ApoER2 Controls Not Only Neuronal Migration in the Intermediate Zone But Also Termination of Migration in the Developing Cerebral Cortex.

Neuronal migration contributes to the establishment of mammalian brain. The extracellular protein Reelin sends signals to various downstream molecules by binding to its receptors, the apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor and exerts essential roles in the neuronal migration and formation of the layered neocortex. However, the cellular and molecular functions of Reelin signaling in the cortical development are not yet fully understood. Here, to gain insight into the role of Reelin signaling during cortical development, we examined the migratory behavior of Apoer2-deficient neurons in the developing brain. Stage-specific labeling of newborn neurons revealed that the neurons ectopically invaded the marginal zone (MZ) and that neuronal migration of both early- and late-born neurons was disrupted in the intermediate zone (IZ) in the Apoer2 KO mice. Rescue experiments showed that ApoER2 functions both in cell-autonomous and noncell-autonomous manners, that Rap1, integrin, and Akt are involved in the termination of migration beneath the MZ, and that Akt also controls neuronal migration in the IZ downstream of ApoER2. These data indicate that ApoER2 controls multiple processes in neuronal migration, including the early stage of radial migration and termination of migration beneath the MZ in the developing neocortex.

First-in-human autologous implantation of genetically modified adipocytes expressing LCAT for the treatment of familial LCAT deficiency.

Background: Familial lecithin: cholesterol acyltransferase (LCAT) deficiency (FLD) is a severe inherited disease without effective treatment. Patients with FLD develop severe low HDL, corneal opacity, hemolytic anemia, and renal injury. Objective: We developed genetically modified adipocytes (GMAC) secreting LCAT (LCAT-GMAC) for ex vivo gene therapy. GMACs were prepared from the patient's adipocytes to express LCAT by retroviral gene transduction to secrete functional enzymes. This study aimed to evaluate the safety and efficacy of LCAT-GMAC implantation in an FLD patient. Methods: Proliferative preadipocytes were obtained from a patient using a ceiling culture and retrovirally transduced with LCAT. After obtaining enough cells by expansion culture of the transduced cells, the resulting LCAT-GMACs were implanted into a patient with FLD. To evaluate the safety and efficacy, we analyzed the outcome of the autologous implantation for 24 weeks of observation and subsequent 240 weeks of the follow-up periods. Results: This first-in-human autologous implantation of LCAT-GMACs was shown to be safe by evaluating adverse events. The LCAT-GMAC implantation increased serum LCAT activity by approximately 50% of the baseline and sustained over three years. Consistent with increased LCAT activity, intermediate-density lipoprotein (IDL) and free cholesterol levels of the small and very small HDL fractions decreased. We found the hemoglobin/haptoglobin complex in the hemolyzed pre-implantation sera of the patient. After one week of the implantation, the hemoglobin/haptoglobin complex almost disappeared. Immediately after the implantation, the patient's proteinuria decreased temporarily to mild levels and gradually increased to the baseline. At 48 weeks after implantation, the patient's proteinuria deteriorated with the development of mild hypertension. By the treatment with antihypertensives, the patient's blood pressure normalized. With the normalization of blood pressure, the proteinuria rapidly decreased to mild proteinuria levels. Conclusions: LCAT-GMAC implantation in a patient with FLD is shown to be safe and appears to be effective, in part, for treating anemia and proteinuria in FLD.

Species differences of macrophage very low-density-lipoprotein (VLDL) receptor protein expression.

Triglyceride-rich lipoproteins (TGRLs) and low-density-lipoprotein (LDL) cholesterol are independent risk factors for coronary artery disease. We have previously proposed that the very low-density-lipoprotein (VLDL) receptor is one of the receptors required for foam cell formation by TGRLs in human macrophages. However, the VLDL receptor proteins have not been detected in atherosclerotic lesions of several animal models. Here we showed no VLDL receptor protein was detected in mouse macrophage cell lines (Raw264.7 and J774.2) or in mouse peritoneal macrophages in vitro. Furthermore, no VLDL receptor protein was detected in macrophages in atherosclerotic lesions of chow-fed apolipoprotein E-deficient or cholesterol-fed LDL receptor-deficient mice in vivo. In contrast, macrophage VLDL receptor protein was clearly detected in human macrophages in vitro and in atherosclerotic lesions in myocardial infarction-prone Watanabe-heritable hyperlipidemic (WHHLMI) rabbits in vivo. There are species differences in the localization of VLDL receptor protein in vitro and in vivo. Since VLDL receptor is expressed on macrophages in atheromatous plaques of both rabbit and human but not in mouse models, the mechanisms of atherogenesis and/or growth of atherosclerotic lesions in mouse models may be partly different from those of humans and rabbits.

