Effect of insulin on the differential expression of VLDL receptor isoforms of SGC7901 cell and its biological implication.

This study examined the effect of insulin on the expression of very low density lipoprotein receptor (VLDLR) subtypes of SGC7901 cells and discussed its biological implication. In vitro, moderately or poorly-differentiated human gastric adenocarcinoma cell line SGC7901 was incubated with insulin for different lengths of time, and then the expression of protein and RNA level in VLDLR subtypes were detected by Western blotting and real-time PCR, respectively. The results showed that, at certain time interval, insulin could down-regulate expression of type I VLDLR and up-regulate the expression of type II VLDLR in SGC7901 cells, at both protein and RNA level. We are led to conclude that insulin serves as a regulator in maintaining the balance between glucose and lipid metabolism in vivo, possibly through its effect on the differential expression of VLDLR subtypes.

The effect of bafilomycin A1 and protease inhibitors on the degradation and recycling of a Class 5-mutant LDLR.

The low-density lipoprotein receptor (LDLR) mediates cholesterol homeostasis through endocytosis of lipoprotein particles, particularly low-density lipoproteins (LDLs). Normally, the lipoprotein particles are released in the endosomes and the receptors recycle to the cell surface. Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the gene encoding the LDLR. These mutations are divided into five functional classes where Class 5 mutations encode receptors that suffer from ligand-induced degradation and recycling deficiency. The aim of this study was to investigate whether it is possible to prevent the fast ligand-induced degradation of Class 5-mutant LDLR and to restore its ability to recycle to the cell surface. E387K is a naturally occurring Class 5 mutation found in FH patients, and in the present study, we used Chinese hamster ovary cells transfected with an E387K-mutant LDLR. Abrogation of endosomal acidification by adding bafilomycin A1 or addition of the irreversible serine protease inhibitors, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) and 3,4-dichloroisocoumarin (DCI), prevented the degradation of the E387K-mutant LDLR. However, the undegraded receptor did not recycle to the cell surface in the presence of LDL. Unexpectedly, AEBSF caused aggregation of early endosome antigen-1- positive endosomes and the intracellular trapped LDLR co-localized with these aggregated early endosomes.

The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions.

The insect low-density lipoprotein (LDL) receptor (LDLR) homolog, lipophorin receptor (LpR), mediates endocytic uptake of the single insect lipoprotein, high-density lipophorin (HDLp), which is structurally related to LDL. However, in contrast to the fate of LDL, which is endocytosed by LDLR, we previously demonstrated that after endocytosis, HDLp is sorted to the endocytic recycling compartment and recycled for re-secretion in a transferrin-like manner. This means that the integrity of the complex between HDLp and LpR is retained under endosomal conditions. Therefore, in this study, the ligand-binding and ligand-dissociation capacities of LpR were investigated by employing a new flow cytometric assay, using LDLR as a control. At pH 5.4, the LpR-HDLp complex remained stable, whereas that of LDLR and LDL dissociated. Hybrid HDLp-binding receptors, containing either the beta-propeller or both the beta-propeller and the hinge region of LDLR, appeared to be unable to release ligand at endosomal pH, revealing that the stability of the complex is imparted by the ligand-binding domain of LpR. The LpR-HDLp complex additionally appeared to be EDTA-resistant, excluding a low Ca(2+) concentration in the endosome as an alternative trigger for complex dissociation. From binding of HDLp to the above hybrid receptors, it was inferred that the stability upon EDTA treatment is confined to LDLR type A (LA) ligand-binding repeats 1-7. Additional (competition) binding experiments indicated that the binding site of LpR for HDLp most likely involves LA-2-7. It is therefore proposed that the remarkable stability of the LpR-HDLp complex is attributable to this binding site. Together, these data indicate that LpR and HDLp travel in complex to the endocytic recycling compartment, which constitutes a key determinant for ligand recycling by LpR.

Low density lipoprotein receptor relatives in chicken ovarian follicle and oocyte development.

The normal development of the chicken oocyte within the ovarian follicle depends on the coordinated expression and function of several members of the low density lipoprotein receptor gene family. The human low density lipoprotein receptor is the prototype of the gene family; since its discovery and the elucidation of the medical significance of mutations in the LDLR gene, many additional family members have been discovered and characterized, and some important advances have resulted from studies in the chicken. I describe the analogies as well as the differences that exist between the molecular genetics of the mammalian and avian members of this important gene family, with emphasis on receptor-mediated oocyte growth. Recent progress in the molecular characterization of the chicken genes whose products mediate oocyte growth, follicle development, and accessory pathways is described in detail, and emerging information of preliminary nature is included. As the availability of chicken genome sequence data has enhanced the rate of progress in the field, our understanding of the physiological roles of members of this receptor family in general has already gained from studies in the avian model system.

