Secondary literature sources for LDLa

The following references were automatically generated.

Kurniawan ND, Aliabadizadeh K, Brereton IM, Kroon PA, Smith R
NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor.
J Mol Biol. 2001; 311: 341-56
Display abstract
The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear 1H-15N NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta-strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. 15N longitudinal and transverse relaxation rates, and [1H]-15N heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85+/-0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides. Copyright 2001 Academic Press.

Gaus K, Basran A, Hall EA
Assessment of the fifth ligand-binding repeat (LR5) of the LDL receptor as an analytical reagent for LDL binding.
Analyst. 2001; 126: 329-36
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The fifth ligand binding repeat (LR5) of the low density lipoprotein (LDL) receptor was assessed ex vivo as an 'analytical reagent' to distinguish LDL state, in atherosclerosis risk monitoring. LR5 was immobilized to mercaptoundecanoic acid modified gold surfaces via a glycine linker. Surface plasmon resonance (SPR) was used to monitor LDL binding. Unfolded LR5 was ineffectual as an affinity ligand for LDL but refolded LR5 showed a high affinity for native LDL but little affinity for oxidized LDL. LR5 refolded in the presence of calcium or EDTA gave the equivalent LDL binding capacity. However, EDTA-LR5 was less stable than Ca-LR5 at pH 5 and, from tryptophan fluorescence evidence, they appeared to involve different regions of LR5 and/or LDL in the binding. Involvement of amino acid residues of the calcium cage of LR5 was tested in LDL binding by monitoring calcium ion release with a calcium ionophore. The results were consistent with approximately 7-8 LR5 binding per LDL, of which only some induce calcium release (a maximum of approximately 25 mol% calcium, based on LR5, was released during LDL binding). For LDL binding to the LDL receptor in vivo more than one ligand-binding repeat is needed and this may be consistent with LR5 acting here also at binding sites which other LRs normally occupy in the LDL-LDL receptor complex. This initial study is encouraging for the use of a minimum peptide repeat array based on the conserved region of the LRs as an affinity surface for atherosclerosis risk monitoring.

Wang QY, Dolmer K, Huang W, Gettins PG, Rong L
Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus.
J Virol. 2001; 75: 2051-8
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Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.

Jeon H, Meng W, Takagi J, Eck MJ, Springer TA, Blacklow SC
Implications for familial hypercholesterolemia from the structure of the LDL receptor YWTD-EGF domain pair.
Nat Struct Biol. 2001; 8: 499-504
Display abstract
The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of cholesterol-carrying particles into cells. The region of the LDLR implicated in receptor recycling and lipoprotein release at low pH contains a pair of calcium-binding EGF-like modules, followed by a series of six YWTD repeats and a third EGF-like module. The crystal structure at 1.5 A resolution of a receptor fragment spanning the YWTD repeats and its two flanking EGF modules reveals that the YWTD repeats form a six-bladed beta-propeller that packs tightly against the C-terminal EGF module, whereas the EGF module that precedes the propeller is disordered in the crystal. Numerous point mutations of the LDLR that result in the genetic disease familial hypercholesterolemia (FH) alter side chains that form conserved packing and hydrogen bonding interactions in the interior and between propeller blades. A second subset of FH mutations are located at the interface between the propeller and the C-terminal EGF module, suggesting a structural requirement for maintaining the integrity of the interdomain interface.

Shi X, Niimi S, Ohtani T, Machida S
Characterization of residues and sequences of the carbohydrate recognition domain required for cell surface localization and ligand binding of human lectin-like oxidized LDL receptor.
J Cell Sci. 2001; 114: 1273-82
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Lectin-like oxidized low-density lipoprotein receptor (LOX-1) has been cloned from human aortic endothelial cells, and has a sequence identical to that from human lung. Previous studies showed that human LOX-1 can recognize modified LDL, apoptotic cells and bacteria. To further explore the relationship between the structure and function of LOX-1, a mutagenesis study was carried out. Our results showed that the carbohydrate recognition domain (CRD) was the ligand-binding domain of human LOX-1. We also investigated the sequences and residues in CRD that were essential for protein cell surface localization and ligand binding. LOX-1s carrying a mutation on each of six Cys in CRD resulted in a variety of N-glycosylation and failed to be transported to the cell surface. This was strong evidence for the involvement of all six Cys in the intrachain disulfide bonds required for proper folding, processing and transport of LOX-1. The C-terminal sequence (KANLRAQ) was also essential for protein folding and transport, while the four final residues (LRAQ) were involved in maintaining receptor function. Both positive charged (R208, R209, H226, R229 and R231) and non-charged hydrophilic (Q193, S198, S199 and N210) residues were involved in ligand binding, suggesting that ligand recognition of LOX-1 is not merely dependent on the interaction of positively charged residues with negatively charged ligands.

Beglova N, North CL, Blacklow SC
Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor.
Biochemistry. 2001; 40: 2808-15
Display abstract
The ligand-binding domain of the LDL receptor consists of seven contiguous LDL-A modules. The fifth of these ligand-binding modules is absolutely required for recognition of both LDL and beta-VLDL particles. A four-residue linker of variable sequence connects each pair of modules, except for modules four and five, which are connected by a 12-residue linker. To provide a more detailed understanding of the structural relationship in a typical pair of functionally important LDL-A repeats of the LDLR, we investigated the backbone dynamics of repeats five (LR5) and six (LR6) alone and in the context of the covalently connected LR5-6 pair. Our results reveal substantial flexibility in the four-residue linker connecting the two repeats in the LR5-6 pair. The intrinsic dynamic behavior of each repeat is essentially unchanged when the repeats are covalently connected. These observations indicate that the relative orientation of repeats in LR5-6 is not fixed. Modeled in an extended conformation, the linker can separate LR5 and LR6 by up to 15 A, a distance that would allow substantial freedom of motion of each repeat with respect to the other in the pair.

Garcia CK et al.
Autosomal recessive hypercholesterolemia caused by mutations in a putative LDL receptor adaptor protein.
Science. 2001; 292: 1394-8
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Atherogenic low density lipoproteins are cleared from the circulation by hepatic low density lipoprotein receptors (LDLR). Two inherited forms of hypercholesterolemia result from loss of LDLR activity: autosomal dominant familial hypercholesterolemia (FH), caused by mutations in the LDLR gene, and autosomal recessive hypercholesterolemia (ARH), of unknown etiology. Here we map the ARH locus to an approximately 1-centimorgan interval on chromosome 1p35 and identify six mutations in a gene encoding a putative adaptor protein (ARH). ARH contains a phosphotyrosine binding (PTB) domain, which in other proteins binds NPXY motifs in the cytoplasmic tails of cell-surface receptors, including the LDLR. ARH appears to have a tissue-specific role in LDLR function, as it is required in liver but not in fibroblasts.

North CL, Blacklow SC
Evidence that familial hypercholesterolemia mutations of the LDL receptor cause limited local misfolding in an LDL-A module pair.
Biochemistry. 2000; 39: 13127-35
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Mutations at conserved sites within the ligand-binding LDL-A modules of the LDL receptor cause the genetic disease familial hypercholesterolemia (FH), and several of these FH mutations in modules five and six prevent the isolated single modules from folding properly to a nativelike three-dimensional structure. Because LDL-A modules occur as a series of contiguous repeats in the LDLR and related proteins, we investigated the impact of two FH mutations in LDL-A module five (D203G and D206E) and two mutations in module six (E219K and D245E) in the context of the covalently connected module five-six pair. HPLC chromatography of the products formed under conditions that efficiently refold the native module five-six pair demonstrate that, for each mutation, a folding defect persists in the module pair. NMR spectroscopy and calcium affinity measurements of the ensemble of misfolded products demonstrate that the unaltered module of each pair can fold to its native structure regardless of the range of misfolded conformations adopted by its mutated neighbor. These findings lend additional support to a model in which individual LDL-A modules of the LDL receptor act as independent structural elements.

Morrow JA, Arnold KS, Dong J, Balestra ME, Innerarity TL, Weisgraber KH
Effect of arginine 172 on the binding of apolipoprotein E to the low density lipoprotein receptor.
J Biol Chem. 2000; 275: 2576-80
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The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136-150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170-183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167-185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg(172) reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Delta167-185 mutant relative to the Arg(172) mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg(172) on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg(172) to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.

Jeon H, Shipley GG
Localization of the N-terminal domain of the low density lipoprotein receptor.
J Biol Chem. 2000; 275: 30465-70
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The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein performing "receptor-mediated endocytosis" of cholesterol-rich lipoproteins. At the N terminus, the LDL receptor has modular cysteine-rich repeats in both the ligand binding domain and the epidermal growth factor (EGF) precursor homology domain. Each repeat contains six disulfide-bonded cysteine residues, and this structural motif has also been found in many other proteins. The bovine LDL receptor has been purified and reconstituted into egg yolk phosphatidylcholine vesicle bilayers. Using gel electrophoresis and cryoelectron microscopy (cryoEM), the ability of the reconstituted LDL receptor to bind its ligand LDL has been demonstrated. After reduction of the disulfide-bonds in the N-terminal domain of the receptor, the reduced LDL receptor was visualized using cryoEM; reduced LDL receptors showed images with a diffuse density region at the distal end of the extracellular domain. Gold labeling of the reduced cysteine residues was achieved with monomaleimido-Nanogold, and the bound Nanogold was visualized in cryoEM images of the reduced, gold-labeled receptor. Multiple gold particles were observed in the diffuse density region at the distal end of the receptor. Thus, the location of the ligand binding domain of the LDL receptor has been determined, and a model is suggested for the arrangement of the seven cysteine-rich repeats of the ligand binding domain and two EGF-like cysteine-rich repeats of the EGF precursor homology domain.

Jorgensen MM et al.
Grp78 is involved in retention of mutant low density lipoprotein receptor protein in the endoplasmic reticulum.
J Biol Chem. 2000; 275: 33861-8
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The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL cholesterol from the plasma. Mutations in the LDL receptor gene cause the disease familial hypercholesterolemia (FH). Approximately 50% of the mutations in the LDL receptor gene in patients with FH lead to receptor proteins that are retained in the endoplasmic reticulum (ER). Misfolding of mutant LDL receptors is a probable cause of this ER retention, resulting in no functional LDL receptors at the cell surface. However, the specific factors and mechanisms responsible for retention of mutant LDL receptors are unknown. In the present study we show that the molecular chaperone Grp78/BiP co-immunoprecipitates with both the wild type and two different mutant (W556S and C646Y) LDL receptors in lysates obtained from human liver cells overexpressing wild type or mutant LDL receptors. A pulse-chase study shows that the interaction between the wild type LDL receptor and Grp78 is no longer detectable after 2(1/2) h, whereas it persists for more than 4 h with the mutant receptors. Furthermore, about five times more Grp78 is co-immunoprecipitated with the mutant receptors than with the wild type receptor suggesting that Grp78 is involved in retention of mutant LDL receptors in the ER. Overexpression of Grp78 causes no major alterations on the steady state level of active LDL receptors at the cell surface. However, overexpression of Grp78 decreases the processing rate of newly synthesized wild type LDL receptors. This indicates that the Grp78 interaction is a rate-limiting step in the maturation of the wild type LDL receptor and that Grp78 may be an important factor in the quality control of newly synthesized LDL receptors.

North CL, Blacklow SC
Solution structure of the sixth LDL-A module of the LDL receptor.
Biochemistry. 2000; 39: 2564-71
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The low-density lipoprotein receptor (LDLR) is the primary mechanism for uptake of plasma cholesterol into cells and serves as a prototype for an entire class of cell surface receptors. The amino-terminal domain of the receptor consists of seven LDL-A modules; the third through the seventh modules all contribute to the binding of low-density lipoproteins (LDLs). Here, we present the NMR solution structure of the sixth LDL-A module (LR6) from the ligand binding domain of the LDLR. This module, which has little recognizable secondary structure, retains the essential structural features observed in the crystal structure of LDL-A module five (LR5) of the LDLR. Three disulfide bonds, a pair of buried residues forming a hydrophobic "mini-core", and a calcium-binding site that serves to organize the C-terminal lobe of the module all occupy positions in LR6 similar to those observed in LR5. The striking presence of a conserved patch of negative surface electrostatic potential among LDL-A modules of known structure suggests that ligand recognition by these repeats is likely to be mediated in part by electrostatic complementarity of receptor and ligand. Two variants of LR6, identified originally as familial hypercholesterolemia (FH) mutations, have been investigated for their ability to form native disulfide bonds under conditions that permit disulfide exchange. The first, E219K, lies near the amino-terminal end of LR6, whereas the second, D245E, alters one of the aspartate side chains that directly coordinate the bound calcium ion. After equilibration at physiologic calcium concentrations, neither E219K nor D245E folds to a unique disulfide isomer, indicating that FH mutations both within and distant from the calcium-binding site give rise to protein-folding defects.

Ashavaid TF, Kondkar AA, Nair KG
Identification of two LDL-receptor mutations causing familial hypercholesterolemia in Indian subjects by a simplified rapid PCR-heteroduplex method.
Clin Chem. 2000; 46: 1183-5

Maletinska L, Blakely EA, Bjornstad KA, Deen DF, Knoff LJ, Forte TM
Human glioblastoma cell lines: levels of low-density lipoprotein receptor and low-density lipoprotein receptor-related protein.
Cancer Res. 2000; 60: 2300-3
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The status of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP) in seven human glioma cell lines was evaluated to extend our knowledge of human glioblastoma multiforme tumor metabolism for future drug design. Cell lines SF-767, SF-763, A-172, U-87 MG, U-251 MG, U-343 MG, and SF-539 were used. Binding of 125I-labeled LDL to these cells at 4 degrees C was carried out to determine the number of LDL receptors on cells and the affinity of LDL for these receptors. The content of LRP was measured by immunoblotting. The presence of specific saturable LDL receptors was proven in six of the cell lines investigated. SF-767 cells revealed high-affinity LDL binding (equilibrium dissociation constant, Kd = 7 nM) and maximum binding capacity approximating 300,000 receptors/cell. Most of the remaining cell lines had relatively lower affinity (Kd = 38-62 nM) but also had very high numbers of receptors (128,000-950,000/cell). All cell lines exhibited LRP, but the expression was variable. The cell lines SF-539, U-87 MG, and U-343 MG were particularly rich in this protein. The data suggest that glioblastoma cells have high numbers of LDL receptors; however, there is considerable variation in binding affinity. Overall, this finding suggests that LDL receptors on glioblastoma cells could potentially be useful for targeting antitumor agents. LRP, a multifunctional receptor expressed on glioblastoma cells, also has the possibility for serving as a therapeutic target.

Sugiyama T et al.
A novel low-density lipoprotein receptor-related protein mediating cellular uptake of apolipoprotein E-enriched beta-VLDL in vitro.
Biochemistry. 2000; 39: 15817-25
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We report here the identification of a novel member of the low-density lipoprotein receptor (the LDL receptor) family through signal sequence trap screening of a mouse lymphocyte cDNA library. The protein was termed LDL receptor-related protein 9 (LRP9). LRP9 is a type I membrane protein predicted to contain 696 amino acids with a calculated molecular mass of 74 764 Da. The NH(2)-terminal half of LRP9 contains two CUB domains separated by a single ligand-binding repeat. The second CUB domain is followed by a cluster of three additional ligand-binding repeats and a transmembrane domain. The COOH-terminal intracellular region contains a proline-rich region. LRP9 mRNA was expressed in the liver, kidney, lung, and heart at high levels, and in the spleen and brain at low levels. In situ hybridization analysis of mouse liver, kidney, and brain detected LRP9 transcripts in hepatocytes, sinusoidal lining cells, peritubular capillaries, choroid plexus, ependyma of the third ventricle, pia matter, and hippocampus. In particular, high levels of expression were observed in the vascular walls. Apolipoprotein E (apoE)-enriched beta-VLDL stimulated cellular cholesteryl ester formation in ldl-A7/LRP9. These results raise the possibility that this newly identified receptor, which is expressed in the liver, may play a physiological role in the uptake of apoE-containing lipoproteins.

Wessel GM, Conner S, Laidlaw M, Harrison J, LaFleur GJ Jr
SFE1, a constituent of the fertilization envelope in the sea urchin is made by oocytes and contains low-density lipoprotein-receptor-like repeats.
Biol Reprod. 2000; 63: 1706-12
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At fertilization in most animals, cortical granules of the egg or oocyte secrete their contents, whose function it is to modify the extracellular matrix. This modified matrix then participates in the block to polyspermy and protection for early embryonic development. In the sea urchin, contents of the cortical granules are secreted within 30 sec of insemination. Several of these content proteins then bind to the nascent vitelline layer of the egg and lift off the cell surface to form a stable, impervious, fertilization envelope. At least six major proteins are present in the envelope, and recently we have identified cDNA clones of two, ovoperoxidase, and SFE9. Here we report on the identification and characterization of SFE1, a constituent of the fertilization envelope of the sea urchin Strongylocentrotus purpuratus, that has revealing characteristics of how the envelope might form and what protein interaction domains might predominate. We present the largest cDNA sequence we were able to identify representing approximately two thirds of the predicted protein coding region. The C-terminal half of the cognate SFE1 protein contains two different amino acid repeat motifs: a cysteine-rich (15%) motif of 40 amino acids that is tandemly repeated 22 times and is followed by a serine/threonine-rich (38%) repeat of 63 amino acids that is tandemly repeated 3.5 times. Surprisingly, just N-terminal to the cysteine-rich repeat region is a sequence of five repeats with similarity to repeats in another cortical granule protein, SFE9, and to the motif originally identified in the receptor of low-density lipoproteins, the LDLr motif. The amino acid composition deduced from the partial SFE1 cDNA is similar also to the composition of proteoliaisin, a protein thought to tether the ovoperoxidase to the vitelline layer of the egg and thereby sequester the crosslinking activity of the ovoperoxidase to a limited population of proteins in the fertilization envelope. However, by use of monoclonal and polyclonal antibodies to SFE1 and proteoliaisin, we show here that they are distinct gene products. We also show that SFE1 is packed selectively into the cortical granules and then is crosslinked into the fertilization envelope following fertilization. In situ RNA hybridization analysis shows that the mRNA of SFE1 (9 kilobases) is present in oocytes selectively and is turned over rapidly in the oocyte following germinal vesicle breakdown. Our findings suggest that the gene encoding this major product of the egg is activated concomitantly with the other cortical granule-specific products already identified, and that a common LDLr-like motif of the fertilization envelope may reveal a structural mechanism for protein interactions in its construction.

Shin JA et al.
Identification of four novel mutations of the low-density lipoprotein receptor gene in Korean patients with familial hypercholesterolemia.
Clin Genet. 2000; 57: 225-9
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To obtain insight into the genetic variation of the low-density lipoprotein (LDL) receptor gene in Korean patients with familial hypercholesterolemia (FH), we used single-strand conformation polymorphism to screen all 18 exons and a promotor of the LDL receptor gene in 20 unrelated Korean FH patients. Four novel point mutations were detected in 5 FH patients and were characterized by sequence analysis. Of them, one is a nonsense mutation, a Glu-->Stop (CAG-->TAG) at codon 161, and results in a large deletion. The other three, which were a Ala-->Glu (GCG-->GAG) mutation at signal peptide, Cys-->Tyr (TGC-->TAC) at codon 210, and Pro-->Leu (CTG-->CCG) at codon 584, were novel missense mutations, which modified the highly conserved region of the LDL receptor gene. All these mutations were absent in normolipidemic controls and were associated in heterozygote carriers with clinical signs of FH. Identification of these novel mutations provides another example of the molecular heterogeneity of the LDL receptor gene mutations causing FH.

