A dot-blot assay for the low density lipoprotein receptor

We describe a new method for detecting the interaction of low density lipoprotein with its receptor using unmodified nitrocellulose as support for membrane protein. The method is specific and sensitive down to 3 micrograms of membrane protein. Unlabeled LDL, but not HDL, competes with 125I-labeled LDL for binding, and binding is abolished by pretreatment of the membranes with pronase and is dependent upon the presence of Ca2+. Furthermore, modification of arginine or lysine residues on LDL abolishes the lipoprotein interaction with the receptor protein supported on the nitrocellulose. When the membranes are solubilized with octyl glucoside, purification steps of the receptor can be directly followed with no interference of the detergent, therefore eliminating the need for its removal. The increased expression of LDL receptors on liver membranes from estradiol-treated rats was also demonstrated. We suggest, therefore, that this method can be used to detect the presence of LDL receptors on minute amounts of membrane protein.     

Self-association of the low density lipoprotein receptor mediated by the cytoplasmic domain

When the low density lipoprotein (LDL) receptor was solubilized from bovine adrenal cortex membranes and subjected to electrophoresis in the absence of reducing agents, a disulfide-bonded dimeric species was demonstrated. Formation of these covalent bonds was blocked when the tissue was homogenized in the presence of sulfhydryl alkylating agents, indicating that the native receptor was self-associated noncovalently and that the disulfide bond formation occurred only after homogenization. The disulfide-linked dimers were disrupted and the receptor was restored to a monomeric form when inside-out adrenal vesicles were treated with trypsin, suggesting that the disulfide bond formation involved the 50-amino acid cytoplasmic domain of the receptor. When the receptor was solubilized from bovine adrenal cortex membranes and then purified by ion exchange and affinity chromatography, it could be covalently coupled into dimers and trimers in the presence of bivalent cross-linking agents. Receptor dimers could also be demonstrated by chemical cross-linking of intact cells that were transfected with an expressible cDNA encoding the normal human LDL receptor. Dimer formation was markedly reduced in transfected cells expressing mutated cDNAs that had premature termination codons at positions 792, 807, and 812, which produced shortened receptors that retained 2, 17, and 22 of the original 50 amino acids of the cytoplasmic domain, respectively. The first two mutant receptors, which did not form oligomers, did not enter coated pits and were not rapidly internalized by cells. However, the mutant receptor that terminates at position 812 was internalized normally even though oligomer formation was greatly reduced. Moreover, a mutant receptor with a cysteine substituted for a tyrosine at position 807, which internalized very slowly, showed a normal susceptibility to chemical cross-linking. Deletion of external domains of the LDL receptor, including the epidermal growth factor homology region and the O-linked sugar domain, did not alter susceptibility to chemical cross-linking. We conclude that the cytoplasmic domain of the LDL receptor is responsible both for self-association into oligomers and for clustering in coated pits, but the available data do not establish a causal connection between these two events.     

The low density lipoprotein receptor

The study of familial hypercholesterolemia at the molecular level has led to its advancement from a clinical syndrome to a fascinating experimental system. FH was first described 50 years ago by Carl Müller who concluded that the disease produces high plasma cholesterol levels and myocardial infarctions in young people, and is transmitted as an autosomal dominant trait determined by a single gene. The existence of two forms of FH, namely heterozygous and homozygous, was recognized by Khachadurian and Fredrickson and Levy much later. The value of FH as an experimental model system lies in the availability of homozygotes, because mutant genes can be studied without interference from the normal gene. The first and most important breakthrough was the realization that the defect underlying FH could be studied in cultured skin fibroblasts. Rapidly, the LDL receptor pathway was conceptualized and its dysfunction in cells from FH homozygotes was demonstrates. Isolation of the normal LDL receptor protein and studies on the biosynthesis and structure of abnormal receptors in mutant cell lines provided essential groundwork for elucidation of defects at the DNA level. The power of the experimental system, FH, became nowhere more obvious than in work that correlated structural information at the protein level with the elucidation of defined defects in the LDL receptor gene. In addition to revealing important structure-function relationships in the LDL receptor polypeptide and delineating mutational events, studies of FH have established several more general concepts. First, the tight coupling of LDL binding to its internalization suggested that endocytosis was not a non-specific process as suggested from early observations. The key finding was that LDL receptors clustered in coated pits, structures that had been described by Roth and Porter 10 years earlier. These investigators had demonstrated, in electron microscopic studies on the uptake of yolk proteins by mosquito oocytes, that coated pits pinch off from the cell surface and form coated vesicles that transport extracellular fluid into the cell. Studies on the LDL receptor system showed directly that receptor clustering in coated pits is the essential event in this kind of endocytosis, and thus established receptor-mediated endocytosis as a distinct mechanism for the transport of macromolecules across the plasma membrane. Subsequently, many additional systems of receptor-mediated endocytosis have been defined, and variations of the overall pathway have been described.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reconstitution of the low density lipoprotein receptor