Polymorphisms in the human apolipoprotein E receptor 2 gene in Japanese sporadic Alzheimer's disease patients.

Apolipoprotein E (apoE) polymorphism is associated with onset of Alzheimer's disease (AD). We found seven polymorphisms in apoE receptor 2 (ApoER2), an apoE-binding receptor, in Japanese sporadic AD patients, but no association of ApoER2 polymorphisms with AD. We consider that the functions of ApoER2 in the brain may be compensated for by those of other apoE-binding receptors such as VLDL receptor.

Involvement of apolipoprotein E in excess fat accumulation and insulin resistance.

Although apolipoprotein E (apoE) is well known to play a major role in lipid metabolism, its role in glucose and energy homeostasis remains unclear. Herein, we established apoE-deficient genetically obese Ay (apoE(-/-);Ay/+) mice. ApoE deficiency in Ay mice prevented the development of obesity, with decreased fat accumulation in the liver and adipose tissues. ApoE(-/-);Ay/+ mice exhibited better glucose tolerance than apoE(+/+);Ay/+ mice. Insulin tolerance testing and hyperinsulinemic-euglycemic clamp study revealed marked improvement of insulin sensitivity, despite increased plasma free fatty acid levels. These metabolic phenotypes were reversed by adenoviral replenishment of apoE protein, indicating circulating apoE to be involved in increased adiposity and obesity-related metabolic disorders. Uptake of apoE-lacking VLDL into the liver and adipocytes was markedly inhibited, but adipocytes in apoE(-/-);Ay/+ mice exhibited normal differentiation, suggesting that apoE-dependent VLDL transport is involved in the development of obesity, i.e., surplus fat accumulation. Interestingly, apoE(-/-);Ay/+ mice exhibited decreased food intake and increased energy expenditure. Pair-feeding experiments indicate these phenomena to both contribute to the obesity-resistant phenotypes associated with apoE deficiency. Thus, apoE is involved in maintaining energy homeostasis. ApoE-dependent excess fat accumulation is a promising therapeutic target for the metabolic syndrome.

In situ measurements of velocity dispersion and attenuation in New Jersey Shelf sediments.

The existence of acoustic velocity dispersion and frequency dependence of attenuation in marine sediments is investigated using in situ measurements from a wideband acoustic probe system during the Shallow Water 2006 experiment. Direct-path pulse propagation measurements show evidence of velocity dispersion within the 10-80 kHz frequency band at two silty-sand sites on the New Jersey Shelf. The measured attenuation in dB/m shows linear frequency dependency within the 10-80 kHz frequency band. The measured velocity dispersion and attenuation curves are in good agreement with those predicted by an extended Biot theory [Yamamoto and Turgut, J. Acoust. Soc. Am. 83, 1744-1751 (1988)] for sediments with a distribution of pore sizes.

Intimal smooth muscle cells up-regulate beta-very low density lipoprotein-mediated cholesterol accumulation by enhancing beta-very low density lipoprotein uptake and decreasing cholesterol efflux.

To clarify the mechanism of smooth muscle cell (SMC)-derived foam cell formation, we investigated beta-very low density lipoprotein (beta-VLDL) cholesterol metabolism in vascular medial SMCs (M-SMCs) from normal rabbits compared with intimal SMCs (I-SMCs) from normal rabbits fed a high-cholesterol diet and LDL receptor-deficient rabbits. For both types of I-SMCs, uptake of [3H]cholesteryl oleate labeled beta-VLDL increased 1.6 times and release of [3H]cholesterol decreased 40% compared with M-SMCs. M-SMCs took up part of the beta-VLDL through the LDL receptor but I-SMCs did not. mRNAs for the VLDL receptor and the LDL receptor relative with 11 ligand binding repeats were expressed at similar levels in all SMCs. M-SMCs expressed more LDL receptor-related protein than I-SMCs. Ligand blotting analysis revealed greater 125I-beta-VLDL binding to a 700-kDa protein in I-SMCs compared with M-SMCs. I-SMCs had higher activities of acid cholesterol esterase and acyl-CoA:cholesterol acyltransferase, and lower activity of neutral cholesterol esterase than M-SMCs in both the absence and the presence of beta-VLDL. These results indicate that I-SMCs accumulate more cholesteryl ester than M-SMCs by taking up more beta-VLDL and by effluxing less cholesterol.