Raungaard B, Jensen HK, Brorholt-Petersen JU, Heath F, Faergeman O
Functional characterization of two low density lipoprotein receptor gene mutations by fluorescence flow cytometric assessment of receptor activity in stimulated human T-lymphocytes.
Clin Genet. 2000; 57: 110-5
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We report a functional characterization of the W23X and W66G low density lipoprotein (LDL) receptor gene mutations. The authors used two-color fluorescence flow cytometry to measure LDL receptor activity in stimulated T-lymphocytes, prepared from patients heterozygous for the W23X or W66G mutation, and compared the results with measurements of LDL receptor activity in stimulated T-lymphocytes prepared from unrelated healthy control subjects. It was found that the W23X mutation significantly reduced LDL receptor expression and LDL binding and internalization, and that the W66G mutation significantly reduced LDL receptor expression and LDL binding. LDL internalization in patients heterozygous for the W66G mutation was not significantly reduced. The data support the concepts that the W23X mutation prevents production of LDL receptors (class I) and that the W66G mutation produces LDL receptors unable to recycle normally in cells (class V).

Kurniawan ND et al.
NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.
Protein Sci. 2000; 9: 1282-93
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The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.

Gotthardt M et al.
Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction.
J Biol Chem. 2000; 275: 25616-24
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The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.

Lombardi MP et al.
Molecular genetic testing for familial hypercholesterolemia: spectrum of LDL receptor gene mutations in The Netherlands.
Clin Genet. 2000; 57: 116-24
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Mutations in the LDL receptor are responsible for familial hypercholesterolemia (FH). At present, more than 600 mutations of the LDL receptor gene are known to underlie FH. However, the array of mutations varies considerably in different populations. Therefore, the delineation of essentially all LDL receptor gene mutations in a population represents a prerequisite for the implementation of nation-wide genetic testing for FH. In this study, the frequency and geographical distribution of 13 known mutations were evaluated in a cohort of 1223 FH patients. We identified 358 mutation carriers, representing 29% of the FH cohort. Four mutations (N543H-2393de19, 1359--1G-->A, 313 + 1 G-->A and W23X) occurred with a relatively high frequency, accounting for 22.4% of the entire study cohort. Two of these common FH mutations (N543H-2393de19 and 1359 - 1G-->A) showed a preferential geographic distribution. Second, to further expand the array of LDL receptor gene mutations, we conducted mutation analysis by denaturing gradient gel electrophoresis (DGGE) in 141 children with definite FH. A mutation was identified in 111 patients, involving 16 new single base substitutions and four small deletions and insertions, which brings the number of different FH-causing mutations in our country up to 61. Our data indicate that an estimate of the prevalence of specific mutations, as well as the compilation of a database of all FH-causing mutations in a given country, can facilitate selection of the most appropriate molecular diagnostic approach.

Parise F et al.
Construction and in vitro functional evaluation of a low-density lipoprotein receptor/transferrin fusion protein as a therapeutic tool for familial hypercholesterolemia.
Hum Gene Ther. 1999; 10: 1219-28
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A cDNA sequence encoding a soluble form of the human low-density lipoprotein receptor (LDL-R) was produced by RT-PCR amplification. This form of the receptor contains the N-terminal cysteine-rich domain, the EGF homology domain, and the serine/threonine-rich domain, but lacks the membrane anchor as well as the cytoplasmic domain. By the same technical approach a cDNA sequence encoding rabbit transferrin was generated. In-frame fusion of the two cDNAs produced a sequence encoding a chimeric protein potentially capable of binding LDL on the N-terminal side and the transferrin receptor on the C-terminal side. It was expected that LDL bound to the chimeric protein could be internalized, targeted to an acidic compartment, and processed through the pathway of the transferrin receptor. Cells transfected with the LDL-R/transferrin cDNA translate, glycosylate, and secrete the corresponding protein in the culture medium. The secreted protein binds LDL in a ligand-blotting experiment. Finally, the chimeric protein mediates the binding and internalization of LDL in mutant cells lacking the LDL receptor. In fact, Watanabe rabbit fibroblasts, incubated with the chimeric protein show a fourfold increase in LDL binding, a fivefold increase in LDL internalization, and a sixfold increase in LDL degradation, with respect to unincubated fibroblasts.

Ebhardt M et al.
Mutation analysis in 46 German families with familial hypercholesterolemia: identification of 8 new mutations. Mutations in brief no. 226. Online.
Hum Mutat. 1999; 13: 257-257
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In order to obtain a survey of the mutations being prevalent in Northern Germany and to enable molecular genetic testing for families with clinically diagnosed familial hypercholesterolemia (FH), we screened 46 unrelated German individuals with elevated LDL levels for mutations in the 18 exons and their flanking intron sequences including the promotor region of the LDL receptor (LDLR) gene. In addition, we tested all patients for the presence of mutations in the gene coding for apolipoprotein B-100 (apoB-100). We detected 15 mutations affecting the LDLR gene, 8 of which, designated A29S, 195insAT, 313+1insG, 553insG, 680insGGACAAATCTG, D200N, E267K and L411V have not yet been reported. One patient is heterozygous for the double mutant N543H and 2393del9Bp. Two patients carried the mutation R3500Q (Arg-->Glu) within the apoB-100 gene.

Paillard F
A novel soluble chimera for the treatment of familial hypercholesterolemia.
Hum Gene Ther. 1999; 10: 1093-4

Cantafora A, Bertolini S, Calandra S
[Familial hypercholesterolemia and mutations of the gene for low-density lipoproteins in Italy]
Ann Ist Super Sanita. 1999; 35: 177-84
Display abstract
The review takes into account molecular bases and major implications for public health in our country of familial hypercholesterolemia (FH) caused by mutations in LDL receptor (LDLR) gene. The relevance of the problem is evidenced by the fact that (a) about 120,000 Italians affected by heterozygous FH face a cardiovascular risk much higher than normal individuals, and (b) their identification is difficult because of the plethora of mutations existing in the country. This report deepens also the relationships between alterations in the coding sequence of LDLR gene and in the function of LDLR protein. Furthermore, it describes, in terms of molecular characteristics and diffusion, the 42 mutations so far detected in Italy that cause about 30% of FH cases. This led us to hypothesize the existence in our country of over hundred different mutations present within defined geographical areas where, in a few cases, they may also have a relatively high incidence (i.e., the so-called FH-clusters). Finally, preliminary studies mentioned here point out the existence of significant correlations between phenotype and genotype of FH and suggest the possibility of realizing on a large scale an early diagnosis and therapy of pathologies caused by a function deficit in LDL receptor.

Bertolini S et al.
Analysis of LDL receptor gene mutations in Italian patients with homozygous familial hypercholesterolemia.
Arterioscler Thromb Vasc Biol. 1999; 19: 408-18
Display abstract
The aim of this study was the characterization of mutations of the LDL receptor gene in 39 Italian patients with homozygous familial hypercholesterolemia, who were examined during the period 1994 to 1996. The age of the patients ranged from 1 to 64 years; one third of them were older than 30. Plasma LDL cholesterol level ranged from 10.8 to 25.1 mmol/L. The residual LDL receptor activity, measured in cultured fibroblasts of 32 patients, varied from <2% to 30% of normal and was inversely correlated with the plasma LDL cholesterol level (r=-0.665; P<0.003). The most severe coronary atherosclerosis was observed in those patients with the lowest residual LDL receptor activity (

Kim SH et al.
Long-distance PCR-based screening for large rearrangements of the LDL receptor gene in Korean patients with familial hypercholesterolemia.
Clin Chem. 1999; 45: 1424-30
Display abstract
BACKGROUND: The LDL receptor is a cell-surface protein that regulates plasma cholesterol by specific uptake of LDL particles from the blood circulation. Familial hypercholesterolemia (FH) results from defective catabolism of LDL, which is caused by mutations in the LDL-receptor gene. METHODS: For the rapid and reliable detection of large rearrangements in the LDL-receptor gene, we established a screening method based on long-distance PCR as an alternative to Southern-blot hybridization. Using long-distance PCR, 45 unrelated Korean subjects heterozygous for FH were screened to assess the frequency and nature of major structural rearrangements in the LDL-receptor gene. RESULTS: Two different deletion mutations, FH6 (same type as FH3 and FH311) and FH 32, were detected in four families by long-distance PCR. Detailed restriction mapping and sequence analysis showed that FH6 was a 5.71-kb deletion extending from intron 8 to intron 12 and that FH32 was a 2-kb deletion extending from intron 6 to intron 7. Sequence analysis for the breakpoints of all deletions detected in Korean FH patients showed that only the left arms of the Alu repetitive sequences were involved in the deletion event. CONCLUSIONS: The screening method based on long-distance PCR provides a powerful strategy for the detection of large rearrangements in the LDL-receptor gene and is a rapid and reliable screening alternative to Southern-blot hybridization.

Chae JJ et al.
Three novel small deletion mutations of the LDL receptor gene in Korean patients with familial hypercholesterolemia.
Clin Genet. 1999; 55: 325-31
Display abstract
The low-density lipoprotein (LDL) receptor gene from 80 unrelated Korean patients with familial hypercholesterolemia (FH) was analyzed to screen for small structural rearrangements that could not be detected by Southern blot hybridization. Three different small deletions were detected in exon 11 of 3 FH patients and were characterized by DNA sequence analysis. Of them two mutations are in-frame 36-bp (FH 2) and 9-bp (FH 34) deletions that result in the loss of twelve amino acids (from Met510 to Ile521) and three amino acids (Thr513, Asp514 and Trp515), respectively. Both mutations are located in the third of the five YWTD motifs of the LDL receptor gene. The third mutation (FH 400) is a 2-bp deletion that shifts the translational reading frame and results in a prematurely terminated receptor protein. The generation of a 36-bp deletion can be explained by the formation of a hairpin-loop structure mediated by inverted repeat sequences. On the other hand, the mechanism responsible for the 9- and the 2-bp deletions is probably strand-slippage mispairing mediated by short direct repeats. All of these three deletions are novel mutations. Each of the three deletions was detected only in a single pedigree out of 80 FH families analyzed.

Katsumata H et al.
Familial hypercholesterolemia in Utah kindred with novel R103W mutations in exon 4 of the LDL receptor gene.
Jpn Heart J. 1999; 40: 443-9
Display abstract
Heterozygous familial hypercholesterolemia (FH) is a serious disorder causing twice normal low-density lipoprotein cholesterol levels early in childhood and very early coronary disease in both men and women. Previously published blood cholesterol criteria greatly under-diagnosed new cases of FH among members of known families with FH and over-diagnosed FH among participants of general population screening. Thus, there is a need for accurate and genetically validated criteria for the early diagnosis of heterozygous FH. In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To carry out molecular genetic diagnosis of the disease, we screened DNA samples for mutations in all 18 exons and the exon- intron boundaries of the low-density lipoprotein (LDL) receptor gene. Novel point mutations were identified in the proband: a C-to-T transversion at nucleotide position 369, causing substitution of Tryptophan for Arginine at codon 103 in exon 4 of the LDL receptor gene. The SSCP method was used to examine seven members of the family recruited for the diagnosis. This method helped to unequivocally diagnose only the proband as heterozygous for this particular LDL receptor mutation while excluding the remaining six individuals from carrier status with FH.

Nobe Y et al.
Familial hypercholesterolemia in Utah kindred with novel 2412-6 Ins G mutations in exon 17 of the LDL receptor gene.
Jpn Heart J. 1999; 40: 435-41
Display abstract
Familial hypercholesterolemia (FH) is a monogenic disorder associated with primary hypercholesterolemia. FH is characterized by autosomal co-dominant inheritance with strikingly elevated LDL-cholesterol, the presence of xanthoma and premature atherosclerosis. In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To determine the genetic etiology of the lipoprotein abnormalities, we screened DNA samples from the family for mutations in all 18 exons and the exon- intron boundaries of the low-density lipoprotein receptor (LDLR) gene. Novel point mutations were identified in the proband: a one-base insertion of G to a five-G stretch at nucleotides 2412-6 (codons 783-785), causing a frameshift in exon 17 of the LDL receptor gene. The direct sequencing method was used to examine six members of the family recruited for the diagnosis. This method helped to unequivocally diagnose the five individuals as heterozygous for this particular LDL receptor mutation. This method also helped us to diagnose with FH, or to exclude from carrier status, three children between ages 6 and 11.

Jensen HK, Jensen LG, Meinertz H, Hansen PS, Gregersen N, Faergeman O
Spectrum of LDL receptor gene mutations in Denmark: implications for molecular diagnostic strategy in heterozygous familial hypercholesterolemia.
Atherosclerosis. 1999; 146: 337-44
Display abstract
Heterozygous familial hypercholesterolemia (FH) is one of the most common potentially fatal single-gene diseases leading to premature coronary artery disease, but the majority of heterozygous FH patients have not been diagnosed. FH is due to mutations in the gene coding for the low-density lipoprotein (LDL) receptor, and molecular genetic diagnosis may facilitate identification of more FH subjects. The Danish spectrum of 29 different mutations, five of which account for almost half of heterozygous FH, is intermediate between that of countries such as South Africa, where three mutations cause 95% of heterozygous FH in the Afrikaners, and Germany or England, where there are many more mutations. In clinical practice, a strategy for the genetic diagnosis of heterozygous FH, tailored to the mutational spectrum of patients likely to be seen at the particular hospital/region of the country, will be more efficient than screening of the whole LDL receptor gene by techniques such as single-strand conformation polymorphism (SSCP) analysis in every heterozygous FH candidate. In Aarhus, Denmark, we have chosen to examine all heterozygous FH candidates for the five most common LDL receptor gene mutations (W23X, W66G, W556S, 313 + 1G --> A, 1846 - 1G --> A) and the apoB-3500 mutation by rapid restriction fragment analysis. Negative samples are examined for other mutations by SSCP analysis followed by DNA sequencing of the exon indicated by SSCP to contain a mutation. If no point mutation or small insertion/deletion is detected, Southern blot or Long PCR analysis is performed to look for the presence of large gene rearrangements. In conclusion, our data suggest that an efficient molecular diagnostic strategy depends on the composition of common and rare mutations in a population.

Hattori H, Nagano M, Iwata F, Homma Y, Egashira T, Okada T
Identification of recurrent and novel mutations in the LDL receptor gene in Japanese familial hypercholesterolemia. Mutation in brief no. 248. Online.
Hum Mutat. 1999; 14: 87-87
Display abstract
We used the denaturing gradient gel electrophoresis (DGGE) method to investigate 120 Japanese patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequence of the low density lipoprotein (LDL) receptor gene. Fourteen aberrant DGGE patterns were found, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations (C317S, F382L A410T, L547V, and E693K), two nonsense mutations (W512X and K790X), four frameshift mutation (355del7, 1246ins5, 1687ins1, and 2035ins1), one splicing mutation (1845+2 T-->C), and two inframe mutations (661ins21 and 1115del9/ins6) were identified. Six of these mutations (L547V, E693K, W512X, 355del7, 1687ins1, and 20354ins1) have not been described before in FH. These newly identified mutations cosegregated in their family members with defective LDL receptor activity and hypercholesterolemia, and are thought to be causal for the FH phenotype. These results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Japanese population.

Marino G et al.
Rapid screening of the LDL receptor point mutation FH-Genoa/Palermo. Mutation in brief no. 238. Online.
Hum Mutat. 1999; 13: 412-412
Display abstract
The LDL-receptor gene point mutation FH-Genoa/Palermo is the most frequent mutation responsible for Familial Hypercholesterolemia in Sicily. The mutation does not introduce or abolish any useful restriction site. We establish a GeneComb-based strategy to identify this mutation in a population of Sicilian unrelated clinically diagnosed FH probands. The method was very sensitive and specific; 12 out of 90 (13.3%) unrelated FH probands were found to carry the FH-Genoa/Palermo mutation. According to these results, the FH-Genoa/Palermo is the more frequent LDL-receptor gene mutation among the Sicilian FH patients. Moreover FH-Genoa/Palermo is the mutation cluster to date more represented in Southern Italy.

Kajinami K
[Genotype-phenotype correlations in familial hypercholesterolemia]
Nippon Rinsho. 1999; 57: 2770-5
Display abstract
The low-density lipoprotein (LDL) receptor gene mutations cause familial hypercholesterolemia (FH), which was characterized by increased levels of LDL cholesterol and premature coronary atherosclerosis. Molecular genetic study of FH showed extreme heterogeneity in their underlying LDL receptor gene mutations, and suggested that this heterogeneity is responsible for the variability of its clinical manifestations. Since 1988, we have identified 11 mutations (10 novel and 1 previously reported) in the LDL receptor gene among 201 unrelated FH families in Hokuriku district, Japan. However, they explained only 38.8% of the patients suggesting that a more efficient mutation screening method should be developed. Through mutation detection, a genetically-determined mild type of FH (homozygotes with relative longevity and normocholesterolemic heterozygotes) was found in a case in which exons 2 and 3 were eliminated by a 10 kb deletion (Tonami-2). In addition, cholesterol-lowering drug therapy was significantly more effective in heterozygous patients with the P664L mutation (Kanazawa-2) compared to those with a 6 kb deletion including exon 15 (Tonami-1). These observations indicate that FH patients should be managed based on the results of their gene analyses.

Grunwald KA et al.
Identification of a novel Arg-->Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia in pigs.
J Lipid Res. 1999; 40: 475-85
Display abstract
We previously carried out genetic and metabolic studies in a partially inbred herd of pigs carrying cholesterol-elevating mutations. Quantitative pedigree analysis indicated that apolipoprotein (apo)B and a second major gene were responsible for the hypercholesterolemia in these animals. In this study, we assessed LDL receptor function by three different methods: ligand blots of liver membranes using beta-very low density lipoprotein (VLDL) as a ligand; low density lipoprotein (LDL)-dependent proliferation of T-lymphocytes; and direct binding of 125I-labeled LDL to cultured skin fibroblasts. All three methods demonstrated that LDL receptor ligands bound with decreased affinity to the LDL receptor in these animals. In skin fibroblasts from the hypercholesterolemic pigs, the Kd of binding was about 4-fold higher than in cells from normal pigs. The cDNA of the pig LDL receptor from normal and hypercholesterolemic pigs was isolated and sequenced. We identified a missense mutation that results in an Arg'Cys substitution at the position corresponding to Arg94 of the human LDL receptor. The mutation is in the third repeat of the ligand binding domain of the receptor. By single-stranded conformational polymorphism (SSCP) analysis, we studied the relationship between LDL receptor genotype and plasma cholesterol phenotype. In contrast to humans, the hypercholesterolemia associated with the LDL receptor mutation in pigs was expressed as a recessive trait. The LDL receptor mutation made a far more significant contribution to hypercholesterolemia than did the apoB mutation, consistent with observations made in human subjects with apoB mutations. Within each genotypic group (mutated apoB or mutated receptor), there was a wide range in plasma cholesterol. As the animals were on a well-controlled low-fat diet, this suggests that there are additional genetic factors that influence the penetrance of cholesterol-elevating mutations.

Norman D, Sun XM, Bourbon M, Knight BL, Naoumova RP, Soutar AK
Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia.
J Clin Invest. 1999; 104: 619-28
Display abstract
Familial hypercholesterolemia (FH) is characterized by a raised concentration of LDL in plasma that results in a significantly increased risk of premature atherosclerosis. In FH, impaired removal of LDL from the circulation results from inherited mutations in the LDL receptor gene or, more rarely, in the gene for apo B, the ligand for the LDL receptor. We have identified two unrelated clinically homozygous FH patients whose cells exhibit no measurable degradation of LDL in culture. Extensive analysis of DNA and mRNA revealed no defect in the LDL receptor, and alleles of the LDL receptor or apo B genes do not cosegregate with hypercholesterolemia in these families. FACS((R)) analysis of binding and uptake of fluorescent LDL or anti-LDL receptor antibodies showed that LDL receptors are on the cell surface and bind LDL normally, but fail to be internalized, suggesting that some component of endocytosis through clathrin-coated pits is defective. Internalization of the transferrin receptor occurs normally, suggesting that the defective gene product may interact specifically with the LDL receptor internalization signal. Identification of the defective gene will aid genetic diagnosis of other hypercholesterolemic patients and elucidate the mechanism by which LDL receptors are internalized.

