Lipophorin receptor of Bombyx mori: cDNA cloning, genomic structure, alternative splicing, and isolation of a new isoform

The cDNA and genomic structure of a putative lipophorin receptor from the silkworm, Bombyx mori (BmLpR), indicated the presence of four isoforms, designated LpR1, LpR2, LpR3, and LpR4. The deduced amino acid sequence of each isoform showed five functional domains that are homologous to vertebrate very low density lipoprotein receptor (VLDLR). All four isoforms seem to have originated from a single gene by alternative splicing and were differentially expressed in a tissue- and stage-specific manner. BmLpR1 harbored an additional 27 amino acids in the O-linked sugar domain, resulting in an extra exon. The silkworm BmLpR gene consisted of 16 exons separated by 15 introns spanning >122 kb and was at least three times larger than the human VLDLR gene. Surprisingly, one of the isoforms, LpR4, was expressed specifically in the brain and central nervous system. Additionally, it had a unique cytoplasmic tail, leading to the proposition that it represents a new candidate LpR for possible brain-related function(s). This is the first report on the genomic characterization of an arthropod lipoprotein receptor gene and the identification of a brain-specific receptor variant from a core member of the low density lipoprotein receptor family in invertebrates.     

Human low density lipoprotein receptor expressed in Xenopus oocytes. Conserved signals for O-linked glycosylation and receptor-mediated endocytosis

The human low density lipoprotein (LDL) receptor is shown to carry out efficient receptor-mediated endocytosis in Xenopus laevis oocytes. Microinjection of mRNAs encoding the human receptor led to synthesis of a 120-kDa precursor possessing high mannose N-linked sugars and core O-linked sugars. During its transport to the cell surface, the protein increased in apparent size to 160 kDa, which is similar to the change that occurs in human cells. This increase was not seen when the receptor lacked the serine/threonine-rich region that undergoes O-linked glycosylation. The surface receptors bound 125I-LDL at 0 degrees C and internalized it with a half-time of 2 min when the cells were warmed to 19 degrees C. The rate of internalization was slowed by 7-fold when a single residue in the cytoplasmic domain (Tyr807) was changed to a cysteine, an alteration that slows incorporation into coated pits in mammalian cells. Deletion of the cytoplasmic domain abolished rapid internalization. We conclude that the signals for O-linked glycosylation and receptor-mediated endocytosis of the LDL receptor have been conserved throughout vertebrate evolution.     

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 effect of ethanol on the lipoprotein metabolism of aortic smooth muscle cells

The effect of ethanol exposure on the binding and metabolism of bovine low density lipoprotein by bovine arterial smooth muscle cells was studied. In cells exposed to ethanol (80 mM) for 48 hr or 14 days and incubated with low density lipoprotein for 24 hr there was a reduction in the amount of low density lipoprotein internalised at all concentrations of lipoprotein. There was no effect on the rate of degradation of the low density lipoprotein and no demonstrable changes in the amount of low density lipoprotein bound to the cell surface at high concentrations of low density lipoprotein. Similar results for internalisation and degradation were obtained in a time dependent study. Binding was shown to be reduced in the ethanol treated cells (48 hr) when low concentrations (5 micrograms/ml) of low density lipoprotein were incubated for short periods (less than 3 hr). Scatchard plot analysis indicated that this reduced binding may be due to a reduction of receptor numbers in these cells.     

Abnormal intracellular sorting of O-linked carbohydrate-deficient interleukin-2 receptors.

The synthesis and intracellular sorting of the interleukin-2 (IL-2) receptor were studied with a line of mutant Chinese hamster ovary (CHO) cells with a reversible defect in protein O glycosylation. Under normal culture conditions the mutant ldlD cannot add N-acetylgalactosamine (Ga1NAc) to proteins. Ga1NAc is the first sugar of mucin-type O-linked oligosaccharides attached to protein. This O-glycosylation defect is rapidly corrected when Ga1NAc is added to the culture mediu. An expression vector for the p55 human IL-2 receptor was transfected into wild-type CHO and ldlD cells and the structure, stability, and cell surface expression of the receptor were examined by immunoprecipitation and antibody-binding assays. Essentially all of the mature form of the normally glycosylated IL-2 receptor in both wild-type CHO cells and ldlD cells incubated with Ga1NAc was expressed on the cell surface. The stability of O-linked carbohydrate-deficient (Od) IL-2 receptors (in ldlD cells without Ga1NAc) was normal; however, missorting of the Od receptors resulted in very little cell surface expression. The sialidase sensitivity and endoglycosidase H resistance of mature Od IL-2 receptors suggest that Od receptor missorting occurred in or beyond the trans Golgi apparatus. The abnormal sorting of the Od IL-2 receptor is compared with the O-glycosylation dependence of the surface expression and stability of the low-density lipoprotein receptor, decay-accelerating factor, and the major antigen envelope protein of Epstein-Barr virus.     