Deficiency of the very low-density lipoprotein (VLDL) receptors in streptozotocin-induced diabetic rats: insulin dependency of the VLDL receptor.

Hyperlipidemia is a common feature of diabetes and is related to cardiovascular disease. The very low-density lipoprotein receptor (VLDL-R) is a member of the low-density lipoprotein receptor (LDL-R) family. It binds and internalizes triglyceride-rich lipoproteins with high specificity. We examined the etiology of hyperlipidemia in the insulin-deficient state. VLDL-R expression in heart and skeletal muscle were measured in rats with streptozotocin (STZ)-induced diabetes. STZ rats showed severe hyperlipidemia on d 21 and 28, with a dramatic decline in VLDL-R protein in skeletal muscle (>90%), heart (approximately 50%) and a loss of adipose tissues itself on d 28. The reduction of VLDL-R protein in skeletal muscle could not be explained simply by a decrease at the transcriptional level, because a dissociation between VLDL-R protein and mRNA expression was observed. The expression of LDL-R and LDL-R-related protein in liver showed no consistent changes. Furthermore, no effect on VLDL-triglyceride production in liver was observed in STZ rats. A decrease in postheparin plasma lipoprotein lipase activity started on d 7 and continued to d 28 at the 50% level even though severe hyperlipidemia was detected only on d 21 and 28. In rat myoblast cells, serum deprivation for 24 h induced a reduction in VLDL-R proteins. Insulin (10(-6) m), but not IGF-I (10 ng/ml), restored the decreased VLDL-R proteins by serum deprivation. These results suggest that the combination of VLDL-R deficiency and reduced plasma lipoprotein lipase activity may be responsible for severe hyperlipidemia in insulin-deficient diabetes.

Etiological significance of apolipoprotein E mutations in lipoprotein glomerulopathy.

Lipoprotein glomerulopathy (LPG) is a newly recognized renal disease characterized by thrombus-like lipoproteins in the glomerular capillaries and abnormal lipoprotein profiles similar to those in type III hyperlipoproteinemia. Recently, these conditions have been shown to be associated with some apolipoprotein E (apoE) mutations. We found an apoE mutation (designated apoE-Sendai) that substitutes arginine 145 with proline. This mutation occurs most frequently in Japanese patients with LPG. To elucidate the etiological role of this mutation in the apoE gene, we established an experimental model for LPG by transducing apoE-Sendai in apoE knockout mice with the use of an adenovirus vector. Based on the findings in patients with LPG and its animal model, we suggest that the glomerular lesions are not only caused by hyperlipidemia, but also by in situ interaction between lipoprotein-containing mutant apoE with the glomerulus. In this review, we outline the clinical features of LPG and discuss the relationship between apoE mutations and LPG.

Reelin receptors ApoER2 and VLDLR are expressed in distinct spatiotemporal patterns in developing mouse cerebral cortex.

In mammalian developing brain, neuronal migration is regulated by a variety of signaling cascades, including Reelin signaling. Reelin is a glycoprotein that is mainly secreted by Cajal-Retzius neurons in the marginal zone, playing essential roles in the formation of the layered neocortex via its receptors, apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR). However, the precise mechanisms by which Reelin signaling controls the neuronal migration process remain unclear. To gain insight into how Reelin signaling controls individual migrating neurons, we generated monoclonal antibodies against ApoER2 and VLDLR and examined the localization of Reelin receptors in the developing mouse cerebral cortex. Immunohistochemical analyses revealed that VLDLR is localized to the distal portion of leading processes in the marginal zone (MZ), whereas ApoER2 is mainly localized to neuronal processes and the cell membranes of multipolar cells in the multipolar cell accumulation zone (MAZ). These different expression patterns may contribute to the distinct actions of Reelin on migrating neurons during both the early and late migratory stages in the developing cerebral cortex.

Seven novel sequence variants in the human low density lipoprotein receptor related protein 5 (LRP5) gene.