Hopkins PN et al.
A novel LDLR mutation, H190Y, in a Utah kindred with familial hypercholesterolemia.
J Hum Genet. 1999; 44: 364-7
Display abstract
Heterozygous familial hypercholesterolemia (FH) is a serious disorder causing twice normal low-density lipoprotein (LDL) cholesterol levels early in childhood and very early coronary disease in both men and women. Treatment with multiple medications together with diet can normalize cholesterol levels in many persons with FH and prevent or delay the development of coronary atherosclerosis. Previously published blood cholesterol criteria greatly under-diagnosed new cases of FH among members of known families with FH and over-diagnosed FH among participants of general population screening. Thus, there is a need for accurate and genetically validated criteria for the early diagnosis of heterozygous FH. In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To carry out molecular genetic diagnosis of the disease, we screened DNA samples for mutations in all 18 exons and the exon-intron boundaries of the LDL receptor gene (LDLR). Novel point mutations were identified in the proband: a C-to-T transversion at nucleotide position 631, causing substitution of tyrosine for histidine at codon 190 in exon 4 of the LDLR gene. The mutant allele-specific amplification method was used to examine 12 members of the family recruited for the diagnosis. This method helped to unequivocally diagnose 7 individuals as heterozygous for this particular LDLR mutation, while excluding the remaining 5 individuals from carrier status with FH.

Loubser O et al.
Founder mutations in the LDL receptor gene contribute significantly to the familial hypercholesterolemia phenotype in the indigenous South African population of mixed ancestry.
Clin Genet. 1999; 55: 340-5
Display abstract
The South African population harbors genes that are derived from varying degrees of admixture between indigenous groups and immigrants from Europe and the East. This study represents the first direct mutation-based attempt to determine the impact of admixture from other gene pools on the familial hypercholesterolemia (FH) phenotype in the recently founded Coloured population of South Africa, a people of mixed ancestry. A cohort of 236 apparently unrelated patients with clinical features of FH was screened for a common mutation causing familial defective apolipoprotein B-100 (FDB) and seven low-density lipoprotein receptor (LDLR) gene defects known to be relatively common in South Africans with FH. Six founder-type 'South African mutations' were responsible for FH in approximately 20% of the study population, while only 1 patient tested positive for the familial defective apolipoprotein B-100 mutation R3500Q. The detection of multiple founder-type LDLR gene mutations originating from European, Indian and Jewish populations provides direct genetic evidence that Caucasoid admixture contributes significantly to the apparently high prevalence of FH in South African patients of mixed ancestry. This study contributes to our knowledge of the biological history of this unique population and illustrates the potential consequences of recent admixture in populations with different disease risks.

Alberto FL, Figueiredo MS, Zago MA, Araujo AG, Dos-Santos JE
The Lebanese mutation as an important cause of familial hypercholesterolemia in Brazil.
Braz J Med Biol Res. 1999; 32: 739-45
Display abstract
Familial hypercholesterolemia (FH) is a common autosomal disorder that affects about one in 500 individuals in most Western populations and is caused by a defect in the low-density-lipoprotein receptor (LDLr) gene. In this report we determined the molecular basis of FH in 59 patients from 31 unrelated Brazilian families. All patients were screened for the Lebanese mutation, gross abnormalities of the LDLr gene, and the point mutation in the codon 3500 of the apolipoprotein B-100 gene. None of the 59 patients presented the apoB-3500 mutation, suggesting that familial defective ApoB-100 (FDB) is not a major cause of inherited hypercholesterolemia in Brazil. A novel 4-kb deletion in the LDLr gene, spanning from intron 12 to intron 14, was characterized in one family. Both 5' and 3' breakpoint regions were located within Alu repetitive sequences, which are probably involved in the crossing over that generated this rearrangement. The Lebanese mutation was detected in 9 of the 31 families, always associated with Arab ancestry. Two different LDLr gene haplotypes were demonstrated in association with the Lebanese mutation. Our results suggest the importance of the Lebanese mutation as a cause of FH in Brazil and by analogy the same feature may be expected in other countries with a large Arab population, such as North American and Western European countries.

Dantuma NP et al.
An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins.
J Lipid Res. 1999; 40: 973-8
Display abstract
A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expressed in locust oocytes, fat body, brain, and midgut. This receptor appeared to be a homolog of the mammalian very low density lipoprotein receptor as it contains eight cysteine-rich repeats in its putative ligand-binding domain. When transiently expressed in COS-7 or stably expressed in LDL receptor-deficient CHO cells, the receptor mediates endocytic uptake of high density lipophorin (HDLp), an abundant lipoprotein in the circulatory compartment of insects. Moreover, in the latter cell line, we demonstrated that an excess of unlabeled HDLp competed with fluorescent labeled HDLp for uptake whereas an excess of human LDL did not affect uptake. Expression of the receptor mRNA in fat body cells is down-regulated during adult development, which is consistent with the previously reported down-regulation of receptor-mediated endocytosis of lipophorins in fat body tissue (Dantuma, N. P., M.A.P. Pijnenburg, J. H. B. Diederen, and D. J. Van der Horst. 1997. J. Lipid Res. 38: 254-265). The expression of this receptor in various tissues that internalize circulating lipophorins and its capability to mediate endocytosis of HDLp indicate that this novel member of the LDL receptor family may function as an endocytic lipophorin receptor in vivo.

North CL, Blacklow SC
Structural independence of ligand-binding modules five and six of the LDL receptor.
Biochemistry. 1999; 38: 3926-35
Display abstract
The low-density lipoprotein receptor (LDLR) is the primary mechanism for the uptake of plasma cholesterol into cells and serves as a prototype for a growing family of cell surface receptors. These receptors all utilize tandemly repeated LDL-A modules to bind their ligands. Each LDL-A module is about 40 residues long, has six conserved cysteine residues, and contains a conserved acidic region near the C-terminus which serves as a calcium-binding site. The structure of the interface presented for ligand binding by these modules, and the basis for their specificity and affinity in ligand binding, is not yet known. We have purified recombinant molecules corresponding to LDL-A modules five (LR5), six (LR6), and the module five-six pair (LR5-6) of the LDL receptor. Calcium is required to establish native disulfide bonds and to maintain the structural integrity of LR5, LR6, and the LR5-6 module pair. Folding studies of the I189D and D206Y mutations within LR5 indicate that each change leads to misfolding of the module, explaining the previous observation that each of these changes mimics the functional effect of deletion of the entire module [Russell, D. W., Brown, M. S., and Goldstein, J. L. (1989) J. Biol. Chem. 264, 21682-21688]. By fluorescence, the affinity of LR5 for calcium, which is crucial for folding and function of these modules, remains approximately 40 nM whether LR6 is attached. Comparison of proton and multidimensional heteronuclear NMR spectra of individual modules to those of the module pair indicates that most of the significant spectroscopic changes lie within the linker region between modules and that little structural interaction occurs between the cores of modules five and six in the 5-6 pair. These findings strongly support a model in which each module is essentially structurally independent of the other.

Huang W, Dolmer K, Gettins PG
NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein.
J Biol Chem. 1999; 274: 14130-6
Display abstract
The low density lipoprotein receptor-related protein is a member of the low density lipoprotein receptor family and contains clusters of cysteine-rich complement-like repeats of about 42 residues that are present in all members of this family of receptors. These clusters are thought to be the principal binding sites for protein ligands. We have expressed one complement-like repeat, CR8, from the cluster in lipoprotein receptor-related protein that binds certain proteinase inhibitor-proteinase complexes and used three-dimensional NMR on the 13C/15N-labeled protein to determine the structure in solution of the calcium-bound form. The structure is very similar in overall fold to repeat 5 from the low density lipoprotein receptor (LB5), with backbone root mean square deviation of 1.5 A. The calcium-binding site also appears to be homologous, with four carboxyl and two backbone carbonyl ligands. However, differences in primary structure are such that equivalent surfaces that might represent the binding interfaces are very different from one another, indicating that different domains will have very different ligand specificities.

Freiberger T et al.
[Direct detection of mutations in the LDL receptor gene in patients with familial hypercholesterolemia]
Cas Lek Cesk. 1998; 137: 750-2
Display abstract
BACKGROUND: Familial hypercholesterolemia is one of the most frequent hereditary metabolic diseases. As a result of the functional disorder of the molecule of the LDL receptor LDL cholesterol is not sufficiently eliminated from the blood stream and exerts an atherogenic effect. The objective of the study was to introduce direct detection of mutations in the gene for the LDL receptor and characterize the spectrum of mutations in the Czech population. METHODS AND RESULTS: The authors analyzed a group of 84 unrelated patients where on the basis of clinical and biochemical criteria the diagnosis of FH was established. From the group 12 patients were eliminated (14.3%) where a mutation 3500 in the gene for apolipoprotein (apo) B-100 was detected. This mutation is most frequently the cause of a familial defect of apo B-100 (FDB), which cannot be differentiated clinically or biochemically from FH. In the LDL receptor gene a total of 11 mutations were found in 14 unrelated patients (16.7%), incl. 7 mutations not described hitherto. CONCLUSIONS: This is the first systematic characteristic of the spectrum of mutations in the LDL receptor gene in the Czech population. Molecular genetic analysis of the gene for the LDL receptor in affected families can contribute towards early assessment of the diagnosis of FH and thus to prevention of life threatening cardiovascular episodes in asymptomatic subjects.

Nissen H, Lestavel S, Hansen TS, Luc G, Bruckert E, Clavey V
Mutation screening of the LDLR gene and ApoB gene in patients with a phenotype of familial hypercholesterolemia and normal values in a functional LDL receptor/apolipoprotein B assay.
Clin Genet. 1998; 54: 79-82
Display abstract
Mutations in the LDL receptor (LDLR) or the apolipoprotein B-100 genes causing familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB), two of the most frequent inherited diseases, are the underlying genetic defects in a small proportion of patients suffering from premature atherosclerotic heart disease. Consequently, secure diagnostic tools for these conditions allowing early preventive measures are needed. Since clinical and biochemical diagnosis often is inaccurate, assays analyzing patient LDLR function and LDL affinity have been established. These assays are, however, not able clearly to differentiate between suspected FH/FDB samples and normal controls. To evaluate if this may be caused by other hitherto undescribed genetic defects or to failure of the functional assays, we undertook denaturing gradient gel electrophoresis based mutation screening of the LDLR gene and the codon 3456 3553 region of the apolipoprotein B gene in six French FH/FDB patients with normal outcomes on functional assays. In all six patients, pathogenic LDLR mutations were found, including three previously undescribed mutations, suggesting that failure of the functional assays explains the normal results found in some phenotypic FH/FDB patients and illustrating the need for DNA based screening techniques for routine genetic diagnosis in FH/FDB.

Morash BA, Tan MH, Nassar BA, Too CK, Guernsey DL
A novel mutation in Exon 4 of the low density lipoprotein receptor gene resulting in heterozygous familial hypercholesterolemia associated with decreased ligand binding.
Atherosclerosis. 1998; 136: 9-16
Display abstract
Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low density lipoprotein (LDL) receptor gene. Currently, diagnosis of heterozygous FH relies on clinical phenotype; however, the use of clinical criteria for the diagnosis of heterozygous FH does not always permit unequivocable diagnosis of the disease. Molecular diagnosis of FH is clinically valuable especially in regions where founder mutations exist or where polygenic hypercholesterolemia is prevalent. In this paper we report the identification of a novel mutation, a cytosine to guanine substitution, at codon 152 in exon 4 of the LDL receptor gene in a Nova Scotian family clinically diagnosed with heterozygous FH. The mutation creates a recognition sequence for the restriction endonuclease BsrI, and can be readily detected by BsrI restriction analysis of a 160 bp amplicon spanning the mutation. This analysis was used to show that the mutation segregated with the disease in this family and is the probable cause of FH in this kindred.

Arca M, Jokinen E
Low density lipoprotein receptor mutations in a selected population of individuals with moderate hypercholesterolemia.
Atherosclerosis. 1998; 136: 187-94
Display abstract
To evaluate mutations in the low density lipoprotein receptor (LDL-R) gene in moderate primary hypercholesterolemia, a combination of polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP) and direct sequencing, was used to screen the LDL-R gene in a selected population of 82 unrelated individuals with moderate elevation of plasma LDL-C [mean 4.55 +/- 0.55 mmol/l (176.4 +/- 21.6 mg/dl)]. Four subjects (5%) were found to be heterozygotes for missense mutations in the LDL-R gene. These mutations were located in four different exons (exons 6, 7, 15 and 17) and all alters highly conserved residues of LDL-R protein. None of these mutations were detected in 79 normocholesterolemic individuals. The mutation in exon 15 (T705I) was previously reported in a compound heterozygote for familial hypercholesterolemia (FH). In the proband carrying the mutation in exon 17 (R793Q), an in vivo LDL turnover study was performed and it demonstrated a reduction of LDL catabolism. These findings demonstrate that mutations in the LDL-R may occur in primary moderate hypercholesterolemia. They also extend the concept that some FH patients may present with a mild phenotype.

Cenarro A et al.
Identification of recurrent and novel mutations in the LDL receptor gene in Spanish patients with familial hypercholesterolemia. Mutations in brief no. 135. Online.
Hum Mutat. 1998; 11: 413-413
Display abstract
We used the single strand conformation polymorphism (SSCP) method to investigate 13 apparently unrelated Spanish patients with familial hypercholesterolemia (FH) for mutations in the promoter region and the 18 exons and their flanking intron sequences of the low density lipoprotein (LDL) receptor gene. We found 16 aberrant SSCP patterns, and the underlying mutations were characterized by DNA sequencing. Five novel missense mutations, Q71E, C74G, C95R, C281Y and D679E, and one nonsense mutation, Q133X, were identified. We also found six missense mutations, S156L, D200Y, D200G, E256K, T413K and C646Y, and one stop codon mutation, W(-18)X, that were previously described in patients from other populations. A new frameshift mutation, 2085del19, was found in one patient. We also identified three splicing mutations; two of them are novel mutations, 1706-10G->A and 2390-1G->A, and the other one has been reported recently, 313+1G->C. Four patients were found to carry two different mutations in the same allele: Q71E and 313+1G->C; C95R and D679E; W(-18)X and E256K, and C281Y and 1706-10G->A. Our results demonstrate that there is a broad spectrum of mutations in the LDL receptor gene in the Spanish population.

Chakir K, Skobeleva NA, Shevtsov SP, Konstantinov VO, Denisenko AD, Schwartz EI
Two novel slavic point mutations in the low-density lipoprotein receptor gene in patients with familial hypercholesterolemia from St. Petersburg, Russia.
Mol Genet Metab. 1998; 63: 31-4
Display abstract
Using PCR-single-strand conformation polymorphism analysis, followed by sequencing of the abnormal samples, two novel point mutations in the 5' end of the fourth exon of the low-density lipoprotein receptor gene were found in two Russian families with familial hypercholesterolemia. These missense mutations consist of C127W and C139G transitions and result in a loss of one of three disulfide bonds in the fourth cysteine-rich repeat of the ligand-binding domain of the low-density lipoprotein receptor. Hypercholesterolemia segregated with the identified mutations.

Raungaard B, Heath F, Brorholt-Petersen JU, Jensen HK, Faergeman O
Flow cytometry with a monoclonal antibody to the low density lipoprotein receptor compared with gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia.
Clin Chem. 1998; 44: 966-72
Display abstract
We used a fluorescence flow cytometry assay with a monoclonal low density lipoprotein (LDL) receptor-specific antibody to detect LDL receptor expression on blood T lymphocytes and monocytes. We prepared peripheral blood mononuclear cells from patients with genetically verified LDL receptor-defective (Trp66-Gly mutation, n = 17) or receptor-negative (Trp23-stop mutation, n = 17) heterozygous familial hypercholesterolemia (FH) and from healthy individuals (n = 24). The cells were stimulated to express the maximum amount of LDL receptor by preincubation in lipoprotein-deficient medium. A dual-labeling technique allowed flow cytometric analysis of LDL receptor expression on cells identified by fluorescently conjugated surface marker antibodies. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of this functional assay with the DNA diagnosis and to validate the assay with molecular genetics instead of clinical indices of heterozygous FH. T lymphocytes expressed more LDL receptors and gave better diagnostic results than monocytes, and cells from patients with either the Trp66-Gly or the Trp23-stop mutation had variable but significantly reduced LDL receptor expression. The data indicate that this fluorescence flow cytometry assay is unsuitable for diagnosis of individual cases of heterozygous FH but that it may be useful for functionally characterizing mutations in the LDL receptor gene.

Patel DD et al.
Analysis of two duplications of the LDL receptor gene affecting intracellular transport, catabolism, and surface binding of the LDL receptor.
J Lipid Res. 1998; 39: 1466-75
Display abstract
Two novel mutations of the low density lipoprotein (LDL)-receptor gene were found in two Italian familial hypercholesterolemia (FH)-heterozygotes. The first mutation was an 18 nucleotide duplication in exon 8 which is preceded by an A-->T transversion. The translation product of the mutant allele was predicted to be a receptor with an in-frame insertion of 6 amino acids in repeat B of the epidermal growth factor precursor homology domain. Analysis of LDL-receptor activity in the proband's fibroblasts showed a 50% reduction of 125I-labeled LDL binding and pulse-chase studies suggested that little, if any, of the mutant protein was processed to the mature form. The second mutation was a 7 kb duplication (from intron 2 to intron 6) of exons 3 through 6, predicted to encode an elongated receptor with the duplication of repeats 2-7 of the ligand binding domain. The elongated receptor was processed slightly more slowly than the normal receptor, but was converted to a mature form of the expected size. This mature, mutant receptor was degraded more rapidly than the normal receptor. On ligand blotting the elongated receptor bound twice as much LDL or beta-very low density lipoprotein (betaVLDL) as the normal receptor. In contrast, maximum binding of LDL to proband's cells was decreased to approximately 70% of the normal cells with a significant increase in apparent affinity. Cell association at 37 degrees C, internalization, and degradation showed a similar reduced maximum. Thus these mutations demonstrate that duplications of amino acid sequences in the low density lipoprotein LDL-receptor may disrupt the LDL-receptor pathway at different levels.

Goel R, Kaul D
Molecular control of the LDL receptor gene in leukemic cells.
Leuk Res. 1998; 22: 89-92

Tashiro J, Endo M, Bujo H, Shinomiya M, Morisaki N, Saito Y
New type of the internalization-defective low-density lipoprotein receptor owing to two-nucleotide deletion (2199delCA or 2201delCA) in Japanese patients with familial hypercholesterolaemia.
Eur J Clin Invest. 1998; 28: 712-9
Display abstract
BACKGROUND: In mutations of the low-density lipoprotein (LDL) receptor gene, the defect of internalization is caused by a mutation in the cytoplasmic domain of the receptor linked with exons 17 and 18, and the O-linked sugar domain linked with exon 15 has been speculated not to affect the function of the receptor. Here, we describe a novel mutation of the O-linked sugar domain of the LDL receptor gene, designated familial hypercholesterolaemia (FH)-Mishima with Japanese pedigree, which resembles but still differs from classical defective internalization cases. METHODS: LDL metabolism was examined in cultured skin fibroblasts from patients. Immunoprecipitation and immunohistochemical techniques were applied for the detection of the receptor protein size and distribution. Screening of the mutant exon(s) of the LDL receptor gene was performed using the polymerase chain reaction-single-strand conformation polymorphism technique (PCR-SSCP), and sequencing of the mutated alleles was carried out using the dideoxy chain termination method. RESULTS: LDL-binding activity at 4 degrees C in skin fibroblasts from patients was similar to normal, but that at 37 degrees C with the ligand decreased time dependently and was lost at 6 h, resulting in the defect of internalization and degradation of LDL. The receptor protein on the cell surface was detected at 4 degrees C by IgG-C7, an anti-LDL receptor antibody, but was not detected after incubation with LDL at 37 degrees C. The size of the receptor was 112 kD as determined by immunoprecipitation analysis. A deletion of two nucleotides in exon 15 was detected in the DNA sequence of the LDL receptor gene. The deletion results in a shift of the reading frame after Thr-713 of the mutant and makes a stop codon at amino acid 759. CONCLUSION: Deletion of the two nucleotides caused novel amino acid sequences after the O-linked sugar domain, which has the ability of sorting on the cell membrane at 4 degrees C, but not at 37 degrees C in vivo, resulting in the complete cessation of activity of the LDL receptor.