The receptor for low density lipoprotein was purified from bovine adrenal cortex in the presence of the nonionic detergent octylglucoside. Receptors were incorporated into the bilayer of egg phosphatidylcholine vesicles by a detergent-dialysis method. Reconstituted receptors were functional in that they bound low density lipoprotein as well as a monoclonal antibody directed against the receptor in a specific, saturable fashion. Binding activity of reconstituted receptors was measured by a gel chromatography assay. The orientation of the receptor molecule within the phospholipid bilayer was investigated by binding assays following proteolytic digestion. Reconstituted receptors showed an orientation that was functionally indistinguishable from that of low density lipoprotein receptors in the plasma membrane of intact human fibroblasts.     

A two-step binding model of PCSK9 interaction with the low density lipoprotein receptor

PCSK9 (proprotein convertase subtilisin-like/kexin type 9) is an emerging target for pharmaceutical intervention. This multidomain protein interacts with the LDL receptor (LDLR), promoting receptor degradation. Insofar as PCSK9 inhibition induces a decrease in plasma cholesterol levels, understanding the nature of the binding interaction between PCSK9 and the LDLR is of critical importance. In this study, the ability of PCSK9 to compete with apoE3 N-terminal domain-containing reconstituted HDL for receptor binding was examined. Whereas full-length PCSK9 was an effective competitor, the N-terminal domain (composed of the prodomain and catalytic domain) was not. Surprisingly, the C-terminal domain (CT domain) of PCSK9 was able to compete. Using a direct binding interaction assay, we show that the PCSK9 CT domain bound to the LDLR in a calcium-dependent manner and that co-incubation with the prodomain and catalytic domain had no effect on this binding. To further characterize this interaction, two LDLR fragments, the classical ligand-binding domain (LBD) and the EGF precursor homology domain, were expressed in stably transfected HEK 293 cells and isolated. Binding assays showed that the PCSK9 CT domain bound to the LBD at pH 5.4. Thus, CT domain interaction with the LBD of the LDLR at endosomal pH constitutes a second step in the PCSK9-mediated LDLR binding that leads to receptor degradation.     

Effect of apolipoprotein M on high density lipoprotein metabolism and atherosclerosis in low density lipoprotein receptor knock-out mice

To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM(-/-)) mice. Plasma apoM was predominantly associated with 10-12-nm alpha-migrating HDL particles. Human apoM overexpression (11-fold) increased plasma cholesterol concentration by 13-22%, whereas apoM deficiency decreased it by 17-21%. The size and charge of apoA-I-containing HDL in plasma were not changed in apoM-Tg or apoM(-/-) mice. However, in plasma incubated at 37 degrees C, lecithin:cholesterol acyltransferase-dependent conversion of alpha- to pre-alpha-migrating HDL was delayed in apoM-Tg mice. Moreover, lecithin: cholesterol acyltransferase-independent generation of pre-beta-migrating apoA-I-containing particles in plasma was increased in apoM-Tg mice (4.2 +/- 1.1%, p = 0.06) and decreased in apoM(-/-) mice (0.5 +/- 0.3%, p = 0.03) versus controls (1.8 +/- 0.05%). In the setting of low density lipoprotein receptor deficiency, apoM-Tg mice with approximately 2-fold increased plasma apoM concentrations developed smaller atherosclerotic lesions than controls. The effect of apoM on atherosclerosis may be facilitated by enzymatic modulation of plasma HDL particles, increased cholesterol efflux from foam cells, and an antioxidative effect of apoM-containing HDL.     