We identified seven novel polymorphisms in the human low density lipoprotein receptor related protein 5 (LRP5) gene. Two of them are predicted to replace amino acid in LRP5 protein (c.314A>G: Q89R and c.4037T>C: V1330A), whereas three are silent mutations in the coding region (c.2268T>C: N740N, c.3405A>G: V1119V, and c.4137C>T: D1363D) and two are polymorphisms in introns (IVS10+6T>C and IVS17-30G>A). Since LRP5 recognizes apolipoprotein E and is genetically linked with type 1 diabetes, these novel polymorphisms will be useful in genetic studies of hyperlipoproteinemia and diabetes. To our knowledge, this is the first report in the literature of sequence variants in the human LRP5 gene.

Receptor-type protein tyrosine phosphatase zeta as a component of the signaling receptor complex for midkine-dependent survival of embryonic neurons.

Midkine (MK), a heparin-binding growth factor, suppresses apoptosis of embryonic neurons in culture, induced by serum deprivation. Receptor-type protein tyrosine phosphatase zeta (PTP zeta) is a chondroitin sulfate proteoglycan with a transmembrane domain and intracellular tyrosine phosphatase domains. The activity of MK was abolished by digestion with chondroitinase ABC, or addition of the antibody to PTP zeta, while digestion with heparitinase showed no significant effect. These results suggested that the survival-promoting signal of MK was received by a receptor complex containing PTP zeta. Low density lipoprotein receptor-related protein (LRP) has been identified as another component of the signaling receptor. Ectodomains of two related proteins expressed on neurons, namely LRP6 and apoE receptor 2, were FLAG-tagged and examined for MK binding, using MK-agarose column. Both the ectodomains were found to exhibit calcium-dependent binding to MK. These proteins may participate in MK signaling in certain cases. The survival-promoting activity of MK was abolished by PP1, an inhibitor of src protein kinase, pertussis toxin, an inhibitor of G protein-linked signaling and sodium orthovanadate, an inhibitor of PTPs.

The very low-density lipoprotein (VLDL) receptor: characterization and functions as a peripheral lipoprotein receptor.

The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor family. In vitro and in vivo studies have shown that VLDL receptor binds triglyceride (TG)-rich lipoproteins but not LDL, and functions as a peripheral remnant lipoprotein receptor. VLDL receptor is expressed abundantly in fatty acid-active tissues (heart, skeletal muscle and fat), the brain and macrophages. It is likely that VLDL receptor functions in concert with lipoprotein lipase (LPL), which hydrolyses TG in VLDL and chylomicron. In contrast to the LDL receptor, VLDL receptor binds apolipoprotein (apo) E2/2 VLDL particles as well as apoE3/3 VLDL, and the expression is not down-regulated by intracellular lipoproteins. Recently, various functions of the VLDL receptor have been reported in lipoprotein metabolism, metabolic syndrome/atherosclerosis, cardiac fatty acid metabolism, neuronal migration and angiogenesis/tumor growth. Gene therapy of VLDL receptor into the liver showed a benefit effect for lipoprotein metabolism in both LDL receptor knockout and apoE mutant mice. Beyond its function as a peripheral lipoprotein receptor, possibilities of its physiological function have been extended to include signal transduction, angiogenesis and tumor growth.

Comparative reactivity of remnant-like lipoprotein particles (RLP) and low-density lipoprotein (LDL) to LDL receptor and VLDL receptor: effect of a high-dose statin on VLDL receptor expression.

BACKGROUND: Comparison of the reactivity of remnant-like lipoprotein particles (RLP) and LDL particles to LDL receptor and VLDL receptor has not been investigated. METHODS: LDL receptor- or VLDL receptor-transfected ldlA-7, HepG2 and L6 cells were used. Human LDL and rabbit ß-VLDL were isolated by ultracentrifugation. Human RLP was isolated using an immunoaffinity mixed gel. The effect of statin on lipoprotein receptors was examined. RESULTS: Both LDL receptor and VLDL receptor recognized RLP. In LDL receptor transfectants, RLP, ß-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-ß-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and ß-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than ß-VLDL because of higher apolipoprotein E in RLP. LDL receptor expression was induced in HepG2 by the low concentration of statin while VLDL receptor expression was induced in L6 myoblasts at higher concentration. CONCLUSIONS: RLP are bound to hepatic LDL receptor more efficiently than LDL, which may explain the mechanism by which statins prevent cardiovascular risk by primarily reducing plasma RLP rather than by reducing LDL. Additionally, a high-dose of statins also may reduce plasma RLP through muscular VLDL receptor.