Rong L, Gendron K, Bates P
Conversion of a human low-density lipoprotein receptor ligandbinding repeat to a virus receptor: identification of residues important for ligand specificity.
Proc Natl Acad Sci U S A. 1998; 95: 8467-72
Display abstract
The amino-terminal region of the low-density lipoprotein receptor (LDLR) contains seven imperfect repeats of a cysteine-rich, roughly 40-aa module (LDL-A module) that are critical for apolipoprotein binding. LDL-A modules are found in numerous cell-surface and secreted proteins and are believed to mediate extracellular protein-protein interactions. The cellular receptor for subgroup A Rous sarcoma virus (RSV) contains a single LDL-A module that binds the RSV envelope protein and allows viral infection. To define residues in an LDL-A module responsible for ligand recognition, we used a gain of function assay by using a chimeric protein in which the LDL-A module of Tva was replaced with a highly homologous module from human LDLR (LDL-A4) and determined whether this chimera or mutants produced in it could mediate RSV infection. LDL-A4 does not function as an RSV receptor; however, systematic replacement of the nonconserved residues of the LDL-A4 module in the chimeric protein with the corresponding residues from Tva identified three residues sufficient to alter ligand specificity and convert LDL-A4 to an efficient viral receptor. Mutations of the corresponding residues in the Tva LDL-A module decreased both envelope binding and viral receptor function, confirming the importance of these residues in ligand recognition by this module. Analysis of the hLDL-A5 structure demonstrates that these three residues are clustered at one end of the LDL-A module. These results demonstrate that using a single LDL-A module in a gain of function assay is a useful model to investigate ligand recognition by this module.

Emi M et al.
Familial hypercholesterolemia kindred in Utah with novel C54S mutations of the LDL receptor gene.
Jpn Heart J. 1998; 39: 785-9
Display abstract
In the course of investigations of coronary artery disease in Utah, we identified a family whose proband showed elevated plasma levels of LDL cholesterol. To determine the genetic etiology of the lipoprotein abnormalities, we screened DNA samples for mutations in all 18 exons and the exon- intron boundaries of the low-density lipoprotein (LDL) receptor gene. Novel point mutations were identified in the proband: a T-to-A transversion at nucleotide position 223, causing substitution of Ser for Cys at codon 54 in exon 3 of the receptor gene. This amino acid replacement would disrupt one of the disulfide bonds necessary for maintenance of the secondary structure of the repeat at the N-terminal of the receptor, prevent correct folding of the receptor, and result in defective intracellular transport of the receptor.

Tomita Y, Kim DH, Magoori K, Fujino T, Yamamoto TT
A novel low-density lipoprotein receptor-related protein with type II membrane protein-like structure is abundant in heart.
J Biochem (Tokyo). 1998; 124: 784-9
Display abstract
We report herein the identification of a novel member of the low-density lipoprotein receptor (LDLR) family termed LDLR-related protein 4 (LRP4). Murine LRP4 cDNA encodes a 1113-amino-acid type II membrane-like protein with eight ligand-binding repeats in two clusters. Southern blot analysis of genomic DNA from several different organisms suggests the presence of LRP4 homologues in chicken lacking the gene encoding apolipoprotein E, which is recognized by the ligand-binding repeats of LDLR. LRP4 transcripts were detected almost exclusively in heart in mouse and humans. Despite the presence of the ligand-binding repeats, COS cells transfected with LRP4 did not show surface-binding of beta-migrating very-low-density lipoprotein, suggesting that LRP4 plays a role in a pathway other than lipoprotein metabolism.

Wilson DJ et al.
A World Wide Web site for low-density lipoprotein receptor gene mutations in familial hypercholesterolemia: sequence-based, tabular, and direct submission data handling.
Am J Cardiol. 1998; 81: 1509-11
Display abstract
Familial hypercholesterolemia is an autosomal dominant inherited condition characterized by a mutation in the low-density lipoprotein receptor (LDLR) gene. A database has been set up on the World Wide Web for mutations in the LDLR gene.

Soutar AK
Update on low density lipoprotein receptor mutations.
Curr Opin Lipidol. 1998; 9: 141-7
Display abstract
Recent research has focused on the rapid detection of new LDL receptor gene variants and large scale screening for known mutations. Whether the nature of the mutation in the LDL receptor gene in familial hypercholesterolaemia determines clinical variability has been examined, as well as the potential value of detecting mutation carriers for clinical practice. There is also evidence that some patients with clinical familial hypercholesterolaemia do not have detectable defects in the LDL receptor or apolipoprotein B.

Hasler-Rapacz J et al.
Identification of a mutation in the low density lipoprotein receptor gene associated with recessive familial hypercholesterolemia in swine.
Am J Med Genet. 1998; 76: 379-86
Display abstract
Elevated blood plasma cholesterol (hypercholesterolemia) is a major risk factor for coronary artery disease (CAD) in humans. Genetic dissection of polygenic lipid and lipoprotein disorders in swine, a key animal model for the study of familial hypercholesterolemia (FH) and CAD, led to the isolation of a monogenic subphenotype (FH-r), that is inherited in the recessive (r) manner. A genome scan mapped the FH-r locus close to the centromere of chromosome 2. Comparative mapping showed that this region shares homology with a part of human chromosome 19 that harbors the low density lipoprotein receptor (LDLR) locus, and therefore suggested LDLR as the prime candidate gene for FH-r. Cloning and sequencing of hepatic LDLR cDNA from two FH-r/r and one normal (N/N) animals disclosed a single missense mutation (R84C) in a region that corresponds to human exon 4. The C84 mutation cosegregates invariantly with hypercholesterolemia, which strongly suggests that this mutation is responsible for the observed hyperlipidemia.

Bieri S, Atkins AR, Lee HT, Winzor DJ, Smith R, Kroon PA
Folding, calcium binding, and structural characterization of a concatemer of the first and second ligand-binding modules of the low-density lipoprotein receptor.
Biochemistry. 1998; 37: 10994-1002
Display abstract
The ligand-binding domain of the low-density lipoprotein (LDL) receptor is comprised of seven tandemly repeated ligand-binding modules, each being approximately 40 amino acids long and containing six conserved cysteine residues. We have expressed and characterized a concatemer of the first two modules (LB1 and LB2) of the human LDL receptor. Oxidative folding of the recombinant concatemer (rLB1-2), in the presence of calcium ions, gave a single dominant isomer with six disulfide bonds. Peptic cleavage of the short linker region that connects the last cysteine residue of LB1 and the first cysteine residue of LB2 yielded two discrete fragments, thus excluding the presence of intermodule disulfide bonds. The N-terminal module, LB1, reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDL receptor. From this, we concluded that the first module was correctly folded, with the same set of disulfide bonds as LB1 of the LDL receptor. The disulfide bond connections of LB2 were identified from mass spectral analysis of fragments formed by digestion of the C-terminal peptic fragment with elastase. These data showed that the disulfide bonds of LB2 connected Cys(I) and Cys(III), Cys(II) and Cys(V), and Cys(IV) and Cys(VI). This pattern is identical to that found for recombinant LB1 and LB2. The concatemer has two high-affinity calcium-binding sites, one per module. An analysis of the secondary chemical shifts of Calpha protons shows that the conformations of LB1 and LB2 in the concatemer are very similar to those of the individual modules, with no evidence for strong interactions between the two modules.

Atkins AR, Brereton IM, Kroon PA, Lee HT, Smith R
Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor.
Biochemistry. 1998; 37: 1662-70
Display abstract
Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.

Bajari TM et al.
A minimal binding domain of the low density lipoprotein receptor family.
Biol Chem. 1998; 379: 1053-62
Display abstract
As more relatives of the low density lipoprotein receptor (LDLR) are discovered, defining their minimal binding domain(s) becomes a challenge. Here we have chosen the multifunctional chicken oocyte receptor for yolk deposition (termed LR8), and the pan-receptor ligand, receptor associated protein (RAP), as model systems to characterize a minireceptor using the phage display approach. Displayed fragments derived from the entire 819 residue LR8 molecule, followed by selection via panning on RAP, led to the definition of an 80 residue stretch LR8 minireceptor. It contains 12 cysteines, and represents parts of the second, the entire third, and parts of the fourth, of the eight clustered 'ligand binding repeats' in LR8; only two of the eight stretches of negatively charged residues of LR8, i.e., EDGSDE and DSGEDEE, are present. The latter sequence is reminiscent of that in the fifth repeat of the human LDLR, thought to be most critical for interaction with positive charge clusters in ligands. Baculovirus-mediated expression of the soluble minireceptor in insect cells showed it to fold as a monomer, and sulfhydryl-reduction-sensitive interaction with RAP was demonstrated for immobilized as well as soluble minireceptor. Furthermore, the LR8-derived minireceptor provided a RAP-responsive surface when covalently coupled to the surface of a gold electrode. In addition to its use in defining minimal binding domains, the phage display approach provides powerful tools for dissection, and consequently, manipulation, of the function of receptors so as to direct their binding activity toward ligands of diagnostic and/or therapeutic interest.

Mak YT et al.
Mutations in the low-density lipoprotein receptor gene in Chinese familial hypercholesterolemia patients.
Arterioscler Thromb Vasc Biol. 1998; 18: 1600-5
Display abstract
It has been reported that in China, patients with heterozygous familial hypercholesterolemia (FH) may go unrecognized because they do not have xanthomata or premature coronary heart disease and their LDL cholesterol levels are lower than those in their Western counterparts. However, in the Chinese patients in Hong Kong, heterozygous FH appears to manifest in a way similar to that seen in Western countries or Japan. We studied sequence variations in the promoter and coding regions of the 18 exons of the LDL receptor gene in 30 Chinese FH patients. Eighteen mutations were identified in 21 patients scattered in the promoter and 10 exons. Eleven of them were first found in this study. We also found 6 polymorphisms with allelic frequencies different from those in whites but similar to the Japanese, indicating some isolation between white and Oriental populations. A total of 29 mutations in the LDL receptor gene are now known in the Chinese. There is no definite common mutation due to a founder effect. Meanwhile, there were no detectable LDL receptor gene mutations in 9 clinically diagnosed FH patients in whom the R3500Q mutation in apolipoprotein B had also been excluded. The gene defects leading to the FH phenotype in these patients may occur somewhere else in the apolipoprotein B or other related genes, or even in the noncoding sequences of the LDL receptor gene.

Vallve JC, Alonso-Villaverde C, Ribalta J, Perez-Jimenez F, Masana L
Exon 4, and in particular codon 152 of the LDL receptor gene, is a hot spot for point mutations.
Atherosclerosis. 1998; 140: 191-2

Runova OL, Mandelshtam MJu, Golubkov VI, Sukonina VE, Denisenko AD, Gaitskhoki VS
Expression of cDNA fragment encoding ligand-binding domain of human low density lipoprotein receptor in Escherichia coli cells.
Biochemistry (Mosc). 1997; 62: 890-7
Display abstract
To study structure-function relationships in low density lipoprotein receptor (LDLR), a key protein in human cholesterol metabolism, it is reasonable to operate with separate protein domains. To obtain highly purified functionally active LDLR ligand-binding domain, we have cloned the corresponding LDLR cDNA fragment in two expression plasmid vectors of Escherichia coli. We have developed methods to purify fusion and practically individual recombinant proteins and characterized the obtained products biochemically. Antibodies raised against fused with beta-galactosidase and individual recombinant protein have been shown to be efficient in identification of LDLR protein in crude lysates of human fibroblasts (cell line HT-1080).

Jensen HK et al.
A common W556S mutation in the LDL receptor gene of Danish patients with familial hypercholesterolemia encodes a transport-defective protein.
Atherosclerosis. 1997; 131: 67-72
Display abstract
In a group of unrelated Danish patients with familial hypercholesterolemia (FH) we recently reported two common low-density lipoprotein (LDL) receptor mutations, W23X and W66G, accounting for 30% of the cases. In this study, we describe another common LDL receptor mutation, a G to C transition at cDNA position 1730 in exon 12, causing a tryptophan to serine substitution in amino acid position 556 (W556S). In the Danish patients, the W556S mutation was present in 12% of 65 possible mutant alleles. The pathogenicity of the W556S mutation, which is located in one of the five conserved motifs Tyr-Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein in the endoplasmic reticulum. The transport-defective W556S mutation and the W23X and W66G mutations seem to account for about 40% of the LDL receptor defects in Danish families with FH.

Vergopoulos A et al.
A xanthomatosis-susceptibility gene may exist in a Syrian family with familial hypercholesterolemia.
Eur J Hum Genet. 1997; 5: 315-23
Display abstract
Familial hypercholesterolemia (FH) is an autosomal-dominant inherited disorder characterized by high serum low-density lipoprotein (LDL)-cholesterol concentrations, xanthoma formation, and premature atherosclerosis. Homozygous individuals die of vascular disease as children or young adults; heterozygous persons are at high risk for premature cardiovascular death. Mutations in the LDL-receptor gene are responsible for FH. We studied 49 members of a consanguineous Syrian kindred containing 6 homozygous individuals from the same pedigree. Half of the homozygotes had giant xanthomas, while half did not, even though their LDL-cholesterol concentrations were elevated to similar degrees (> 14 mmol/l). Heterozygous FH individuals from this family were also clearly distinguishable with respect to xanthoma size. We performed DNA analysis and were successful in identifying a hitherto not described mutation in this family's LDL receptor. DNA sequence analysis of the LDL-receptor gene revealed a T to C substitution at nucleotide 1,999 in codon 646 of exon 14. We next conducted a segregation analysis, which suggests that a susceptibility gene may explain the formation of giant xanthomas in this family. We raise the hypothesis that the appearance of giant xanthomas in this FH family is controlled by a second gene acting in an autosomal-dominant or recessive fashion. Elucidation of this 'xanthoma' gene may shed additional light on LDL-cholesterol deposition.

Kotze MJ et al.
CpG hotspot mutations at the LDL receptor locus are a frequent cause of familial hypercholesterolaemia among South African Indians.
Clin Genet. 1997; 51: 394-8
Display abstract
Mutation analysis of genomic DNA samples obtained from seven unrelated South African Indians with familial hypercholesterolaemia (FH) revealed two novel and two recurrent missense mutations in the low density lipoprotein receptor (LDLR) gene. The novel mutations are transversions of C to G and A to T at nucleotide positions 1215 (N384K) and 2356 (S765C), respectively. The known mutations were detected in CpG dinucleotides at bases 661 and 682, respectively, in the mutation-rich exon 4 of the LDLR gene. Mutation D200Y was found in a single FH family, while mutation E207K was detected in two apparently unrelated Indian families on a new mutual haplotype. Analysis of published mutations including our new data has shown that more than 50% of the different LDLR gene mutations identified to date in South African Indians occur at CpG hotspots.

Bujo H, Saito Y
A new member with eleven binding repeats: novel insights into the LDL receptor gene family.
Ann N Y Acad Sci. 1997; 811: 185-90

Varret M, Rabes JP, Collod-Beroud G, Junien C, Boileau C, Beroud C
Software and database for the analysis of mutations in the human LDL receptor gene.
Nucleic Acids Res. 1997; 25: 172-80
Display abstract
The low-density lipoprotein receptor (LDLr) plays a pivotal role in cholesterol homeostasis. Mutations in the LDLr gene (LDLR), which is located on chromosome 19, cause familial hypercholesterolemia (FH), an autosomal dominant disorder characterized by severe hypercholesterolemia associated with premature coronary atherosclerosis. To date almost 300 mutations have been identified in the LDLR gene. To facilitate the mutational analysis of the LDLR gene, and promote the analysis of the relationship between genotype and phenotype, a software package along with a computerized database (currently listing 210 entries) have been created.

Urdal P, Leren TP, Tonstad S, Lund PK, Ose L
Flow cytometric measurement of low density lipoprotein receptor activity validated by DNA analysis in diagnosing heterozygous familial hypercholesterolemia.
Cytometry. 1997; 30: 264-8
Display abstract
We have evaluated whether low density lipoprotein (LDL) receptor activity of stimulated lymphocytes, as measured by an improved flow cytometric assay, may be used to diagnose familial hypercholesterolemia (FH). Cells were isolated from 75 children suspected from strict clinical criteria to be FH heterozygotes and from 29 normal children. DNA from the FH patients were also subjected to molecular genetic analysis of the LDL receptor gene in order to confirm the clinical diagnosis. A molecular genetic diagnosis of FH was obtained in 68 of the 75 patients; 67 of these had a low (below 70% of normal) receptor activity and 1 had a borderline (71%) activity. By contrast, 28 of the normal children showed a normal (above 80%) and 1 a borderline (78%) receptor activity. Of the 7 patients in whom no mutation in the LDL receptor gene was found, 4 showed a normal, 1 a borderline, and 2 showed a low activity. In summary, measurement of LDL receptor activity allowed us to separate between genetically diagnosed FH heterozygotes and healthy children. The combined use of LDL receptor activity measurements and molecular genetic analysis allows us both to diagnose and exclude FH in children suspected to suffer from this disease.

Kotze MJ et al.
Mutation analysis reveals an insertional hotspot in exon 4 of the LDL receptor gene.
Hum Genet. 1996; 98: 476-8
Display abstract
Mutation analysis of the low density lipoprotein receptor (LDLR) gene revealed a novel 8-bp duplication after nucleotide 681 in a Costa Rican patient with familial hypercholesterolaemia. The frameshift caused by this mutation results in a premature termination codon in the EGF precursor homology domain of the mature LDLR, whereby a truncated protein of the first 206 residues with an additional 39 abnormal residues would be created. The insertion overlaps with previously described duplications of 18 bp and 21 bp, thus revealing an insertional hotspot in exon 4 of the LDLR gene. We propose that the structural features of this region of the LDLR gene contribute significantly to genetic instability and the subsequent DNA duplication via an endogenous sequence-directed mechanism of mutagenesis.

Novak S, Hiesberger T, Schneider WJ, Nimpf J
A new low density lipoprotein receptor homologue with 8 ligand binding repeats in brain of chicken and mouse.
J Biol Chem. 1996; 271: 11732-6
Display abstract
The blood-brain barrier necessitates disparate macromolecular transport systems in the brain and central nervous system. We now report the discovery of a new member of the low density lipoprotein receptor (LDLR) family whose expression is highly restricted to the brain. The full-length cDNA specifying the chicken receptor (open reading frame, 2754 base pairs) as well as a cDNA for the major portion of its murine homologue have been obtained. The novel receptor shows the greatest similarity to the group of LDLR relatives with 8 ligand binding repeats, in chicken termed LR8 and in mammals, very low density lipoprotein receptors. Thus, in addition to 8 tandemly arranged ligand binding repeats, the five-domain receptor contains an O-linked sugar region and the internalization signal, Phe-Asp-Asn-Pro-Val-Tyr, typical for all LDLR gene family members. In chicken, the 6.5-kb receptor transcript is present at high levels in brain and at much lower levels in extraoocytic cells of the ovary; in mouse, the same transcript of 6.5 kb was detected in brain, but not in heart (the major site of very low density lipoprotein receptor expression), lung, liver, kidney, and ovary. An antibody directed against the predicted carboxyl terminus of the avian receptor detected a 130-kDa protein in brain extracts. The apparent size of the immunoreactive protein is compatible with extensive glycosylation of the 894-residue mature form of the receptor. The presence of this novel receptor in brain of a bird and a rodent suggests an important and evolutionary conserved function.