An expression system for human apolipoprotein B100 in a rat hepatoma cell line

We have designed an in vitro expression system for human apolipoprotein (apo) B. A full-length human apoB minigene was constructed from cDNA and genomic apoB clones and inserted into a vector where its expression was directed by the cytomegalovirus promoter. The apoB minigene was expressed in a rat hepatoma cell line, McA-RH7777. Human apoB100, which is the ligand for the low density lipoprotein receptor, was secreted in low density lipoprotein or very low density lipoprotein particles, depending on the composition of the medium. A protein with the mobility of apoB48, a structurally related protein involved in cholesterol metabolism, was also produced from the human apoB minigene. This in vitro expression system for human apoB will enable investigators to identify which domains of this protein are involved in processes such as lipoprotein assembly and secretion. This system should also allow investigators to identify definitively the domain in apoB that enables the protein to bind to the low density lipoprotein receptor.     

A new class mutation of low density lipoprotein receptor with altered carbohydrate chains

In a monensin-resistant mutant (Monr-31) of Chinese hamster ovary cells, the O-linked sugar chains of the low density lipoprotein (LDL) receptor are altered, suggesting a mutation at a Golgi apparatus gene. In a compactin-resistant mutant (MF-2) of Chinese hamster V79 cells, the mature LDL receptor is apparently 5000 daltons smaller; the difference is due to altered glycosylation of O-linked sugar chains. Hybrids between MF-2 and Monr-31 still produced LDL receptor molecules with aberrant sugar chains; thus both mutants are in the same complementation group. Krieger and his colleagues (Krieger, M., Kingsley, D., Sege, R., Hobbie, L., and Kozarsky, K. (1985) Trends. Biochem. Sci. 10, 447-452) have classified Chinese hamster ovary cell mutants with altered LDL receptor structure into four groups: ldlA, ldlB, ldlC, and ldlD. Cell-cell hybrids between their ldl mutants and Monr-31 produced wild type mature LDL receptors with normal molecular sizes, suggesting that these compactin- and monensin-resistant mutants define a new class of LDL receptor mutant. Since both of our mutants are defective in internalization of LDL, we assign them as int mutants. This may imply a further etiology for hypercholesterolemia, and cases can now be examined for such a class.     

Regulatory role of insulin in the degradation of low density lipoprotein by cultured human skin fibroblasts.

The degradation of 125I-labeled low density lipoprotein by cultured human skin fibroblasts was enhanced 25% by preincubation of cells with insulin. This effect of insulin appeared to be mediated via stimulation of low density lipoprotein binding to its cell surface receptor, since binding and subsequent internalization of low density lipoprotein were stimulated to a similar extent as was degradation. In addition, insulin enhanced binding of low density lipoprotein at 4 degrees C, at which temperature internalization of the lipoprotein does not occur. A similar effect of insulin on the interaction of very low density lipoprotein with cultured fibroblasts was observed. Insulin-induced changes in the degradation of low density lipoprotein and very low density lipoprotein appeared to be a function of the change in lipoprotein binding. Thus, insulin may play a role in the regulation of low density lipoprotein and very low density lipoprotein degradation by peripheral cells by influencing the receptor-mediated transport of these lipoproteins.     

The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA

The nucleotide sequence of a cloned 5.3 kilobase cDNA for the human low density lipoprotein receptor revealed five domains in the 839 amino acid protein: 322 NH2-terminal amino acids, extremely rich in disulfide-bonded cysteine residues (15%) and including an 8-fold repeat of 40 residues that may contain the LDL binding site; 350 residues homologous to the precursor of mouse epidermal growth factor; a region immediately outside the plasma membrane, rich in serine and threonine and the site of O-linked glycosylation; 22 hydrophobic amino acids, spanning the plasma membrane; and 50 COOH-terminal amino acids, projecting into the cytoplasm. The mRNA for the receptor contains a 3' untranslated region of 2.5 kilobases that includes multiple copies of the Alu family of repetitive DNAs. Transfection of simian COS cells with the human LDL receptor cDNA linked to the SV40 early promoter resulted in expression of functional cell surface receptors.     