Fasting-induced hypothermia and reduced energy production in mice lacking acetyl-CoA synthetase 2.

Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.

SOX6 suppresses cyclin D1 promoter activity by interacting with beta-catenin and histone deacetylase 1, and its down-regulation induces pancreatic beta-cell proliferation.

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Identification of N-arachidonylglycine, U18666A, and 4-androstene-3,17-dione as novel insulin Secretagogues.

The glucose-induced insulin secretion is fine-tuned by numerous factors. To systematically identify insulinotropic factors, we optimized a primary beta-cell-based functional assay to monitor intracellular Ca2+ flux ([Ca2+]i). By this assay system, we successfully identified several insulinotropic peptides including cholecystokinin, gastrin releasing peptide, vasopressin, and oxytocin from tissue extracts. Screening of an assortment of chemical compounds, we determined three novel insulin secretagogues: N-arachidonylglycine (NAGly), 3beta-(2-diethylamino-ethoxy) androstenone hydrochloride (U18666A), and 4-androstene-3,17-dione. The NAGly increased [Ca2+]i through stimulation of the voltage-dependent Ca2+ channels and it was dependent on extracellular glucose level. On the other hand, U18666A and 4-androstene-3,17-dione increased [Ca2+]i in the presence of K ATP channel opener diazoxide while it was inhibited by the presence of Ca2+ channel blocker nitrendipine, suggesting that their effects are independent of K ATP channel. These unique features will be useful for further development of insulinotropic factors and drugs for treating type 2 diabetes.

SOX6 attenuates glucose-stimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice.

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing beta-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca(2+) mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in beta-cells may contribute to the beta-cell adaptation in obesity-related insulin resistance.

The very low density lipoprotein (VLDL) receptor--a peripheral lipoprotein receptor for remnant lipoproteins into fatty acid active tissues.

The VLDL (very low density lipoprotein) receptor is a member of the LDL (low density lipoprotein) receptor family. The VLDL receptor binds apolipoprotein (apo) E but not apo B, and is expressed in fatty acid active tissues (heart, muscle, adipose) and macrophages abundantly. Lipoprotein lipase (LPL) modulates the binding of triglyceride (TG)-rich lipoprotein particles to the VLDL receptor. By the unique ligand specificity, VLDL receptor practically appeared to function as IDL (intermediate density lipoprotein) and chylomicron remnant receptor in peripheral tissues in concert with LPL. In contrast to LDL receptor, the VLDL receptor expression is not down regulated by lipoproteins. Recently several possible functions of the VLDL receptor have been reported in lipoprotein metabolism, atherosclerosis, obesity/insulin resistance, cardiac fatty acid metabolism and neuronal migration. The gene therapy of VLDL receptor into the LDL receptor knockout mice liver showed a benefit effect for lipoprotein metabolism and atherosclerosis. Further researches about the VLDL receptor function will be needed in the future.

The important role for betaVLDLs binding at the fourth cysteine of first ligand-binding domain in the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor (LDLR) is a crucial role for binding and uptaking apolipoprotein (apo) B-containing lipoproteins, such as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and LDL. The defect function of the LDLR causes familial hypercholesterolemia (FH), the phenotype of which is elevated plasma cholesterol and premature coronary heart disease (CHD). In the present study, we characterize the role of the cysteine residue of the ligand-binding domain of the LDLR. The mutant LDLR protein of cysteine for serine at codon 25 (25S-LDLR) was expressed in Chinese hamster ovary (CHO) cell line, ldl-A7. By Western blot analysis, the 25S-LDLR was detected with monoclonal antibody IgG-12D10, which reacts with the linker site of the LDLR but not with IgG-C7, which reacts with the NH2 terminus of the receptor. The 25S-LDLR bound LDL similarly to the wild-type LDLR, but the rate of uptake of LDL by the mutant receptor was only about half of that by the wild-type receptor. In contrast, the 25S-LDLR bound and internalized beta VLDL more avidly than LDL. These results suggest that the fourth cysteine residue of the first ligand-binding domain of the LDLR might be important for the internalization of atherogenic lipoproteins by vascular cells despite reduced LDL uptake, leading to atherosclerosis and premature cardiovascular disease.