Dirlam KA, Gretch DG, LaCount DJ, Sturley SL, Attie AD
Expression and characterization of a truncated, soluble, low-density lipoprotein receptor.
Protein Expr Purif. 1996; 8: 489-500
Display abstract
The low-density lipoprotein (LDL) receptor mediates the clearance of apolipoprotein B- and E-containing lipoproteins from the bloodstream. In the current study, we characterized the binding properties of the amino terminus of the LDL receptor. We produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human LDL receptor. This truncated receptor protein (LDL-R354) was secreted from recombinant baculovirus-infected Spodoptera frugiperda (Sf-21) cells. Upon electrophoresis, LDL-R354 migrated with a mobility of 55,000. Treatment of cells with tunicamycin decreased the size of the truncated receptor, suggesting the presence of asparagine-linked carbohydrates. Nonreducing SDS-PAGE resulted in at least three discernible bands with M(r)s consistent with the truncated receptor existing as monomers and multimers, suggesting the possibility of intermolecular disulfide cross-linking. All forms of the truncated receptor bound LDL on a ligand blot in a calcium-dependent manner. The purified truncated receptor bound 125I-LDL with high affinity in a competitive binding assay (IC50 = 0.8 microgram/ml). LDL-R354 also bound calcium. This interaction was sensitive to the conformation of the ligand binding domain, as reduction of the disulfide bonds greatly decreased the affinity of the receptor for calcium. The ligand and calcium binding activities of this truncated receptor protein demonstrate that the ligand binding domain of the LDL receptor can fold into a functionally active protein.

Mehta KD, Chang R, Norman J
Chiloscyllium plagiosum low-density lipoprotein receptor: evolutionary conservation of five different functional domains.
J Mol Evol. 1996; 42: 264-72
Display abstract
All five functional domains of the low-density lipoprotein (LDL) receptor were assembled in their modern form more than 450 million years ago, as revealed from the cloning and sequencing of an LDL receptor cDNA from Chiloscyllium plagiosum (banded cat shark). The shark LDL receptor has the same overall architecture as the mammalian and amphibian counterparts. Each of the seven cysteine-rich repeats in the ligand binding domain resembles its counterpart in the human LDL receptor more than it does the other repeats in the shark receptor as suggested by the presence of unique "signature" sequences, indicating that these repeats had already acquired their independent structures by the time of shark development. Furthermore, amino acid sequences of the entire ligand binding domain of shark LDL receptor show 35% identity over a stretch of 294 residues with a Lymnaea stagnalis G-protein-linked receptor (LSGLR). The region of homology between these unrelated proteins includes conservation of most of the unique characteristics of the cysteine-rich repeats of LDL receptor at the expected positions in LSGLR. The results presented are consistent with the hypothesis that all seven repeats in the ligand binding domain of LDL receptor may have been lifted directly from an ancestral gene instead of being evolutionary duplications of a single repeat recruited by the primitive LDL receptor from another gene.

Djordjevic JT, Bieri S, Smith R, Kroon PA
A deletion in the first cysteine-rich repeat of the low-density-lipoprotein receptor leads to the formation of multiple misfolded isomers.
Eur J Biochem. 1996; 239: 214-9
Display abstract
The ligand-binding domain of the low-density-lipoprotein (LDL) receptor comprises seven cysteine-rich repeats, each approximately 40 amino acids long. The deletion of two amino acids (Asp26 and Gly27) from the first of these repeats (LB1), leads to a defective LDL receptor, and the clinical syndrome of familial hypercholesterolemia [Leitersdorf, E., Hobbs, H. H., Fourie, A. M., Jacobs, M., van der Westhuyzen, D.R. & Coetzee, G.A. (1988) Proc. Natl Acad. Sci. USA 85, 7912-7916]. Receptors which reach the cell surface fail to bind IgG-C7, a conformation-specific monoclonal antibody directed to LB1. To determine the effects of the two-amino-acid deletion on the folding of the LB1 of the LDL receptor, we have expressed LB1 and the mutant repeat, des-Asp26, Gly27-LB1, as recombinant (rLB1 and des-Asp26, Gly27-rLB1) peptides, and have determined their ability to fold in vitro. Unlike rLB1, which folded into a single isomer that was recognized by IgG-C7 and had three disulfide bonds, des-Asp26, Gly27-rLB1 folded into an equilibrium mixture of four isomers. Each of these isomers contained three disulfide bonds, but none were recognized by IgG-C7. We suggest that LDL receptors in the endoplasmic reticulum (ER) of the cell also fold into an equilibrium mixture of distinct receptor molecules, each with an abnormally folded isomer of des-Asp26, Gly27-LB1, and that the retarded transport of receptors to the cell surface arises because only a subset of the isomers reaches the cell surface.

Kim DH et al.
Human apolipoprotein E receptor 2. A novel lipoprotein receptor of the low density lipoprotein receptor family predominantly expressed in brain.
J Biol Chem. 1996; 271: 8373-80
Display abstract
Isolation and characterization of a human cDNA demonstrated a novel lipoprotein receptor designated apolipoprotein E receptor 2 (apoER2). The new receptor consists of five functional domains resembling the low density lipoprotein (LDL) and very low density lipoprotein (VLDL) receptors. LDL receptor deficient Chinese hamster ovary cells expressing human apoER2 bound apoE rich beta-migrating VLDL with high affinity and internalized. LDL was bound with much lower affinity to these cells. The 4.5- and 8.5-kb mRNAs for the receptor were most highly expressed in human brain and placenta. In rabbit tissues, multiple species of the mRNA with 4, 4.5, 5.5, 8.5, and 11 kb were detected most intensely in brain and testis and, to a much lesser extent, in ovary, but were undetectable in other tissues. In rat adrenal pheochromocytoma PC12 cells, the receptor mRNA was induced by treatment of the cells with nerve growth factor. The receptor transcripts were detectable most intensely in the cerebellar cortex, choroid plexus, ependyma, hippocampus, olfactory bulb and, to a much lesser extent, in the cerebral cortex as revealed by in situ hybridization histochemistry. In the cerebellar cortex, the receptor transcripts were densely deposited in Purkinje cell somata.

Jingami H, Yamamoto T
The VLDL receptor: wayward brother of the LDL receptor.
Curr Opin Lipidol. 1995; 6: 104-8
Display abstract
The VLDL receptor binds lipoproteins that contain apolipoprotein E and consists of five functional domains that resemble the LDL receptor. Although the LDL- and VLDL-receptor genes are located on different chromosomes, their gene structure and organization are almost the same. Despite the presence of sterol regulatory element-1-like sequences in the VLDL-receptor gene, the transcription of the gene is not down-regulated by sterols. The VLDL-receptor messenger RNA is highly expressed in tissues that actively metabolize fatty acids as their source of energy. The physiological role of the chicken cognate of the VLDL receptor has been revealed recently, but its function in mammals is still debatable.

Theart L et al.
Screening for mutations in exon 4 of the LDL receptor gene: identification of a new deletion mutation.
J Med Genet. 1995; 32: 379-82
Display abstract
DNA from 14 unrelated New Zealand familial hypercholesterolaemia (FH) heterozygotes, originating from the United Kingdom, was screened for mutations in exon 4 of the low density lipoprotein receptor (LDLR) gene. One patient was heterozygous for mutation D206E, which was initially identified in South Africa. The chromosomal background of this mutant allele was compatible with that described previously in Afrikaner and English patients, suggesting that this mutation originated in the United Kingdom. The 2 bp deletion in codon 206 and mutations D154N and D200G, previously reported in English FH patients, were not detected in this sample. In one of the patients, however, a new deletion of 7 bp was identified after nucleotide 581 (or 582) in exon 4 of the LDLR gene.

Yamamoto T, Hoshino A, Takahashi S, Kawarabayasi Y, Iijima H, Sakai J
The role of the very low density lipoprotein receptor in the metabolism of plasma lipoproteins containing ApoE.
Ann N Y Acad Sci. 1995; 748: 217-24

Bieri S, Djordjevic JT, Jamshidi N, Smith R, Kroon PA
Expression and disulfide-bond connectivity of the second ligand-binding repeat of the human LDL receptor.
FEBS Lett. 1995; 371: 341-4
Display abstract
The human LDL receptor (LDLR) has a binding domain which consists of seven contiguous ligand-binding (LB) repeats, each approximately 40 amino acids long with three disulfide bonds. The second LB repeat, which is required for full binding of LDL, has been expressed, purified and folded to yield a single, fully oxidized isomer. By selective reduction and alkylation, we have shown that the cysteine residues have a I-III, II-V, IV-VI connectivity, matching that recently determined for the amino-terminal repeat. We suggest that the first two LB repeats of the LDLR, with their unique disulfide-bonding pattern, serve as a structural paradigm for other LB repeats.

Bieri S, Djordjevic JT, Daly NL, Smith R, Kroon PA
Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain.
Biochemistry. 1995; 34: 13059-65
Display abstract
The low density lipoprotein (LDL) receptor is the prototype of a family of structurally related cell surface receptors that mediate the endocytosis of multiple ligands in mammalian cells. Its ligand-binding domain consists of seven cysteine-rich ligand-binding repeats, each approximately 40 amino acid residues long. Ligand-binding repeats occur in other members of the LDL receptor (LDLR) gene family and in a number of functionally unrelated proteins. As a first step toward an understanding of the structure and function of LB repeats, we have expressed the amino-terminal ligand-binding repeat (LB1) of the human LDLR as a recombinant peptide (rLB1) and have determined its disulfide-pairing scheme. Oxidative folding of rLB1 yielded a single isomer which contained three disulfide bonds. This isomer reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDLR, suggesting that rLB1 was correctly folded. rLB1 was resistant to digestion with trypsin, chymotrypsin, and V8 protease, consistent with a tightly folded structure. Disulfide bond connections were established using two separate approaches. Digestion with the nonspecific proteolytic enzyme proteinase K yielded an 8 amino acid peptide with a single disulfide bond which connected Cys(IV) and Cys(VI). In the second approach, disulfide bonds were sequentially reduced with tris(2-carboxyethyl)phosphine and the resulting cysteine residues alkylated with iodoacetamide. An analysis of peptides which contained two cysteinylacetamide residues, derived from a single reduced disulfide bond, showed that Cys(I) and Cys(III) were disulfide-bonded and confirmed the presence of a disulfide bond between Cys(IV) and Cys(VI). We infer that the remaining disulfide bond bridges Cys(II) and Cys(V).(ABSTRACT TRUNCATED AT 250 WORDS)

Ullman CG, Haris PI, Smith KF, Sim RB, Emery VC, Perkins SJ
Beta-sheet secondary structure of an LDL receptor domain from complement factor I by consensus structure predictions and spectroscopy.
FEBS Lett. 1995; 371: 199-203
Display abstract
Low density lipoprotein receptor domains (LDLrs) represent a large cell surface receptor superfamily of consensus length 39 residues. Alignment of 194 sequences indicated highly conserved Cys and Asp/Glu residues, and a consensus secondary structure with three beta-strands was predicted. Sequence threading against known protein folds indicated consistency with small beta-sheet proteins. Complement factor I contains two LDLrs, and the second of these was successfully expressed using a bacterial pGEX system. FT-IR spectroscopy on this indicated a small amount of beta-sheet together with turns and loops. LDLr is proposed to have a beta-sheet structure in which the five biologically important Asp/Glu residues are located on an exposed loop.

Hodits RA et al.
An antibody fragment from a phage display library competes for ligand binding to the low density lipoprotein receptor family and inhibits rhinovirus infection.
J Biol Chem. 1995; 270: 24078-85
Display abstract
Recently antibodies with a wide range of binding specificities have been isolated from large repertoires of antibody fragments displayed on filamentous phage, including those that are difficult to raise by immunization. We have used this approach to isolate an antibody fragment against chicken very low density lipoprotein (VLDL) receptor. It binds to the receptor with good affinity (Kaff = 2 x 10(8) M-1) as measured by plasmon surface resonance, and competes for binding of natural ligands (vitellogenin, VLDL, and receptor-associated protein). The antibody also binds to other members of the low density lipoprotein (LDL) receptor family including rat LDL receptor and human and rat low density lipoprotein receptor-related protein (LRP/alpha 2MR), and it competes for binding of receptor-associated protein to LRP/alpha 2MR. Moreover, the antibody fragment inhibits infection of human fibroblasts deficient in LDL-R but expressing LRP/alpha 2MR by human rhinovirus. Binding of the antibody is abolished upon reduction of the receptors and is strictly Ca2+ dependent. The phage antibody thus recognizes the ligand binding site(s) of several members of the LDL receptor family, in contrast to antibodies produced by hybridoma technology.

Sass C, Giroux LM, Lussier-Cacan S, Davignon J, Minnich A
Unexpected consequences of deletion of the first two repeats of the ligand-binding domain from the low density lipoprotein receptor. Evidence from a human mutation.
J Biol Chem. 1995; 270: 25166-71
Display abstract
Heterozygosity for a 5-kilobase (kb) deletion of the first two ligand-binding repeats (exons 2 and 3) of the low density lipoprotein (LDL) receptor (R) gene (LDL-R delta 5kb) confers familial hypercholesterolemia (FH). The FH phenotype is unexpected based on previous site-directed mutagenesis showing that deletion of exons 2 and 3 resulted in little or no defect in LDL-R activity. In the present study, we took unique advantage of the ability to distinguish the LDL-R delta 5kb from the normal receptor on the basis of size, in order to resolve this apparent discrepancy. Fibroblasts from heterozygotes for the LDL-R delta 5kb displayed 50% of normal capacity to bind LDL and beta-VLDL, apparently due to lower receptor number. Cellular mRNA for the delta 5kb allele was at least as abundant as that for the normal allele. Immunoblotting and cell binding assays with anti-LDL-R antibody IgG-4A4 demonstrated normal synthesis and transport of the delta 5kb receptor. Ligand blotting demonstrated that the delta 5kb receptor displayed minimal or no ability to bind LDL or beta-VLDL. Thus, in contrast to transfected cell lines, in human fibroblasts, the first two cysteine rich repeats of the LDL-R appear functionally necessary. These characteristics of the LDL-R delta 5kb in human fibroblasts explain the in vivo phenotype of carriers.

Daly NL, Djordjevic JT, Kroon PA, Smith R
Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor.
Biochemistry. 1995; 34: 14474-81
Display abstract
The ligand-binding domain of the low-density lipoprotein receptor comprises seven cysteine-rich repeats, which have been highly conserved through evolution. This domain mediates interactions of the receptor with two lipoprotein apoproteins, apo E and apo B-100, putatively through a calcium-dependent association of the ligands with a cluster of acidic residues on the receptor. The second repeat (rLB2) of the receptor binding domain has been expressed as a thrombin-cleavable GST fusion protein, cleaved, and purified. On oxidation the protein refolded to give a single peak on reverse-phase HPLC. The aqueous solution structure of rLB2 has been determined using two-dimensional 1H NMR spectroscopy. In contrast to the amino-terminal repeat, rLB1, rLB2 has a very flexible structure in water. However, the conformation of rLB2 is markedly more ordered in the presence of a 4-fold molar excess of calcium chloride; the proton resonance dispersion and the number of NOESY cross-peaks are greatly enhanced. The three-dimensional structure of rLB2, obtained from the NMR data by molecular geometry and restrained molecular dynamics methods, parallels that of rLB1, with an amino-terminal hairpin structure followed by a succession of turns. However, there are clear differences in the backbone topology and structural flexibility. As for rLB1, the acidic residues are clustered on one face of the module. The side chain of Asp 37, which is part of a completely conserved SDE sequence thought to be involved in ligand binding, is buried, as is its counterpart (Asp 36) in rLB1. These results provide the first experimental support for the hypothesis that each of the repeats in the ligand-binding domain has a similar global fold but also highlight significant differences in structure and internal dynamics.

Belanger C, Zingler K, Young JA
Importance of cysteines in the LDLR-related domain of the subgroup A avian leukosis and sarcoma virus receptor for viral entry.
J Virol. 1995; 69: 1019-24
Display abstract
The extracellular domain of the subgroup A avian sarcoma and leukemia virus (ALSV-A) receptor contains a region that is related in sequence to the ligand-binding motifs of the low-density lipoprotein receptor (LDLR). This domain contains six cysteines that are highly conserved between different members of the LDLR protein superfamily, and these residues are presumed to participate in intrachain disulfide bonds. To assess the importance of each cysteine in the ALSV-A receptor, individual or multiple cysteines were mutated to alanines and the altered receptors were tested for the ability to confer susceptibility to viral infection. Receptors bearing single mutations allowed subgroup A viral entry, albeit at less than wild-type levels. Receptors containing two or three substitutions were completely inactive if one of the changed residues was Cys-35 or Cys-50. Of the altered receptors tested, the only exception to this rule was a functional receptor which lacked both Cys-35 and Cys-50, an activity that was dependent on the presence of other cysteines in this protein. Most interestingly, a receptor containing both Cys-35 and Cys-50 but lacking the other four cysteines was completely functional. These results demonstrate the importance of Cys-35 and Cys-50 for viral entry mediated by the ALSV-A receptor and show that in the presence of these two residues, all of the other cysteines in this protein can be removed without loss of this function.

Dyer CA, Cistola DP, Parry GC, Curtiss LK
Structural features of synthetic peptides of apolipoprotein E that bind the LDL receptor.
J Lipid Res. 1995; 36: 80-8
Display abstract
Apolipoprotein (apo) E, via its receptor binding domain contained in residues 140-150, mediates hepatic and peripheral tissue binding of cholesterol-rich lipoproteins. Previously, we reported that a synthetic peptide representing a linear tandem repeat of amino acids 141-155, the 141-155 dimer, binds the low density lipoprotein (LDL) receptor. To define the structural features essential for LDL receptor binding of the 141-155 dimer, a series of modified peptides were synthesized. The secondary structure content of the modified apoE peptides was assessed by circular dichroism (CD) and the receptor activity was studied in cellular LDL receptor binding assays. alpha-Helix content was necessary but not sufficient for receptor activity because both a 129-162 monomer and the 141-155 dimer peptides had comparable CD spectra and helix contents, but only the 141-155 dimer was receptor active. Deletion of the charged amino terminal residues including arg142 and lys143 in the 145-155 or 144-150 dimers had no effect on alpha-helix content, yet abolished their receptor activities. Helical net models of all receptor active peptides indicated that the LDL-receptor binding activity of the 141-155 dimer is dependent on at least two clusters of basic amino acids present on the hydrophilic face of the amphipathic alpha-helix of the 141-155, 141-150, 141-155 (lys143-->ala) and 141-155 (arg150-->ala) dimer peptides.

Sun XM, Neuwirth C, Wade DP, Knight BL, Soutar AK
A mutation (T-45C) in the promoter region of the low-density-lipoprotein (LDL)-receptor gene is associated with a mild clinical phenotype in a patient with heterozygous familial hypercholesterolaemia (FH).
Hum Mol Genet. 1995; 4: 2125-9
Display abstract
We have identified a rare mutation (T-45C) in the low density lipoprotein (LDL)-receptor gene in a Welsh patient with a clinical diagnosis of heterozygous familial hypercholesterolaemia (FH). The mutation is in the proximal Sp1 binding site in repeat 3 of the 42 bp region of the promoter required for sterol-dependent regulation of transcription, but the substituted nucleotide is not a strongly conserved base in the consensus sequence for Sp1 binding. Normal and mutant promoter fragments (from base -600 to -5) were linked to a luciferase reporter gene, and transient expression in COS cells showed that the mutation reduced transcriptional activity to approximately 43% of normal in the presence, and 25% in the absence of sterols in the medium. Competitive gel-shift mobility assays showed that the mutation reduced the binding affinity for transcription factor Sp1. Analysis of a neutral polymorphism in the LDL-receptor mRNA from the patient's lymphoblasts showed that expression of one allele was reduced. Since Southern blotting of genomic DNA and sequencing of the entire coding region of the LDL-R gene did not reveal any other potential defects, we infer that the T-45 C mutation is the underlying cause of hypercholesterolaemia in the proband.