Lipoprotein receptors in extraembryonic tissues of the chicken

Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the main yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via a member of the low density lipoprotein receptor gene family termed LR8 (Bujo, H., Hermann, M., Kaderli, M. O., Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T., and Schneider, W. J. (1994) EMBO J. 13, 5165-5175). This endocytosis is accompanied by partial degradation of the yolk precursor protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. The receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed at ratios that vary with embryonic age in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allantois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labeled VLDL in cultured EEC. Addition to the incubation medium of the 39-kDa receptor-associated protein, which inhibits all known LR8/ligand interactions, blocks the uptake of VLDL by EEC. The levels of endogenous receptor-associated protein correspond to those of LR8+ but not LR8-, suggesting that it may play a role in the modulation of surface presentation of LR8+. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Because the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which affects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo.     

Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts

A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts.     

Differential glycosylation regulates processing of lipoprotein receptors by gamma-secretase

The low density lipoprotein (LDL) receptor-related protein 1 (LRP1) belongs to a growing number of cell surface proteins that undergo regulated proteolytic processing that culminates in the release of their intracellular domain (ICD) by the intramembranous protease gamma-secretase. Here we show that LRP1 is differentially glycosylated in a tissue-specific manner and that carbohydrate addition reduces proteolytic cleavage of the extracellular domain and, concomitantly, ICD release. The apolipoprotein E (apoE) receptor-2 (apoER2), another member of the LDL receptor family with functions in cellular signal transmission, also undergoes sequential proteolytic processing, resulting in intracellular domain release into the cytoplasm. The penultimate processing step also involves cleavage of the apoER2 extracellular domain. The rate at which this cleavage step occurs is determined by the glycosylation state of the receptor, which in turn is regulated by the alternative splicing of an exon encoding several O-linked sugar attachment sites. These findings suggest a role for differential and tissue-specific glycosylation as a physiological switch that modulates the diverse biological functions of these receptors in a cell-type specific manner.     

The transmembrane domain and PXXP motifs of ApoE receptor 2 exclude it from carrying out clathrin-mediated endocytosis

The low density lipoprotein (LDL) receptor family comprises several proteins with similar structures including the LDL receptor and apoE receptor 2 (apoER2). The human brain expresses two major splice variants of apoER2 mRNA, one of which includes an additional exon that encodes 59 residues in the cytoplasmic domain. This exon is absent from the LDL receptor and contains three proline-rich (PXXP) motifs that may allow apoER2 to function as a signal transducer. To investigate the role of this insert, we took advantage of the well characterized low density lipoprotein receptor pathway. Chimeras comprising the ectodomain and transmembrane domain of the LDL receptor fused to the cytoplasmic domain of apoER2 lacking the PXXP-containing residues are able to mediate clathrin-dependent endocytosis of LDL as effectively as cells expressing the LDL receptor but not if the PXXP insert is present in the protein. Although expressed on the cell surface, the PXXP-containing chimeric receptor is excluded from clathrin vesicles as judged by its failure to co-localize with adaptor protein-2 possibly due to interaction with intracellular adaptors or scaffolding proteins. Chimeras with the transmembrane domain of apoER2, predicted to be longer than that of the LDL receptor by several residues, fail to mediate endocytosis of LDL or to co-localize with adaptor protein-2 regardless of the presence or absence of the PXXP insert. Thus features of apoER2 that distinguish it as a signaling receptor, rather than as an endocytosis receptor like the LDL receptor, reside in or near the transmembrane domain and in the proline-rich motifs.     

The putative tumor suppressor LRP1B, a novel member of the low density lipoprotein (LDL) receptor family, exhibits both overlapping and distinct properties with the LDL receptor-related protein

The low density lipoprotein receptor-related protein-deleted in tumor (LRP1B, initially referred to as LRP-DIT) was cloned and characterized as a candidate tumor suppressor. It is a new member of the low density lipoprotein receptor gene family. Its overall domain structure and large size (approximately 600 kDa) are similar to LRP and suggest that it is a multifunctional cell surface receptor. Herein, we characterize a series of ligands for the receptor using cell lines that stably express it as a domain IV minireceptor (mLRP1B4). Ligands of LRP including receptor-associated protein, urokinase plasminogen activator, tissue-type plasminogen activator, and plasminogen activator inhibitor type-1 each demonstrate binding, internalization, and degradation via mLRP1B4. Interestingly, the kinetics of ligand endocytosis is distinctly different from that of LRP, with LRP1B exhibiting a markedly diminished internalization rate. In addition, tissue expression analysis reveals that the LRP1B gene is expressed in brain, thyroid, and salivary gland. These studies thus extend the physiological roles of members of the LDL receptor family.