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.     

Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.     

Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor

Studies of low density lipoprotein (LDL) metabolism in nonhuman model systems have indicated that the mammalian liver has dual mechanisms for the uptake and regulation of the concentration of plasma LDL. Heretofore, direct evaluation of lipoprotein uptake mechanisms in human hepatocytes has not been possible. In order to compare hepatocyte LDL uptake with fibroblast LDL metabolism, human hepatocytes were isolated and cultured from small biopsy specimens obtained from normolipidemic and homozygous familial hypercholesterolemic patients. Cells cultured in serum-free culture medium retained the morphological and biochemical characteristics of hepatocytes for at least 7 days. The uptake and degradation of LDL by hepatocytes was compared to that of the cultured human fibroblasts. Like fibroblasts, hepatocytes bound, internalized, and degraded LDL. In both cell types, uptake approached saturation at a concentration of 50 micrograms of LDL protein/ml. Competition for LDL binding by LDL, high density lipoprotein, and modified LD revealed that the hepatocyte binding was specific for LDL. Cellular cholesterol loading by incubation in LDL-enriched culture medium resulted in diminished LDL uptake in both cell types. Chemical modification of LDL by acetoacetylation, acetylation, and reductive methylation abolished LDL uptake and degradation by fibroblasts. However, hepatocytes bound and degraded the modified LDL at 30-50% the level of native LDL. Homozygous familial hypercholesterolemic hepatocytes were devoid of the LDL receptor pathway but metabolized native LDL to the extent observed with modified LDL uptake by normal hepatocytes. In contrast to the classic LDL receptor pathway, the second or alternate pathway does not lead to regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity. These findings indicate the presence of two separate pathways of LDL uptake in human hepatocytes which have different effects on hepatocytic cholesterol metabolism.     

Mechanisms by which lipoprotein lipase alters cellular metabolism of lipoprotein(a), low density lipoprotein, and nascent lipoproteins. Roles for low density lipoprotein receptors and heparan sulfate proteoglycans.

We sought to investigate effects of lipoprotein lipase (LpL) on cellular catabolism of lipoproteins rich in apolipoprotein B-100. LpL increased cellular degradation of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) by 277% +/- 3.8% and 32.5% +/- 4.1%, respectively, and cell association by 509% +/- 8.7% and 83.9% +/- 4.0%. The enhanced degradation was entirely lysosomal. Enhanced degradation of Lp(a) had at least two components, one LDL receptor-dependent and unaffected by heparitinase digestion of the cells, and the other LDL receptor-independent and heparitinase-sensitive. The effect of LpL on LDL degradation was entirely LDL receptor-independent, heparitinase-sensitive, and essentially absent from mutant Chinese hamster ovary cells that lack cell surface heparan sulfate proteoglycans. Enhanced cell association of Lp(a) and LDL was largely LDL receptor-independent and heparitinase-sensitive. The ability of LpL to reduce net secretion of apolipoprotein B-100 by HepG2 cells by enhancing cellular reuptake of nascent lipoproteins was also LDL receptor-independent and heparitinase-sensitive. None of these effects on Lp(a), LDL, or nascent lipoproteins required LpL enzymatic activity. We conclude that LpL promotes binding of apolipoprotein B-100-rich lipoproteins to cell surface heparan sulfate proteoglycans. LpL also enhanced the otherwise weak binding of Lp(a) to LDL receptors. The heparan sulfate proteoglycan pathway represents a novel catabolic mechanism that may allow substantial cellular and interstitial accumulation of cholesteryl ester-rich lipoproteins, independent of feedback inhibition by cellular sterol content.     