Jensen HK et al.
An alanine29-serine variant in exon 2 of the low density lipoprotein receptor gene: no association with hypercholesterolemia.
Clin Genet. 1994; 46: 214-5

Schuster H, Humphries S
European workshop on LDL receptor defects. European Working Group on Familial Hypercholesterolaemia.
Clin Investig. 1994; 72: 898-907

Oka K et al.
Mouse very-low-density-lipoprotein receptor (VLDLR) cDNA cloning, tissue-specific expression and evolutionary relationship with the low-density-lipoprotein receptor.
Eur J Biochem. 1994; 224: 975-82
Display abstract
The very-low-density-lipoprotein receptor (VLDLR) is a recently described lipoprotein receptor that shows considerable similarity to the low-density-lipoprotein receptor (LDLR). This receptor has been suggested to be important for the metabolism of apoprotein-E-containing triacylglycerol-rich lipoproteins, such as very-low-density-lipoprotein (VLDL), beta-migrating VLDL and intermediate-density lipoprotein. cDNA clones that code for the VLDLR were isolated from a mouse heart cDNA library. The deduced amino acid sequence predicts a mature protein of 846 amino acids preceded by a 27-residue signal peptide. Three mRNA species for the VLDLR with sizes of 3.9, 4.5 and 7.9 kilobases were present in high concentration in heart and muscle, which utilize triacylglycerols as an energy source. VLDLR mRNA is also detected in decreasing amounts in kidney, brain, ovary, testis, lung and adipose tissue. It is essentially absent in liver and small intestine. The amino acid sequence of the VLDLR is highly conserved among rabbit, human and mouse. VLDLR contains five structural domains very similar to those in LDLR, except that the ligand-binding domain in VLDLR has an eightfold repeat instead of a sevenfold repeat in LDLR. Sequence conservation among animal species is much higher for the VLDLR than the LDLR. Sequences of the VLDLR from three vertebrate species and the LDLR from five vertebrate species were aligned and a phylogenetic tree was reconstructed. Although both receptors contain five domains and share amino acid sequence similarity, our computations showed that they diverged before the divergence between mammals and amphibians. In addition, sequence comparison of both receptor sequences suggests that the rabbit is evolutionarily closer to man than to the mouse. These results are consistent with the hypothesis that the VLDLR and the LDLR have evolved from a common ancestral gene to play distinct roles in lipoprotein metabolism and that the metabolic handling of triacylglycerol by the body via the VLDLR is a highly conserved mechanism.

Yokode M, Kita T
[Molecular analysis of the LDL receptor]
Nippon Rinsho. 1994; 52: 3170-6
Display abstract
The LDL receptor is a cell surface glycoprotein that regulates plasma cholesterol by mediating endocytosis of LDL, the major cholesterol transport protein in human plasma. Mutations in the LDL receptor gene cause familial hypercholesterolemia (FH). The LDL receptor was purified in 1982, its cDNA was cloned in 1984 by Yamamoto et al, and its gene was isolated in 1985 by Sudhof et al. This review attempts to focus on the molecular basis of the LDL receptor pathway and its regulatory roles in cholesterol homeostasis in the body.

Hofer F et al.
Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus.
Proc Natl Acad Sci U S A. 1994; 91: 1839-42
Display abstract
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.

Koivisto UM, Palvimo JJ, Janne OA, Kontula K
A single-base substitution in the proximal Sp1 site of the human low density lipoprotein receptor promoter as a cause of heterozygous familial hypercholesterolemia.
Proc Natl Acad Sci U S A. 1994; 91: 10526-30
Display abstract
We have identified a Finnish family with a typical phenotype of heterozygous familial hypercholesterolemia (FH) due to a single-base substitution in the proximal Sp1 binding site of the low density lipoprotein (LDL) receptor gene promoter. The mutation, a C-->T substitution at nucleotide -43, cosegregated with the FH phenotype in six available family members and abolished binding of Sp1 transcription factor to this site. As a consequence, transcriptional activity of the mutated LDL receptor promoter was only about 1/20th of that of the wild-type promoter, as judged by transfection studies in HeLa cells. Studies of primary fibroblast cultures established from a family member revealed a markedly reduced LDL receptor mRNA concentration as well as reduction of binding, internalization, and degradation of 125I-labeled LDL to values < 50% of those in normal fibroblasts. This DNA alteration is thus a naturally occurring promoter mutation causing a severe disorder of human lipoprotein metabolism.

Gudnason V, Day IN, Humphries SE
Effect on plasma lipid levels of different classes of mutations in the low-density lipoprotein receptor gene in patients with familial hypercholesterolemia.
Arterioscler Thromb. 1994; 14: 1717-22
Display abstract
We used the single-strand conformational polymorphism method to screen 311 patients with familial hypercholesterolemia from London lipid clinics and Southampton and South West Hampshire health district for mutations in the 3' part of exon 4 of the low-density lipoprotein (LDL) receptor gene. This part of the gene codes for repeat 5 of the binding domain of the LDL receptor, which is known to be critical for the receptor-mediated removal of both triglyceride-rich lipoprotein remnants and LDL. Six previously described mutations were identified in 29 apparently unrelated individuals (9.3%), with the mutations all lying within a 50-bp fragment of the gene. Three of the mutations are null alleles producing no protein, and the other three lead to production of a defective protein. The effect of the different gene mutations on lipid levels was examined, after the data were combined with information on previously reported mutations in this patient group. Mean LDL cholesterol levels were highest in those individuals with a mutation creating a null allele (9.54 mmol/L) and were similar to levels in those individuals with a mutation affecting repeat 5 that resulted in the production of a defective protein (9.37 mmol/L). In this sample, previously identified patients with a defective protein mutation outside repeat 5 had lower mean levels of LDL cholesterol (7.78 mmol/L), which were similar to levels seen in patients in whom the specific mutation had not been identified (7.31 mmol/L). Overall, these differences were highly statistically significant (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Sakai J et al.
Structure, chromosome location, and expression of the human very low density lipoprotein receptor gene.
J Biol Chem. 1994; 269: 2173-82
Display abstract
Isolation and characterization of cDNAs encoding human very low density lipoprotein (VLDL) receptor revealed the presence of two forms of the receptor: one consists of five domains that resemble the low density lipoprotein (LDL) receptor, and a variant form lacks an O-linked sugar domain. More than 96% of amino acids in the human and rabbit VLDL receptors are identical, whereas those in the LDL receptors are less conserved between the two species (76%). The human VLDL receptor gene contains 19 exons spanning approximately 40 kilobases. The exon-intron organization of the gene is almost the same as that of the LDL receptor gene, except for an extra exon that encodes an additional repeat in the ligand binding domain of the VLDL receptor. Analysis of DNA from human-rodent hybrid cells revealed that the gene is located on chromosome 9. Although the 5'-flanking region of the VLDL receptor gene contains two copies of a sterol regulatory element-1 like sequence, the levels of mRNA for the receptor in THP-1 human monocytic leukemia cells were unchanged by sterols. The 5'-untranslated region of the receptor mRNA contains a polymorphic triplet repeat found also in the fragile X syndrome gene.

Gagne C, Moorjani S, Torres AL, Brun D, Lupien PJ
Homozygous familial hypercholesterolaemia.
Lancet. 1994; 343: 177-177

Bujo H et al.
Chicken oocyte growth is mediated by an eight ligand binding repeat member of the LDL receptor family.
EMBO J. 1994; 13: 5165-75
Display abstract
Deposition of the yolk mass components of chicken oocytes, very low density lipoprotein (VLDL) and vitellogenin (VTG), is mediated by a 95 kDa plasma membrane protein, termed VLDL/VTG receptor (VLDL/VTGR). Molecular characterization of the VLDL/VTGR revealed that it is a member of the LDLR gene superfamily, and harbours eight complement-type, cysteine-rich ligand binding repeats at the N-terminus. This ligand binding domain structure is the hallmark of the recently discovered mammalian so-called VLDLRs, whose true physiological function remains to be elucidated. Northern blot analysis revealed that this receptor is expressed almost exclusively in oocytes, with very much lower levels of hybridizing transcripts present in heart and skeletal muscle. Heterologous expression of the cloned receptor demonstrated its ability to bind both VLDL and VTG. The receptor gene is located on the avian sex chromosome Z, in agreement with the sex linkage of a single-gene defect in animals that fail to reproduce because of the lack of expression of functional VLDL/VTGR. In situ hybridization analysis of oocytes suggested that VLDL/VTGR mRNA may relocalize during oocyte growth. Thus, the current study has identified and characterized the first non-mammalian VLDLR. Its key role in avian reproduction and extremely high evolutionary conservation shed new light on VLDLR function in mammals, which also express the gene in ovaries.

Bruckert E, Chapman J
[Low density lipoprotein receptor and hypercholesterolemia]
Rev Prat. 1994; 44: 1287-93
Display abstract
Familial hypercholesterolaemia is the most frequent of the monogenic genetic disorders. The studies of Brown, Goldstein and collaborators have facilitated decisive progress in our understanding of the pathophysiology of this hypercholesterolaemia, and more specifically in our knowledge of the metabolism of the low-density lipoproteins (LDL) and of cholesterol at the cellular level. Indeed, they have documented the existence of a ubiquitous receptor which facilitates cellular uptake of LDL-cholesterol; such cholesterol is subsequently available to satisfy the metabolic requirements of the cell. The description of numerous mutations of the LDL receptor protein have allowed an exemplary approach to the study of the relationship between its structure and function. Familial hypercholesterolaemia represents a classical illustration of the atherogenic role of LDL-cholesterol in a clinical context in man. This disorder has already featured three minor therapeutic revolutions: LDL apheresis, inhibitors of HMG-CoA reductase (statins) and most recently, gene therapy.

Yamakawa-Kobayashi K, Kobayashi T, Yanagi H, Shimakura Y, Satoh J, Hamaguchi H
A novel complex mutation in the LDL receptor gene probably caused by the simultaneous occurrence of deletion and insertion in the same region.
Hum Genet. 1994; 93: 625-8
Display abstract
A novel complex mutation with the presence of both deletion and insertion in very close proximity in the same region was detected in exon 8 of the LDL receptor gene from two apparently unrelated Japanese families with familial hypercholesterolemia (FH). In this mutant LDL receptor gene, the nine bases from nucleotide (nt) 1115 to nt 1123 (AGGGTGGCT) were replaced by six different bases (CACTGA), and consequently the four amino acids from codon 351 to 354, Glu-Gly-Gly-Tyr, were replaced by three amino acids, Ala-Leu-Asn, in the conserved amino acid region of the growth factor repeat B of the LDL receptor. The nature of the amino acid substitution and data on the families suggest that this mutation is very likely to affect the LDL receptor function and cause FH. The generation of this complex mutation can be explained by the simultaneous occurrence of deletion and insertion through the formation of a hairpin-loop structure mediated by inverted repeat sequences. This this mutation supports the hypothesis that inverted repeat sequences influence the stability of a given gene and promote human gene mutations.

Naggert JK, Mu JL
The mouse very low density lipoprotein receptor (Vldlr) gene maps to chromosome 19.
Mamm Genome. 1994; 5: 453-5

Ellsworth JL, Carlstrom AJ, Deikman J
Ketoconazole and 25-hydroxycholesterol produce reciprocal changes in the rate of transcription of the human LDL receptor gene.
Biochim Biophys Acta. 1994; 1210: 321-8
Display abstract
Sterol-dependent regulation of low-density lipoprotein (LDL) receptor gene expression was studied in the human hepatoma HepG2 cell line. Incubation of HepG2 cells with 20 microM ketoconazole increased the level of LDL receptor mRNA. After a lag of approx. 1.0 h the level rose 6.5-fold within 8.0 h and remained elevated for up to 24 h. Incubation with 10 micrograms 25-hydroxycholesterol/ml for 24 h produced a 40-50% reduction in the level of LDL receptor mRNA. Ketoconazole- and 25-hydroxycholesterol-induced changes in LDL receptor mRNA accumulation were due to alterations in the relative rate of LDL receptor gene transcription as measured by nuclear run-on transcription. Incubation with 20 microM ketoconazole for 4 h or 10 micrograms 25-hydroxycholesterol/ml for 24 h produced a 3.6-fold increase and a 40% reduction, respectively, in the transcription rate of LDL receptor gene. Removal of the Alu-like sequence elements within the LDL receptor cDNA was required to consistently measure changes in LDL receptor gene transcription. No significant changes were noted in the half-life of LDL receptor mRNA in ketoconazole or 25-hydroxycholesterol-treated cells. These data demonstrate that sterol-dependent changes in the level of LDL receptor mRNA can be completely accounted for by changes in the rate of LDL receptor gene transcription.

Fischer DG, Novick D, Cohen B, Rubinstein M
Isolation and characterization of a soluble form of the LDL receptor, an interferon-induced antiviral protein.
Proc Soc Exp Biol Med. 1994; 206: 228-32
Display abstract
Interferons (IFNs) act by inducing several intracellular antiviral proteins. We report here that IFNs also induce an extracellular soluble protein that inhibits vesicular stomatitis virus (VSV) infection. This protein accounts for 25%-50% of the total antiviral activity elicited by IFN. The antiviral protein was purified to homogeneity from culture supernatants of IFN-treated cells by several chromatographic steps, to give a single 28-kDa active polypeptide. Upon sequencing, this novel protein corresponded to the N-terminal ligand-binding domain of the human 160-kDa low-density lipoprotein receptor (LDLR). In addition, we find that IFN induces the cell surface LDLR and this phenomenon may explain previous reports on reduction of serum cholesterol in IFN-treated patients. Viruses produce soluble cytokine receptors that inhibit their respective cytokines, thereby assisting virus infection. It appears now that host cells employ similar molecules for the opposite role of controlling virus infections.

Tensen CP et al.
A G protein-coupled receptor with low density lipoprotein-binding motifs suggests a role for lipoproteins in G-linked signal transduction.
Proc Natl Acad Sci U S A. 1994; 91: 4816-20
Display abstract
We have isolated and analyzed a cDNA from the central nervous system of the mollusc Lymnaea stagnalis encoding a putative receptor, which might be a natural hybrid between two different classes of receptor proteins. Preceded by a signal peptide, two types of repeated sequences are present in the N-terminal part of the protein. The first repeat displays a high sequence similarity to the extracellular binding domains of the low density lipoprotein receptor, which binds and internalizes cholesterol-containing apolipoproteins. The second repeat and the C-terminal part of the Lymnaea receptor are very similar to regions of a specific class of guanine nucleotide-binding protein-coupled receptors, the mammalian glycoprotein hormone receptors. The mRNA encoding the receptor is predominantly expressed in a small number of neurons within the central nervous system and to a lesser extent in the heart.

Solberg K, Rodningen OK, Tonstad S, Ose L, Leren TP
Familial hypercholesterolaemia caused by a non-sense mutation in codon 329 of the LDL receptor gene.
Scand J Clin Lab Invest. 1994; 54: 605-9
Display abstract
Analysis of single-strand conformation polymorphisms (SSCP) was employed to screen familial hypercholesterolaemia (FH) subjects for point mutations in exon 7 of the low density lipoprotein receptor (LDLR) gene. An abnormal band pattern was found in one out of 100 unrelated FH subjects. The underlying mutation was found by DNA sequencing to be due to heterozygosity (C/T) at nucleotide 1048. Nucleotide 1048 is the first nucleotide of codon 329, and is located within the domain that has a high degree of homology with the precursor for epidermal growth factor. The C-->T transition, referred to as FH-Fossum, changes codon 329 from CGAArg to TGAStop. The mutation is expected to cause a class 1 receptor defect.

Sakai J, Yamamoto T
[VLDL receptor]
Nippon Rinsho. 1994; 52: 3184-90
Display abstract
The LDL receptor was discovered by Goldstein and Brown in 1974. This discovery has led to dramatic progress toward understanding the mechanisms of cholesterol homeostasis. Recently we have found a second lipoprotein receptor that specifically binds apoE-containing lipoproteins. This new lipoprotein receptor designated VLDL receptor is composed of five functional domains that resemble the LDL receptor. The VLDL mRNA is abundant in heart, muscle and adipose tissue, suggesting that the receptor mediates the uptake of triglyceriderich lipoproteins thereby providing muscle and fat cells with fatty acid.

Schluter G, Wick U
An 87 bp deletion in exon 5 of the LDL receptor gene in a mother and her son with familial hypercholesterolemia.
Clin Genet. 1994; 45: 84-7
Display abstract
DNA analysis of the low density lipoprotein receptor (LDLR) gene was performed in two persons with familial hypercholesterolemia (FH). Southern blot experiments indicated the heterozygous loss of an EcoRI site in exon 5 of the LDLR gene. Upon amplification and sequencing of exon 5 in both probands, an 87-bp deletion in a heterozygous state could be evaluated. This is a novel mutation, most probably leading to the formation of a nonfunctional LDLR. Analysis of the deletion breakpoints revealed the presence of a six-base-pair consensus sequence 5'TGA/GA/GG/TA/C3', which is characteristic of small deletions in different genetic defects.

Bertolini S et al.
Partial duplication of the EGF precursor homology domain of the LDL-receptor protein causing familial hypercholesterolemia (FH-Salerno).
J Lipid Res. 1994; 35: 1422-30
Display abstract
A novel mutation of low density lipoprotein (LDL)-receptor gene was found in an Italian family hypercholesterolemia (FH) patient during a screening of 300 FH patients. The proband as well as her daughter were found to be heterozygotes for the mutation. Binding, internalization, and degradation of 125I-labeled LDL by the proband's fibroblasts were reduced to approximately 50% compared to values found in control cells. DNA analysis by Southern blotting showed that the mutant allele was characterized by an insertion of about 10 kb, which resulted from a duplication of exons 9-14 of the LDL-receptor gene. In addition, Northern blot analysis of the proband's RNA showed, besides the normal sized LDL-receptor mRNA (5.3 kb), an additional mRNA of about 6.2 kb. The junction between exon 14 and the duplicated exon 9 was amplified by polymerase chain reaction (PCR) from the cDNA. The sequence of the amplified fragment showed that exon 14 joined the duplicated exon 9 without changing the reading frame. The derived amino acid sequence indicated that the mutated receptor protein had a partial duplication of the EGF precursor homology domain. Ligand and immunoblotting revealed that proband's fibroblasts contained one-half of the normal amount of LDL-receptor protein (molecular mass 130 kDa) and an abnormally large receptor of approximately 160 kDa. The amount of this abnormal receptor as detected by two monoclonal antibodies (10A2 and 4B3) was found to be approximately 30% that of the normal LDL-receptor present in the same cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lombardi P et al.
An acceptor splice site mutation in intron 16 of the low density lipoprotein receptor gene leads to an elongated, internalization defective receptor.
Atherosclerosis. 1993; 104: 117-28
Display abstract
In this report, we describe the characterization of a mutation in the low density lipoprotein (LDL) receptor gene of a true homozygous familial hypercholesterolemic (FH) patient. The combined use of denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis revealed a unique A to G transition in the penultimate 3'-nucleotide of intron 16 of the LDL receptor gene, which disrupts the acceptor splice site. cDNA sequence analysis indicated that a cryptic splice site was activated in intron 16, upstream from the original splice site, leading to the inclusion of 62 nucleotides and a reading frame-shift. The resulting new translation product contains a stretch of 154 amino acids at the carboxy-terminal that have no resemblance to the normal receptor protein. To elucidate the biological effects of the mutation, the structural and functional properties of the mutated LDL receptor protein were studied. Immunoprecipitation of the newly synthesized LDL receptors showed that an aberrant precursor form of the LDL receptor protein was synthesized, about 10 kDa larger than normal, which is not further processed to the mature form. Some 50% of the normal LDL binding activity was found on the cell surface of the patient's fibroblasts, whereas internalization and degradation of LDL were abolished.

Robinson A
No prescription for despair.
CMAJ. 1993; 149: 639-43

Koivisto PV, Koivisto UM, Kovanen PT, Gylling H, Miettinen TA, Kontula K
Deletion of exon 15 of the LDL receptor gene is associated with a mild form of familial hypercholesterolemia. FH-Espoo.
Arterioscler Thromb. 1993; 13: 1680-8
Display abstract
We describe a mutation of the low-density lipoprotein (LDL) receptor gene, designated familial hypercholesterolemia (FH)-Espoo, which deletes exon 15 of the LDL receptor gene. The mutant receptor is predicted to lack 57 amino acids, including 18 serine and threonine residues, which are the sites of the clustered O-linked sugars of the receptor. Studies on 10 carriers of this gene revealed that FH-Espoo is associated with an exceptionally mild form of FH. Thus, in conditions in which cell proliferation was rendered dependent on the function of LDL receptors, lymphocytes from the patients with the FH-Espoo allele had a growth rate intermediate between those from healthy subjects and patients with the FH-Helsinki gene, a mutation known to abolish LDL receptor function. The in vivo fractional catabolic rate of LDL apolipoprotein B was lower than normal in the two FH-Espoo heterozygotes studied. Although higher than those in healthy controls, the serum LDL cholesterol concentrations in patients with the FH-Espoo gene were significantly lower than those in patients with the FH-Helsinki mutation. The thickness of the Achilles tendons was within the normal limits in subjects with the FH-Espoo gene. Our study suggests that moderate varieties of hypercholesterolemia, ie, those not considered to represent FH, may occasionally be due to subtle LDL receptor gene mutations.