Liver heparan sulfate proteoglycans mediate clearance of triglyceride-rich lipoproteins independently of LDL receptor family members.

We examined the role of hepatic heparan sulfate in triglyceride-rich lipoprotein metabolism by inactivating the biosynthetic gene GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) in hepatocytes using the Cre-loxP system, which resulted in an approximately 50% reduction in sulfation of liver heparan sulfate. Mice were viable and healthy, but they accumulated triglyceride-rich lipoprotein particles containing apoB-100, apoB-48, apoE, and apoCI-IV. Compounding the mutation with LDL receptor deficiency caused enhanced accumulation of both cholesterol- and triglyceride-rich particles compared with mice lacking only LDL receptors, suggesting that heparan sulfate participates in the clearance of cholesterol-rich lipoproteins as well. Mutant mice synthesized VLDL normally but showed reduced plasma clearance of human VLDL and a corresponding reduction in hepatic VLDL uptake. Retinyl ester excursion studies revealed that clearance of intestinally derived lipoproteins also depended on hepatocyte heparan sulfate. These findings show that under normal physiological conditions, hepatic heparan sulfate proteoglycans play a crucial role in the clearance of both intestinally derived and hepatic lipoprotein particles.

The NMR solution structure of the relaxin (RXFP1) receptor lipoprotein receptor class A module and identification of key residues in the N-terminal region of the module that mediate receptor activation.

The receptors for the peptide hormones relaxin and insulin-like peptide 3 (INSL3) are the leucine-rich repeat-containing G-protein-coupled receptors LGR7 and LGR8 recently renamed as the relaxin family peptide (RXFP) receptors, RXFP1 and RXFP2, respectively. These receptors differ from other LGRs by the addition of an N-terminal low density lipoprotein receptor class A (LDLa) module and are the only human G-protein-coupled receptors to contain such a domain. Recently it was shown that the LDLa module of the RXFP1 and RXFP2 receptors is essential for ligand-stimulated cAMP signaling. The mechanism by which the LDLa module modulates receptor signaling is unknown; however, it represents a unique paradigm in understanding G-protein-coupled receptor signaling. Here we present the structure of the RXFP1 receptor LDLa module determined by solution NMR spectroscopy. The structure is similar to other LDLa modules but shows small differences in side chain orientations and inter-residue packing. Interchange of the module with the second ligand binding domain of the LDL receptor, LB2, results in a receptor that binds relaxin with full affinity but is unable to signal. Furthermore, we demonstrate via structural studies on mutated LDLa modules and functional studies on mutated full-length receptors that a hydrophobic surface within the N-terminal region of the module is essential for activation of RXFP1 receptor signal in response to relaxin stimulation. This study has highlighted the necessity to understand the structural effects of single amino acid mutations on the LDLa module to fully interpret the effects of these mutations on receptor activity.

Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members.

Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.

Function and significance of very low density lipoprotein receptor subtype II.

To explore the functions of very low density lipoprotein receptor (VLDL-R) subtype II in lipoprotein metabolism and foam cells formation, the recombinant plasmid with the two subtypes cDNA was constructed respectively, the 1dl-A7 cell lines were transfected and two cell lines expressing VLDL-R were obtained: one stably expressing the VLDLR with the O-linked sugar region (type I VLDLR) and the other without the O-linked sugar region (type II VLDLR). In the study on binding of VLDLR to their nuclein labeled natural ligands (VLDL and beta-VLDL), it was found that surface binding of 25 I-VLDL or 125 I-beta-VLDL of 1dl-A7 cells transfected with type I VLDL R recombinant (1dl-A7-VRI) was more higher than that of 1dl-A7 cells transfected with type II VLDLR recombinant (1dl-A7-VRII). After being incubated with VLDL for different time, the contents of triglyceride and total cholesterol in cells were mensurated, and the formation of foam cells and accumulation of lipid in cells was observed by oil-red O staining. The results showed that the contents of triglyceride and total cholesterol in 1dl-A7-VR I were much higher than those in 1dl-A7-VR II, and 1dl-A7-VR I could transform into foam cells notably. It was suggested that type I VLDLR binds with relative higher affinity to VLDL and beta-VLDL, and internalizes much more lipoprotein into cells. As a result, we can conclude that type I VLDLR plays a more important role in lipoprotein metabolism and foam cells formation than type II VLDLR.

Degradation of the LDL receptor class 2 mutants is mediated by a proteasome-dependent pathway.