Mechanisms of inhibition by apolipoprotein C of apolipoprotein E-dependent cellular metabolism of human triglyceride-rich lipoproteins through the low density lipoprotein receptor pathway

The mechanism of inhibition by apolipoprotein C of the uptake and degradation of triglyceride-rich lipoproteins from human plasma via the low density lipoprotein (LDL) receptor pathway was investigated in cultured human skin fibroblasts. Very low density lipoprotein (VLDL) density subfractions and intermediate density lipoprotein (IDL) with or without added exogenous recombinant apolipoprotein E-3 were used. Total and individual (C-I, C-II, C-III-1, and C-III-2) apoC molecules effectively inhibited apoE-3-mediated cell metabolism of the lipoproteins through the LDL receptor, with apoC-I being most effective. When the incubation was carried out with different amounts of exogenous apoE-3 and exogenous apoC, it was shown that the ratio of apoE-3 to apoC determined the uptake and degradation of VLDL. Excess apoE-3 overcame, at least in part, the inhibition by apoC. ApoC, in contrast, did not affect LDL metabolism. Neither apoA-I nor apoA-II, two apoproteins that do not readily associate with VLDL, had any effect on VLDL cell metabolism. The inhibition of VLDL and IDL metabolism cannot be fully explained by interference of association of exogenous apoE-3 with or displacement of endogenous apoE from the lipoproteins. IDL is a lipoprotein that contains both apoB-100 and apoE. By using monoclonal antibodies 4G3 and 1D7, which specifically block cell interaction by apoB-100 and apoE, respectively, it was possible to assess the effects of apoC on either apoprotein. ApoC dramatically depressed the interaction of IDL with the fibroblast receptor through apoE, but had only a moderate effect on apoB-100. The study thus demonstrates that apoC inhibits predominantly the apoE-3-dependent interaction of triglyceride-rich lipoproteins with the LDL receptor in cultured fibroblasts and that the mechanism of inhibition reflects association of apoC with the lipoproteins and specific concentration-dependent effects on apoE-3 at the lipoprotein surface.     

Low density lipoprotein receptor regulation. Kinetic models

The macromolecular species distribution in a receptor-mediated endocytotic pathway was computer simulated based on kinetic data reported in the literature. In the proposed model, the rapidity with which the recycled receptor is shuttled to the cell surface is indicated by the magnitude of k-3, the shuttling constant. The magnitude of k-3 will vary with the experimental conditions, but when this value is large, the internalized receptor is shuttled back to the cell surface with a traverse time of 14 min. Under steady-state conditions, after the cells have been incubated in the presence of LDL for 5 h (M.S. Brown and J.L. Goldstein, Cell 9 (1976) 663), the time required for a receptor to traverse the entire endocytotic pathway is 52 min. Our simulation suggests that normal LDL binding in such a short-term experiment may be independent of receptor synthesis. Thus, the degradation of LDL and resultant build-up of cholesterol would have no apparent inhibitory effect on the down-regulation of receptor synthesis.     

A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein.

The internalization of oxidized low density lipoprotein (OxLDL) by macrophages is hypothesized to contribute to foam cell formation and eventually to atherosclerotic lesion formation. OxLDL is a ligand for the acetylated low density lipoprotein (AcLDL) receptor, however, our data show that this receptor accounts for less than half of OxLDL uptake by mouse macrophages, suggesting additional receptors for OxLDL. We have developed a novel expression cloning strategy in order to isolate clones encoding OxLDL receptors. In addition to the AcLDL receptor, we isolated a molecular clone for a structurally unrelated receptor capable of mediating the high affinity uptake of OxLDL following transfection into cells. This receptor has been identified as the mouse Fc gamma RII-B2, a member of a family of receptors known to mediate immune complex uptake through recognition of the Fc region of IgG. The uptake of OxLDL by cells transfected with the Fc gamma RII-B2 clone is not blocked by AcLDL but is blocked by the anti-Fc gamma RII monoclonal antibody, 2.4G2.     

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.     

Superinduction of the human gene encoding low density lipoprotein receptor

Expression of human LDL receptor mRNA and protein is induced in human glioblastoma-astrocytoma cells upon transfer into lipoprotein-deficient medium, a mode of induction likely to be physiological. The presence of cycloheximide (CHX) leads to up to 7.5-fold superinduction of LDL receptor mRNA within 4 hr and, upon removal of this inhibitor, to superinduction of LDL receptor protein as well. The extent of superinduction of LDL receptor mRNA reaches over 40-fold beyond the level expressed in medium containing regular serum. Despite its extensive superinduction, LDL receptor mRNA decays rapidly in the presence of CHX. Stabilization of LDL receptor mRNA is thus unlikely to account for the observed superinduction. These results show that normally the LDL receptor gene is expressed to only a small fraction of its full potential.