Suzuki T, Riggs AF
Linker chain L1 of earthworm hemoglobin. Structure of gene and protein: homology with low density lipoprotein receptor.
J Biol Chem. 1993; 268: 13548-55
Display abstract
The extracellular hemoglobins (Hbs) of annelids and tube worms are giant multisubunit proteins of up to approximately 200 polypeptides and molecular masses to at least 3,900 kDa. They differ from all other Hbs in having both O2-binding chains and "linker" chains. The latter are required for assembly and structural integrity of the protein and are deficient in or lack heme. We have determined the nucleotide sequences of the cDNA and gene for linker chain L1 of the hemoglobin of Lumbricus terrestris. The cDNA-derived amino acid sequence has 225 residues and a calculated molecular mass of 25,847 Da. The chain is 21-28% identical to linker chains of the related annelid Tylorrhynchus heterochaetus and the deep-sea tube worm Lamellibrachia sp. A remarkable feature of the linker chains is a conserved 38-39-residue segment that contains a repeating pattern of cysteinyl residues: (Cys-X6)3-Cys-X5-Cys-X10-Cys. This pattern, not present in any globin sequence, corresponds exactly to the cysteine-rich repeats of the ligand binding domains of the low density lipoprotein (LDL) receptors of man and Xenopus laevis. Furthermore, the cysteine-rich segment of linker chain L1 has the sequence Asp-Gly-Ser-Asp-Glu which is characteristic of LDL receptor repeats. Similar cysteine-rich sequences also occur in two other mammalian proteins, complement C9 and renal glycoprotein GP330. The results support the conclusion that the cysteine-rich motif of the LDL receptor and annelid Hbs is a multipurpose protein-binding unit of ancient origin which has been incorporated into diverse unrelated proteins, presumably by the process of exon shuffling.

Boguslawski W
[Structure and function of LDL receptors in familial hypercholesterolemia]
Postepy Hig Med Dosw. 1993; 47: 55-65
Display abstract
Structure and function of the LDL-receptor in genetic disorder--familial hypercholesterolemia is described as well as the structure of receptor coding gene. General outline of therapy in FH is also described.

Hoffer MJ et al.
The mouse low density lipoprotein receptor gene: cDNA sequence and exon-intron structure.
Biochem Biophys Res Commun. 1993; 191: 880-6
Display abstract
The low density lipoprotein (LDL) receptor plays a central role in the cholesterol metabolism. The cDNA sequence of the mouse low density lipoprotein receptor (Ldlr) gene has been determined and shows 76% homology with the human gene. The exon-intron structure has been determined for the 129/J mouse strain. The gene is composed of 18 exons and spans a region of 28 kb. In addition, the promoter regions of the mouse and human genes are homologous. Northern blot analysis revealed an mRNA of approximately 5 kb. The cloning of the Ldlr gene will enhance the usefulness of the mouse for the study of cholesterol metabolism and, in particular, for carrying out gene targeting experiments.

Yamamoto T
[Lipoprotein receptors]
Tanpakushitsu Kakusan Koso. 1993; 38: 821-30

Defesche JC, Lansberg PJ, Reymer PW, Lamping RJ, Kastelein JJ
Analysis of the Afrikaner mutation in exon 9 of the low-density lipoprotein receptor gene in a large Dutch kindred suffering from familial hypercholesterolaemia.
Neth J Med. 1993; 42: 53-60
Display abstract
Familial hypercholesterolaemia (FH) is the most common genetic metabolic disorder, affecting about 1 in 500 persons in the general population. With novel techniques, it is possible to identify the molecular defects underlying FH in the gene coding for the low-density lipoprotein (LDL) receptor, thereby confirming the diagnosis of FH. In this study we present a large family with a specific mutation in exon 9 of the LDL-receptor gene (an Afrikaner mutation) and we demonstrate that by a large-scale case-finding study in this family, carriers of such a mutation can be detected. Of 63 family members, 13 were shown to be at risk for cardiovascular disease as judged by their lipoprotein profile or the presence of the Afrikaner mutation. Two persons were detected, affected with a dyslipidaemia other than FH. Medical management was initiated in order to reduce the high cardiovascular risk associated with this disorder.

Soutar AK
Familial hypercholesterolaemia and LDL receptor mutations.
J Intern Med. 1992; 231: 633-41
Display abstract
Inherited defects in the gene for the low density lipoprotein (LDL)-receptor give rise to familial hypercholesterolaemia (FH), a disorder in which defective catabolism of LDL causes a marked increase in its concentration in plasma. As a result, there is excessive deposition of cholesterol in the arterial wall leading to accelerated atherosclerosis and premature coronary heart disease in most patients, although there are differences in its severity. Many different mutations have been found in the LDL receptor genes of FH patients, and although this heterogeneity has provided information about the relationship between structure and function in different domains of the protein, it makes simple DNA-based diagnosis of the disease impossible. When sufficient groups of patients with defined mutations are available it will be possible to determine the relative importance of any particular mutation compared with other genetic or environmental factors in relation to the severity of their symptoms or their response to treatment.

Polvino WJ, Dichek DA, Mason J, Anderson WF
Molecular cloning and nucleotide sequence of cDNA encoding a functional murine low-density-lipoprotein receptor.
Somat Cell Mol Genet. 1992; 18: 443-50
Display abstract
The low-density-lipoprotein (LDL) receptor is an important mediator of mammalian cholesterol metabolism; its congenital absence in humans is characterized by hypercholesterolemia, atherosclerosis, and coronary artery disease. We report here the identification and cloning of a cDNA encoding the murine LDL receptor. The cDNA insert is 4467 base pairs in length and the deduced amino acid sequence bears 78.2% homology with the reported human sequence. This murine cDNA was subcloned into a retroviral-based expression vector, LmLSN1, and transfected into 3T3 cells. The production of functional LDL receptor was confirmed by ligand binding of DiI-LDL cholesterol.

Yokode M, Pathak RK, Hammer RE, Brown MS, Goldstein JL, Anderson RG
Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice.
J Cell Biol. 1992; 117: 39-46
Display abstract
When expressed in livers of transgenic mice, the human low density lipoprotein (LDL) receptor is specifically targeted to the basolateral (sinusoidal) surface of hepatocytes as determined by immunofluorescence and immunoelectron microscopy. The COOH-terminal cytoplasmic domain of the receptor (residues 790-839) contains a signal for this targeting. A mutant receptor truncated at residue 812 was localized exclusively to the apical (bile canalicular) surface. A mutant receptor terminating at residue 829 showed the normal basolateral distribution, as did a receptor in which alanine was substituted for serine 833, which was previously shown to be a site for phosphorylation in vitro. These data localize the basolateral targeting signal to the 17-residue segment between residues 812 and 828. A 10-amino acid stretch within this segment shows a 4/10 match with a sequence within a previously identified basolateral sorting motif for the receptor for polymeric IgA/IgM in MDCK cells. The four shared residues are spaced at intervals of three, raising the possibility that they all face the same side of an alpha-helix. We conclude that this 10-amino acid stretch may contain a signal that directs certain proteins, including the LDL receptor and the polymeric IgG/IgM receptor, to the basolateral surface of polarized epithelia.

Miyake Y, Tajima S, Funahashi T, Yamamura T, Yamamoto A
A point mutation of low-density-lipoprotein receptor causing rapid degradation of the receptor.
Eur J Biochem. 1992; 210: 1-7
Display abstract
The exons of the low-density-lipoprotein-(LDL)-receptor gene from a Japanese patient with homozygous familial hypercholesterolemia were amplified by the polymerase chain reaction (PCR), and their nucleotide sequences were determined. A point mutation from G to C was found in exon 9, which was expected to change Asp at position 412 to His. This amino acid change occurred within the epidermal-growth-factor-precursor homology domain of the LDL receptor, slightly impairing the processing from the precursor to the mature form and causing rapid degradation of the mature form in the fibroblasts of the patient. The mutant LDL-receptor gene transfected into COS-1 cells expressed a LDL-receptor protein with the same properties as the protein expressed in the fibroblasts of the patient; impaired processing and rapid degradation of the synthesized receptor protein. The mutation was identified in family members of the patient by dot-blot hybridization of PCR-amplified DNA with the mutant oligonucleotide. The family members carrying the mutant gene showed higher serum cholesterol levels than the others. However, their cholesterol levels were also greatly influenced by the apolipoprotein-E phenotype.

Takahashi S, Kawarabayasi Y, Nakai T, Sakai J, Yamamoto T
Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor-like protein with distinct ligand specificity.
Proc Natl Acad Sci U S A. 1992; 89: 9252-6
Display abstract
A cDNA that expresses a receptor for very low density lipoprotein (VLDL) was isolated from a rabbit heart cDNA library and characterized. The deduced amino acid sequence of the cDNA revealed that the cDNA encodes a protein with striking homology to the low density lipoprotein (LDL) receptor. Like the LDL receptor, the mature protein consists of the following five domains spanning 846 amino acids: 328 N-terminal amino acids including an 8-fold repeat of 40 amino acids homologous to the ligand binding repeat of the LDL receptor; 396 amino acid residues homologous to the epidermal growth factor precursor including three cysteine-rich repeats; a region immediately outside of the plasma membrane rich in serines and threonines; 22 amino acids traversing the plasma membrane; and 54 amino acids including the NPVY sequence that is required for clustering of the LDL receptor in coated pits and that projects into the cytoplasm. LDL-receptor-deficient Chinese hamster ovary cells transfected with the cDNA bound and internalized VLDL, beta-migrating VLDL, and intermediate density lipoprotein but did not bind LDL with high affinity. The 3.6- and 9.5-kilobase mRNAs for the VLDL receptor are highly abundant in heart, muscle, and adipose tissue. Barely detectable amounts of the mRNAs were present in liver. Based on the structural features, ligand specificity, and tissue expression of the mRNAs, we suggest that this VLDL receptor may mediate uptake of apolipoprotein E-containing lipoproteins enriched with triglyceride in nonhepatic tissues that are active in fatty acid metabolism.

Hobbs HH, Brown MS, Goldstein JL
Molecular genetics of the LDL receptor gene in familial hypercholesterolemia.
Hum Mutat. 1992; 1: 445-66
Display abstract
The low density lipoprotein (LDL) receptor is a cell surface transmembrane protein that mediates the uptake and lysosomal degradation of plasma LDL, thereby providing cholesterol to cells. Mutations disrupting the function of this receptor produce autosomal dominant familial hypercholesterolemia (FH). Affected individuals have elevated plasma levels of LDL, which causes premature coronary atherosclerosis. To date, 71 mutations in the LDL receptor gene have been characterized at a molecular level. In this report, we describe 79 additional mutations and review the insights that all 150 mutations have provided into the structure/function relationship of the receptor protein and the clinical manifestations of FH.

Gudnason V, Humphries S
PCR-based method to create a specific mutation as a reference for oligomelting.
Clin Chem. 1991; 37: 2137-8

Wilson C, Wardell MR, Weisgraber KH, Mahley RW, Agard DA
Three-dimensional structure of the LDL receptor-binding domain of human apolipoprotein E.
Science. 1991; 252: 1817-22
Display abstract
Human apolipoprotein E, a blood plasma protein, mediates the transport and uptake of cholesterol and lipid by way of its high affinity interaction with different cellular receptors, including the low-density lipoprotein (LDL) receptor. The three-dimensional structure of the LDL receptor-binding domain of apoE has been determined at 2.5 angstrom resolution by x-ray crystallography. The protein forms an unusually elongated (65 angstroms) four-helix bundle, with the helices apparently stabilized by a tightly packed hydrophobic core that includes leucine zipper-type interactions and by numerous salt bridges on the mostly charged surface. Basic amino acids important for LDL receptor binding are clustered into a surface patch on one long helix. This structure provides the basis for understanding the behavior of naturally occurring mutants that can lead to atherosclerosis.

Bansal A, Gierasch LM
The NPXY internalization signal of the LDL receptor adopts a reverse-turn conformation.
Cell. 1991; 67: 1195-201
Display abstract
Peptides corresponding to the proposed coated pit internalization signal of the native low density lipoprotein receptor, NPVY, take up in aqueous solution a reverse-turn conformation with the Asn in position i and the Tyr in position i + 3. By contrast, peptides derived from receptors that are defective for endocytosis do not adopt the reverse turn. These internalization-defective receptors include those with a nonaromatic residue substituted for the Tyr and those with Asn----Ala or Pro----Ala substitutions. While the tendency of an Asn-Pro sequence to induce a reverse turn was anticipated, the structural importance of an aromatic residue in position i + 3 was not. The sequences associated with the internalization signal thus appear to play a critical conformational role that is required for endocytosis, probably by enabling binding to adaptor molecules. With the results presented in the accompanying paper on lysosomal acid phosphatase, we now have direct evidence for previous proposals of a general correlation of internalization signals with a turn conformational motif.

Aalto-Setala K, Kontula K
Molecular genetics of familial hypercholesterolemia.
Adv Exp Med Biol. 1991; 285: 33-7

Collawn JF et al.
Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis.
Cell. 1990; 63: 1061-72
Display abstract
Using detailed functional studies on 24 human transferrin receptor mutants, we identified YXRF as the internalization sequence. Provided that at least 7 residues separate this tetrapeptide from the transmembrane region, changing the tetrapeptide position within the TR cytoplasmic domain does not reduce internalization activity. Thus, any conformational determinant for internalization must be localized to the YXRF sequence. Twenty-eight tetrapeptide analogs of YXRF, found by an unbiased search of all known three-dimensional protein structures, significantly favored tight turns similar to a type I turn. Of the ten tetrapeptides most closely related to YXRF, eight were surface exposed and had tight-turn conformations, as were four of five tetrapeptides with sequences related to the low density lipoprotein receptor internalization motif, NPXY. The internalization sequences of both receptors contain aromatic residues with intervening hydrogen-bonding residues. Thus, two distinct internalization sequences favor a common structural chemistry and implicate an exposed tight turn as the recognition motif for high efficiency endocytosis.

Leitersdorf E, Tobin EJ, Davignon J, Hobbs HH
Common low-density lipoprotein receptor mutations in the French Canadian population.
J Clin Invest. 1990; 85: 1014-23
Display abstract
Familial hypercholesterolemia (FH) has a frequency of 0.2% in most populations of the world. In selected populations such as the Afrikaners in South Africa, the Christian Lebanese, and the French Canadians, the disease is more frequent due to the founder effect. Previous studies demonstrated that a single mutation at the LDL receptor locus, the so-called French Canadian deletion, makes up 60% of the mutant genes responsible for FH in the French Canadian population. In this study, efforts were directed to determine if there were other common LDL receptor mutations in this population. Three missense mutations were identified and each mutation was reproduced and expressed in vitro. Two of the three mutations result in the production of an LDL receptor protein that is not processed to its mature form at a normal rate. Molecular assays were developed to detect the mutations directly, and the LDL receptor genes of 130 French Canadian FH heterozygotes were screened for the presence of the three missense mutations as well as two deletions. LDL receptor mutations were detected in 76% of individuals and 14% had one of the three missense mutations.

Aalto-Setala K
Molecular genetics of hypercholesterolemia.
Ann Med. 1990; 22: 1-2

Kuwano M, Ono M
Somatic cell mutants of low density lipoprotein receptor.
Cell Struct Funct. 1989; 14: 501-7

Raychowdhury R, Niles JL, McCluskey RT, Smith JA
Autoimmune target in Heymann nephritis is a glycoprotein with homology to the LDL receptor.
Science. 1989; 244: 1163-5
Display abstract
The pathogenesis of Heymann nephritis, a rat model of human membranous glomerulonephritis, depends on the interaction of autoantibodies with a renal glycoprotein (GP330) on glomerular podocytes. Partial complementary DNAs coding for GP330 were isolated and sequenced. The deduced amino acid sequence from 4.3 kilobases of complementary DNA contains the sequences identical to two peptides derived from the isolated glycoprotein. The deduced amino acid sequence of this protein contains regions with homology to the human low density lipoprotein (LDL) receptor, an indication that GP330 and the LDL receptor may be members of the same gene family. Autoantibodies from the kidneys of rats with Heymann nephritis reacted with a nonglycosylated segment of GP330 that contains cysteine-rich 40-amino acid repeats, which are also features of the LDL receptor. GP330 is also similar in some regions to the mouse epidermal growth factor precursor.

Russell DW, Brown MS, Goldstein JL
Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins.
J Biol Chem. 1989; 264: 21682-8
Display abstract
Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.

Chen GQ
[Biochemistry of LDL receptors]
Sheng Li Ke Xue Jin Zhan. 1989; 20: 167-70

Lee LY et al.
Nucleotide sequence of the rat low density lipoprotein receptor cDNA.
Nucleic Acids Res. 1989; 17: 1259-60

Schneider WJ
An update on uptake: the LDL receptor.
Can J Cardiol. 1989; 5: 0-0

Russell DW, Esser V, Hobbs HH
Molecular basis of familial hypercholesterolemia.
Arteriosclerosis. 1989; 9: 813-813
Display abstract
Familial hypercholesterolemia (FH) is a genetic disease characterized by an elevated level of low density lipoprotein (LDL), xanthomas, and an increased frequency of heart attacks. One of the first descriptions of this disease was reported some 50 years ago by the Norwegian physician, Carl Muller. Research and clinical studies in the ensuing half century have shown that FH is caused by mutations in the gene for the LDL receptor. In this article, we review our studies of the last 5 years that have focused on the molecular genetics of the LDL receptor locus and its pathogenesis in FH.

Ebert DL, Maeda N, Lowe SW, Hasler-Rapacz J, Rapacz J, Attie AD
Primary structure comparison of the proposed low density lipoprotein (LDL) receptor binding domain of human and pig apolipoprotein B: implications for LDL-receptor interactions.
J Lipid Res. 1988; 29: 1501-9
Display abstract
Apolipoprotein B (apoB) is the predominant protein in low density lipoprotein (LDL) and is responsible for LDL binding to the LDL receptor. Although the primary amino acid sequence of human apoB has been determined, little is known about the structural domains involved in mediating apoB binding to the LDL receptor. Amino acid sequence comparisons across species lines provide a means of defining structures that are essential for function. We have sequenced a l.l kb fragment of pig apoB genomic DNA, corresponding to a 363 amino acid segment proposed to mediate human apoB binding to the LDL receptor. In human apoB this domain contains two regions enriched in positively charged amino acids flanking two disulfide-linked cysteine residues. The pig amino acid sequence shared 72% identity with the human sequence. However, there were differences that have significant structural and functional implications. Human apoB arginine-3,359 corresponds to a critical arginine (position 142) residue in the apoE LDL receptor binding domain. In the pig, this arginine residue was not conserved. Also, the two disulfide-linked cysteine residues found near the proposed apoB binding domain were not conserved in the pig. Despite these differences, pig LDL had a higher affinity than human LDL for both the pig and human LDL receptor. Thus, these features are not required for high affinity binding of pig LDL to the LDL receptor, and may not be necessary for the binding of human LDL to the LDL receptor.

Leitersdorf E, Hobbs HH, Fourie AM, Jacobs M, van der Westhuyzen DR, Coetzee GA
Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia.
Proc Natl Acad Sci U S A. 1988; 85: 7912-6
Display abstract
The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each approximately 40 amino acids long. Previous studies by van Driel et al. [van Driel, I. R., Goldstein, J. L., Sudhof, T. C. & Brown, M. S. (1987) J. Biol. Chem. 262, 17443-17449] showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of 125I-labeled LDL but not 125I-labeled IgG-C7. Previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new PstI restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate. Thus, deletion of two amino acids within the first cysteine-rich repeat retards receptor transport from the endoplasmic reticulum to the cell surface, in contrast to deletion of the entire first repeat, which has no effect on receptor maturation.