Familial hypercholesterolemia is a genetic disorder that results from various gene mutations, primarily within the LDL receptor (LDLR). Approximately 50% of the LDLR mutations are defined as class 2 mutations, with the mutant proteins partially or entirely retained in the endoplasmic reticulum. To determine the degradation pathway of the LDLR class 2 mutants, we examined the effects of inhibition of several potential pathways on the levels of the wild-type LDLR and its four representative class 2 mutants (S156L, C176Y, E207K, and C646Y) stably expressed in Chinese hamster ovary (CHO) cells. We found that proteasome inhibitors MG132 and lactacystin blocked the degradation of the LDLR mutants, but not that of the wild-type LDLR. Treatment of CHO cells with these proteasome inhibitors led to a significant accumulation of the mutants at steady state. Furthermore, cell surface levels of the LDLR mutants were significantly increased upon inhibition of the proteasome degradation pathway. In contrast to the proteasome inhibitors, inhibitors of trypsin-like proteases, chymotrypsin-like proteases, and lysosomal pathway inhibitors did not affect the levels of the LDLR mutants. Taken together, these data demonstrate that the proteasome is the principal degradation pathway for LDLR class 2 mutants.

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit.

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.

Molecular characterization of the VLDL receptor homolog mediating binding of lipophorin in oocyte of the mosquito Aedes aegypti.

Lipophorin (Lp) functions as a yolk protein precursor in the mosquito Aedes aegypti and it is internalized via receptor-mediated endocytosis (Insect Biochem. Mol. Biol., 30 (2000) 1161). We cloned and molecularly characterized a putative mosquito ovarian lipophorin receptor (AaLpRov) cDNA. The cDNA has a length of 3468 bp coding for a 1156-residue protein with a predicted molecular mass of 128.9 kDa. The deduced amino acid sequence of the cDNA revealed that it encodes a protein homolog of the LDL receptor superfamily, and that it harbors eight cysteine-rich ligand binding repeats at the N-terminus like vertebrate VLDL receptors. The deduced amino acid sequence of this mosquito ovarian receptor is most similar to that of the locust lipophorin receptor (LmLpR) (64.3%), and is only distantly related to the mosquito vitellogenin receptor (VgR) (18.3%), another ovarian LDLR homolog with a different ligand. The AaLpRov cDNA was expressed in a TnT Coupled Reticulocyte Lysate system, and co-immunoprecipitation experiments confirmed that the receptor protein specifically binds Lp. Developmental expression profiles clearly showed that AaLpRov transcripts are present in the vitellogenic ovary, with peak expression at 24-36 h post blood meal. In situ hybridization indicated that AaLpRov transcripts are present only in female germ line cells. Distance-based phylogenetic analyses suggest that the insect LpR and vertebrate LDL/VLDL receptor lineages separated after divergence from the insect VgR lineage.

Investigation of the distribution and changes of VLDLR subtype in fibrotic cardiac muscles.

Very low-density lipoprotein receptor (VLDLR) is the major receptor with which cells can uptake the triacylglycerol from blood. It is divided into two subtypes according to presence of O-linked sugar domain located in the VLDLR receptor immediately outside of the membrane. Type I VLDLR contains the O-link domain, while type II has no such domain. The type I VLDLR are mainly found on the surface of human myocardial cells. The result of our quantitative polymerase chain reaction on the normal and fibrotic cardiac muscles showed that both subtypes and expression level of VLDLR on the myocardial cell surface did not vary significantly between the normal and the fibrotic cardiac muscles despite the presence of malfunction due to fibrosis. This finding suggests that fibrosis doesn't exert significant influence on the subtype and the expression of VLDLR on the surface of myocardial cells. Such inconsistence with the changes found in other fibrotic tissues is awaiting further studies.

Receptors for oxidized low density lipoprotein.

An increasing body of evidence indicates that oxidized low density lipoprotein (LDL) is involved in the pathogenesis of atherosclerosis. One of the first biologic actions of oxidized LDL to be identified in vitro was its ability to interact with the 'acetyl LDL receptor' discovered by Goldstein and Brown. Over the past decade, considerable progress has been made in identifying and characterizing cell-surface receptors for oxidized LDL. Most of these receptors are thought to be multifunctional because they interact with several structurally different ligands, and accordingly have been termed 'scavenger receptors'. The objective of this article is to review the most important publications dealing with structure, ligand specificity, regulation, and function of scavenger receptors.

Molecular characterization of the mosquito vitellogenin receptor reveals unexpected high homology to the Drosophila yolk protein receptor.

The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.

Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus.

A protein binding to a minor-group human rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino acid sequences of tryptic peptides showed identity with the human low density lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding sites on human fibroblasts. Cells down-regulated for LDLR expression yielded much less HRV2 upon infection than cells with up-regulated LDLR. Virus also bound to the large subunit of the alpha 2-macroglobulin receptor/LDLR-related protein (alpha 2MR/LRP). LDLR-deficient fibroblasts yielded considerably less virus in the presence of receptor-associated protein (RAP), providing evidence that alpha 2MR/LRP also acts as a minor group HRV receptor.