in the apolipoprotein B (apoB) gene (for reviews, see Refs. 1-4). These genetic disorders clearly underscore the importance of these two proteins in lipoprotein biogenesis. ApoB is the structural protein required for lipoprotein assembly and secretion. MTP, an endoplasmic reticulum-localized protein, is a heterodimer of a unique 97-kDa subunit and a 58-kDa ubiquitous folding enzyme, protein disulfide isomerase. MTP is known to enhance the rate of lipid transfer between donor and acceptor vesicles in vitro (for a recent review, see Wetterau et al. (5)). Further evidence for the importance of MTP in lipoprotein assembly was obtained by co-expressing apoB and MTP in nonhepatic and nonintestinal cells that do not express endogenous apoB or MTP (6 -8). Expression of apoB alone in most studies resulted in the intracellular synthesis and degradation of apoB polypeptides, but no secretion (6 -8). In contrast, co-expression of apoB with MTP resulted in the synthesis and secretion of apoB polypeptides as lipoprotein particles (6 -8). The requirement for lipid transfer activity of MTP in the assembly of apoBcontaining lipoproteins was established by the use of inhibitors (9 -14). Compounds that inhibited lipid transfer activity in vitro decreased secretion of apoB-containing lipoproteins in cultured cells. Recently, evidence has been presented for protein-protein interactions between apoB and MTP, which may be important for achieving net transfer of lipids to apoB during its translation or may reflect a separate chaperone-like activity for MTP in apoB folding and assembly (15-18). ApoB and MTP bind with high affinity, and these interactions are affected by the length and degree of lipidation of apoB (17). Lysine and arginine residues in the N-terminal 18% of apoB are critical for these interactions (18). However, the binding sites involved in these protein-protein interactions are not known. In the present study, we have used fusion proteins containing defined sequences of apoB to map an MTP binding site. EXPERIMENTAL PROCEDURESMaterials-MTP was purified to homogeneity and assayed as described earlier (17, 19 -22). Antibodies used for enzyme-linked immunosorbent assay have been described previously (23,24). Antibodies against purified MTP were kindly provided by Dr. Haris Jamil of Bristol-Myers Squibb, Princeton, NJ. An anti-FLAG monoclonal antibody, M2, and other reagents were purchased from Sigma. AGI-S17 was kindly provided by Drs. Russell Medford and Uday Saxena of Atherogenics Inc., Norcross, GA.Expression of FLAG/ApoB Chimeras in COS Cells-To identify a putative MTP binding site in apoB, several FLAG/apoB chimeras were constructed. Various apoB DNA sequences were amplified by the polymerase chain reaction and cloned into the NotI and ApaI site of the pCMV/FLAG expression vector (25,26). Th...

Microsomal triglyceride transfer protein (MTP), a heterodimer of 97 kDa and protein disulfide isomerase, is required for the assembly of apolipoprotein B (apoB)-containing triglyceride-rich lipoproteins. These proteins have been shown to interact with each other during early stages of lipoprotein biosynthesis. Our studies indicated that binding between apoB and heterodimeric MTP was of high affinity (Kd 10-30 nM) due to ionic interactions. In contrast to MTP, protein disulfide isomerase alone interacted very poorly with lipoproteins, indicating the importance of the heterodimer in these bindings. Preincubation of lipoproteins with detergents enhanced their interaction with MTP. Native VLDL bound poorly to MTP, but its preincubation with Tween-20 resulted in significantly increased binding to MTP. Furthermore, binding of LDL was enhanced by preincubation with taurocholate, indicating that partial delipidation of apoB-containing lipoproteins results in increased binding to MTP. Subsequently, attempts were made to study interactions between C-terminally truncated apoB polypeptides and MTP. Binding of all the polypeptides to MTP was enhanced in the presence of taurocholate. Comparisons revealed that the binding of different apoB polypeptides to MTP was in the order of apoB18 > apoB28 > apoB42 > apoB100. These studies indicated that optimum interactions occur between apoB18 and MTP, and that the increase in apoB length beyond apoB18 has a negative effect on these interactions. Since apoB18 does not assemble triglyceride-rich lipoproteins, these studies suggest that apoB may interact with MTP before its lipidation. It is proposed that steps in lipoprotein biosynthesis may be dictated by the sequential display of different functional domains on the apoB polypeptide.

We have standardized simple but sensitive enzyme-linked immunoassays to understand a relationship between intracellular levels and secretion rates of apoB. The assays were based on commercially available antibodies and were specific to human apoB. A monoclonal antibody, 1D1, was immobilized on microtiter wells and incubated with different amounts of low density lipoproteins to obtain a standard curve. Conditioned media were added to other wells in parallel, and the amount of apoB was quantitated from a linear regression curve. To standardize conditions for the measurement of intracellular apoB, cells were homogenized and solubilized with different concentrations of taurocholate. We found that 0.5% taurocholate was sufficient to solubilize all the apoB in HepG2, Caco-2, and McA-RH7777 cells. Next, a standard curve was prepared in the presence of taurocholate and used to determine intracellular levels of apoB in different cell lines. The intracellular levels (pmol/mg cell protein) and the rates of secretion (pmol/mg/h) of apoB100 were positively correlated (r2 = 0.81, P = 0.0009) in HepG2 cells. Furthermore, a positive correlation (r2 = 0.88, P < 0.0001) was found between intracellular and secreted apoB42 in stably transfected McA-RH7777 cells. In contrast, no correlation was observed for human apoB28 and apoB18 in stably transfected cells that were secreted either partially associated or completely unassociated with lipoproteins. These studies indicated that the rate of secretion of lipid-associated apoB, but not the lipid-free apoB, was tightly controlled.

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