Aalto-Setala K
The Finnish type of the LDL receptor gene mutation: molecular characterization of the deleted gene and the corresponding mRNA.
FEBS Lett. 1988; 234: 411-6
Display abstract
In one third of Finnish patients with the heterozygous form of familial hypercholesterolemia the disease is due to a gross deletion at the 3'-end of the LDL receptor gene. The present study demonstrates that an 8-kb deletion completely eliminates exons 16 and 17 and a part of exon 18. Cloning and partial sequencing of a DNA fragment from the mutated allele indicated that the 5'-boundary of the deletion lies within intron 15 while the 3'-breakpoint is located at nucleotide 3390 in exon 18. RNA blot hybridization studies revealed that the mutated allele encodes a truncated 4.2 kb mRNA (normal, 5.3 kb). This type of mutation has not been reported in other ethnic groups.

Funahashi T, Miyake Y, Yamamoto T
[LDL receptor abnormality]
Tanpakushitsu Kakusan Koso. 1988; 33: 779-82

Esser V, Russell DW
Transport-deficient mutations in the low density lipoprotein receptor. Alterations in the cysteine-rich and cysteine-poor regions of the protein block intracellular transport.
J Biol Chem. 1988; 263: 13276-81
Display abstract
Certain individuals with familial hypercholesterolemia (FH) produce mutant forms of the low density lipoprotein (LDL) receptor that fail to move from the endoplasmic reticulum to the Golgi complex. Here, we describe the cloning and expression of one such mutant allele, FH 429. The mutation causes a substitution of a Val for a Gly at residue 544. When recreated in an expressible cDNA, this substitution gives rise to an LDL receptor that is not transported to the cell surface and is rapidly degraded. Three previously mapped transport-deficient alleles of the LDL receptor were traced to the cysteine-rich repeats of the protein, suggesting that the generation of non-disulfide-bonded (free) cysteines might cause the block in transport. The FH 429 mutation is not located in a cysteine-rich region, however. We have attempted to test the role of cysteine by expressing mutant cDNAs that encode proteins blocked in transport and predicted to contain free cysteines. The results suggest that free cysteines are not obligatory for the blocked intracellular movement of mutant LDL receptors.

Esser V, Limbird LE, Brown MS, Goldstein JL, Russell DW
Mutational analysis of the ligand binding domain of the low density lipoprotein receptor.
J Biol Chem. 1988; 263: 13282-90
Display abstract
The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.

Yamamoto A
[LDL-receptor]
Kokyu To Junkan. 1988; 36: 833-40

Sunaga T, Takashima T, Matsui Y
[Lipoprotein metabolism and the molecular structure of the LDL receptor]
Nippon Rinsho. 1987; 45: 213-29

Lehrman MA, Russell DW, Goldstein JL, Brown MS
Alu-Alu recombination deletes splice acceptor sites and produces secreted low density lipoprotein receptor in a subject with familial hypercholesterolemia.
J Biol Chem. 1987; 262: 3354-61
Display abstract
A Japanese subject with homozygous familial hypercholesterolemia was found to have a 7.8-kilobase deletion in the gene for the low density lipoprotein receptor. The deletion joins intron 15 to the middle of exon 18, which encodes the 3' untranslated region, thereby removing all 3' splice acceptor sites distal to intron 15. By S1 nuclease mapping, we demonstrated that the 5' splice donor site of intron 15 is no longer used. Instead a continuous transcript is produced in which exon 15 is followed by the remaining segments of intron 15 and exon 18. The translational reading frame of exon 15 continues for 165 nucleotides into intron 15 before a termination codon is reached. This mRNA should produce a truncated receptor that lacks the normal membrane-spanning region and cytoplasmic domain and that has 55 novel amino acids at its COOH terminus. A cDNA expression vector containing this sequence produced a receptor that behaved similarly to the truncated protein produced by the Japanese patient, i.e. greater than 90% of the receptor was secreted from the cell, and the receptors remaining on the surface showed defective internalization. The deletion in this subject resulted from a recombination between two repetitive sequences of the Alu family, one in intron 15 and the other in exon 18. To date, Alu sequences have been observed at the deletion joints of all four gross deletions in the low density lipoprotein receptor gene that have been characterized. Within these Alu sequences, six out of the seven breakpoints have occurred in the left arm. These data suggest that recombination between Alu sequences may be a frequent cause of deletions in the human genome.

Lehrman MA et al.
The Lebanese allele at the low density lipoprotein receptor locus. Nonsense mutation produces truncated receptor that is retained in endoplasmic reticulum.
J Biol Chem. 1987; 262: 401-10
Display abstract
We here describe a mutant low density lipoprotein receptor gene that produces a shortened receptor protein lacking three domains: the region of clustered O-linked carbohydrates, the membrane-spanning region, and the cytoplasmic tail. The defect is attributable to a single nucleotide substitution that creates a premature termination codon at amino acid 660, eliminating 180 residues from the mature protein. The truncated protein retains only two domains: a complete ligand-binding region (residues 1-292) and a partial epidermal growth factor precursor homology region (residues 293-659). The termination codon occurs in the middle of a cysteine-rich sequence that is part of the epidermal growth factor precursor homology domain. The mutant protein is present in markedly reduced amounts and may be translated at a reduced rate. After synthesis, most of the receptor remains within the cell for several hours with its N-linked carbohydrate in an unprocessed endoglycosidase H-sensitive form. This finding suggests that the shortened receptor leaves the endoplasmic reticulum at an abnormally slow rate, which is likely attributable to abnormal folding of the truncated protein. The mutation creates a new restriction site for the enzyme HinfI, thus permitting diagnosis by Southern blotting of genomic DNA. Two copies of this mutant gene were present in each of four unrelated Arab patients with homozygous familial hypercholesterolemia (three from Lebanon and one from Syria). We believe that this mutation, hereafter referred to as the "Lebanese allele," is responsible for the extraordinarily high incidence of familial hypercholesterolemia in Lebanon.

Davis CG, Goldstein JL, Sudhof TC, Anderson RG, Russell DW, Brown MS
Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region.
Nature. 1987; 326: 760-5
Display abstract
A domain in the low-density lipoprotein receptor contains three cysteine-rich 'growth factor' repeats like those that occur in many proteins. When this domain is deleted, the receptor no longer releases its ligand at acid pH, it is no longer recycled efficiently and it is rapidly degraded after ligand binding.

Davis CG
The LDL receptor: oligonucleotide-directed mutagenesis of the cytoplasmic domain.
Ciba Found Symp. 1987; 130: 34-51
Display abstract
Mutations at many different sites in the gene for the low density lipoprotein (LDL) receptor can cause the disease familial hypercholesterolaemia. A particularly interesting class of mutations includes those producing 'internalization-defective' receptors-receptors which are expressed on the cell surface and which bind LDL normally, but which fail to cluster in coated pits. This defect was first observed in fibroblasts from patient J.D. Cloning and sequencing of the terminal exons of J.D.'s internalization-defective LDL receptor gene revealed a single point mutation which caused the substitution of cysteine for tyrosine at residue 807. On the basis of this observation, we have used techniques of oligonucleotide-directed mutagenesis to make an extensive series of mutations in the full-length LDL receptor cDNA. Stable cell-lines expressing these mutant receptors have been analysed for receptor function. Of 13 different amino acids expressed at residue 807, only the aromatics tyrosine, phenylalanine and tryptophan allowed rapid internalization. Position 807 seems to be a particularly sensitive site, since neither substitution of a cysteine for residue 806 or 808 nor deletion of two triplets downstream had any effect on receptor internalization. In addition, a series of truncations localize the signals for internalization to the membrane-proximal 22 amino acid residues.

Russell DW
Protein domains of the low density lipoprotein receptor.
Acta Med Scand Suppl. 1987; 715: 39-44

Kingsley DM, Kozarsky KF, Hobbie L, Krieger M
Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant.
Cell. 1986; 44: 749-59
Display abstract
We previously isolated an unusual hamster cell mutant (ldlD) that does not express LDL receptor activity unless it is cocultivated with other cells or grown in high concentrations of serum. We now show that ldlD cells are deficient in the enzyme UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) 4-epimerase. When ldlD cells are grown in glucose-based media, they cannot synthesize enough UDP-galactose and UDP-GalNAc to allow normal synthesis of glycolipids and glycoproteins. The 4-epimerase deficiency accounts for all glycosylation defects previously observed in ldlD cells, including production of abnormal LDL receptors. All abnormal phenotypes of ldlD cells can be fully corrected by exogenous galactose and GalNAc. The separate effects of these sugars on LDL receptor activity suggest that O-linked carbohydrate chains are crucial for receptor stability. ldlD cells may be useful for structural and functional studies of many proteins, proteoglycans, and glycolipids containing galactose or GalNAc.

Yamamoto T, Bishop RW, Brown MS, Goldstein JL, Russell DW
Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit.
Science. 1986; 232: 1230-7
Display abstract
The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal with familial hypercholesterolemia, produces a mutant receptor for plasma low-density lipoprotein (LDL) that is not transported to the cell surface at a normal rate. Cloning and sequencing of complementary DNA's from normal and WHHL rabbits, shows that this defect arises from an in-frame deletion of 12 nucleotides that eliminates four amino acids from the cysteine-rich ligand binding domain of the LDL receptor. A similar mutation, detected by S1 nuclease mapping of LDL receptor messenger RNA, occurred in a patient with familial hypercholesterolemia whose receptor also fails to be transported to the cell surface. These findings suggest that animal cells may have fail-safe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues.

Auwerx J, de Loof H, Verhoeven G
Alu sequences in the LDL receptor messenger RNA.
Nature. 1986; 321: 733-4

Hobbs HH, Brown MS, Goldstein JL, Russell DW
Deletion of exon encoding cysteine-rich repeat of low density lipoprotein receptor alters its binding specificity in a subject with familial hypercholesterolemia.
J Biol Chem. 1986; 261: 13114-20
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The proposed ligand binding domain of the low density lipoprotein (LDL) receptor consists of a 40-amino acid cysteine-rich unit that is repeated with some variation seven times. We describe here a mutant allele at the LDL receptor locus in which one of the seven repeats has been deleted. This mutation was found in a patient with the clinical syndrome of homozygous familial hypercholesterolemia. By molecular cloning, we show that the deletion arose by homologous recombination between repetitive Alu sequences in intron 4 and intron 5 of the gene. The deletion removes exon 5, which normally encodes the sixth repeat of the ligand binding domain. In the resultant mRNA, exon 4 is spliced to exon 6, preserving the reading frame. This mRNA produces a shortened protein that reaches the cell surface and reacts with anti-receptor antibodies but does not bind LDL, which contains apoprotein B-100 as its major protein component. Surprisingly, the deleted protein retains the ability to bind and internalize beta-migrating very low density lipoprotein, a lipoprotein that contains apoprotein E as well as apoprotein B-100. These data support the hypothesis that the seven repeated sequences in the receptor constitute the LDL binding domain. The data further indicate that the sixth repeat is required for binding of LDL, but not beta-migrating very low density lipoprotein, and that deletion of a single cysteine-rich repeat can alter the binding specificity of the LDL receptor.

Davis CG, Lehrman MA, Russell DW, Anderson RG, Brown MS, Goldstein JL
The J.D. mutation in familial hypercholesterolemia: amino acid substitution in cytoplasmic domain impedes internalization of LDL receptors.
Cell. 1986; 45: 15-24
Display abstract
Genomic DNA encompassing the terminal exons of the gene for the low density lipoprotein (LDL) receptor was isolated from J.D., a patient with familial hypercholesterolemia whose receptor fails to cluster in coated pits. The DNA sequence revealed a substitution of a cysteine codon for a tyrosine codon at residue 807 in the cytoplasmic domain of the receptor. We reproduced this substitution through oligonucleotide-directed mutagenesis of the normal human receptor cDNA. Upon transfection into receptor-deficient hamster cells, the cDNA specified a receptor that bound LDL normally, but entered the cell slowly. Electron microscopy showed that this receptor was distributed diffusely over the cell surface, whereas the receptor produced by the normal cDNA was concentrated in coated pits. These results support the hypothesis that cytoplasmic domains direct receptors to coated pits, thereby determining the high rate of receptor internalization in animal cells.

De Loof H, Rosseneu M, Brasseur R, Ruysschaert JM
Use of hydrophobicity profiles to predict receptor binding domains on apolipoprotein E and the low density lipoprotein apolipoprotein B-E receptor.
Proc Natl Acad Sci U S A. 1986; 83: 2295-9
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We have used mean hydrophobicity and hydrophobic moment calculations to predict the receptor binding domains in apolipoprotein E and in the low density lipoprotein apolipoprotein B-E receptor. In apolipoprotein E, two receptor binding domains, residues 136-160 and 214-236, having a high hydrophilicity and a high mean helical hydrophobic moment, were identified. The first domain has been located experimentally and mutations influencing the hydrophobicity parameters of the binding site have been shown to affect the receptor binding. The second domain is probably, either separately or in combination with the first domain, involved in receptor binding or in heparin binding. In the low density lipoprotein apolipoprotein B-E receptor, six protein domains were identified. In the first domain (residues 1-371), eight hydrophilic maxima, organized in pairs through disulfide bonds, form the four experimentally observed receptor binding sites. These sites consist of repeats of 26 amino acids but differ from those reported by others [Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. W. & Russell, D. W. (1984) Cell 39, 27-38]. The second, more hydrophobic, domain (residues 372-640) forms the core of the receptor, explaining its homology with the precursor of mouse epidermal growth factor, while the cysteine residues in the third domain (residues 641-699), interacting with those of the first domain, further stabilize the molecule. Beyond the fourth hydrophilic domain (residues 700-767), to which carbohydrates are linked, a very hydrophobic membrane spanning region (residues 768-789) could be detected easily. The last domain (residues 790-839), situated in the cytoplasma, contains hydrophilic maxima, as this region might interact with clathrin-related proteins. These data suggest that hydrophobicity analysis can detect and predict protein domains: hydrophilic receptor sites as well as hydrophobic core-forming and membrane-spanning regions.

Russell DW, Yamamoto T
Molecular cloning of bovine LDL receptor cDNAs.
Methods Enzymol. 1986; 128: 895-909
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Using methods described above, a partial cDNA clone for the bovine LDL receptor has been isolated. DNA sequence analysis and Northern blotting experiments are used to confirm the identity of pLDLR-1. Further DNA sequence analysis of pLDLR-1 reveals that the partial cDNA insert encodes 264 amino acids corresponding to the carboxy-terminal 25% of the bovine LDL receptor. Antipeptide antibodies directed against regions of the predicted protein sequence specifically recognize the purified bovine receptor. These findings provide an independent confirmation of the identity of pLDLR-1.

Sege RD, Kozarsky KF, Krieger M
Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor.
Mol Cell Biol. 1986; 6: 3268-77
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The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

Lehrman MA, Goldstein JL, Brown MS, Russell DW, Schneider WJ
Internalization-defective LDL receptors produced by genes with nonsense and frameshift mutations that truncate the cytoplasmic domain.
Cell. 1985; 41: 735-43
Display abstract
Certain mutant alleles at the low density lipoprotein (LDL) receptor locus produce receptors that bind LDL normally, but fail to cluster in coated pits and therefore cannot transport LDL into cells. We prepared genomic DNA libraries from cells of two individuals with this phenotype (internalization-defective familial hypercholesterolemia) and isolated the segment of the gene encoding the COOH-terminal cytoplasmic domain of the receptor. One mutant gene contains a single base substitution that changes a tryptophan codon (TGG) to a termination codon (TGA). This produces a receptor with only two amino acids in the cytoplasmic domain. The second mutant gene contains a four-base duplication, producing a frameshift that alters the reading frame. The cytoplasmic tail of this receptor has six of the normal amino acids plus eight additional amino acids. These data suggest that the signal for targeting the LDL receptor to coated pits resides in the cytoplasmic domain of the molecule.

Sudhof TC, Goldstein JL, Brown MS, Russell DW
The LDL receptor gene: a mosaic of exons shared with different proteins.
Science. 1985; 228: 815-22
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The multifunctional nature of coated pit receptors predicts that these proteins will contain multiple domains. To establish the genetic basis for these domains (LDL) receptor. This gene is more than 45 kilobases in length and contains 18 exons, most of which correlate with functional domains previously defined at the protein level. Thirteen of the 18 exons encode protein sequences that are homologous to sequences in other proteins: five of these exons encode a sequence similar to one in the C9 component of complement; three exons encode a sequence similar to a repeat sequence in the precursor for epidermal growth factor (EGF) and in three proteins of the blood clotting system (factor IX, factor X, and protein C); and five other exons encode nonrepeated sequences that are shared only with the EGF precursor. The LDL receptor appears to be a mosaic protein built up of exons shared with different proteins, and it therefore belongs to several supergene families.

Goldstein JL, Brown MS
The LDL receptor and the regulation of cellular cholesterol metabolism.
J Cell Sci Suppl. 1985; 3: 131-7

Stanley KK, Kocher HP, Luzio JP, Jackson P, Tschopp J
The sequence and topology of human complement component C9.
EMBO J. 1985; 4: 375-82
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A partial nucleotide sequence of human complement component C9 cDNA representing 94% of the coding region of the mature protein is presented. The amino acid sequence predicted from the open reading frame of this cDNA concurs with the amino acid sequence at the amino-terminal end of three proteolytic fragments of purified C9 protein. No long stretches of hydrophobic residues are present, even in the carboxy-terminal half of the molecule which reacts with lipid-soluble photoaffinity probes. Monoclonal antibody epitopes have been mapped by comparing overlapping fragments of C9 molecule to which the antibodies bind on Western blots. Several of these epitopes map to small regions containing other surface features (e.g., proteolytic cleavage sites and N-linked oligosaccharide). The amino-terminal half of C9 is rich in cysteine residues and contains a region with a high level of homology to the LDL receptor cysteine-rich domains. A model for C9 topology based on these findings is proposed.

Lehrman MA, Schneider WJ, Sudhof TC, Brown MS, Goldstein JL, Russell DW
Mutation in LDL receptor: Alu-Alu recombination deletes exons encoding transmembrane and cytoplasmic domains.
Science. 1985; 227: 140-6
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The molecular size of the plasma LDL (low density lipoprotein) receptor synthesized by cultured fibroblasts from a patient with the internalization-defective form of familial hypercholesterolemia (FH 274) was smaller by 10,000 daltons than the size of the normal LDL receptor. The segment of the gene encoding the truncated portion of the FH 274 receptor was cloned into bacteriophage lambda. Comparison of the nucleotide sequences of the normal and FH 274 genes revealed a 5-kilobase deletion, which eliminated the exons encoding the membrane-spanning region and the carboxyl terminal cytoplasmic domain of the receptor. The deletion appeared to be caused by a novel intrastrand recombination between two repetitive sequences of the Alu family that were oriented in opposite directions. The truncated receptors lack membrane-spanning regions and cytoplasmic domains; they are largely secreted into the culture medium, but a small fraction remains adherent to the cell surface. The surface-adherent receptors bind LDL, but they are unable to cluster in coated pits, thus explaining the internalization-defective phenotype.

Coetzee GA, van Wingerden JJ, van der Westhuyzen DR
Low-density lipoprotein receptor deficiency resulting in familial hypercholesterolaemia in a black man. A follow-up study.
S Afr Med J. 1985; 67: 259-61
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The binding and catabolism of low-density lipoprotein (LDL) was measured in cultured skin fibroblasts and Epstein-Barr virus-transformed lymphoblastoid cells from a South African black man. On the basis of LDL receptor activity, the patient was classified as a familial hypercholesterolaemic homozygote of the LDL receptor-defective type.

Cai HJ
[Progress in LDL receptor research]
Sheng Li Ke Xue Jin Zhan. 1984; 15: 79-82

Schneider WJ, Slaughter CJ, Goldstein JL, Anderson RG, Capra JD, Brown MS
Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts.
J Cell Biol. 1983; 97: 1635-40
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The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti-receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface.