Retention of mutant low density lipoprotein receptor in endoplasmic reticulum (ER) leads to ER stress

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and retention of mutant LDLR are poorly understood. In the present study we show that the G544V mutant LDLR associates with the chaperones Grp78, Grp94, ERp72, and calnexin in the ER of transfected Chinese hamster ovary cells. Retention of the mutant LDLR was shown to cause ER stress and activation of the unfolded protein response. We observed a marked increase in the activity of two ER stress sensors, IRE1 and PERK. These results show that retention of mutant LDLR in ER induces cellular responses, which might be important for the clinical outcome of familial hypercholesterolemia.     

Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex

In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.     

Low-density lipoprotein receptor point mutation results in expression of both active and inactive surface forms of the same mutant receptor.

LDL receptors, expressed in cultured fibroblasts from patients homozygous for the FH Afrikaner-1 (FH1) mutation (Asp206 to Glu), are transported from the endoplasmic reticulum (ER) to the Golgi apparatus more slowly than in normal cells. In the present study, binding characteristics of FH1 cells for lipoprotein ligands (LDL and beta VLDL) and for receptor-specific monoclonal antibodies pointed to the existence of two surface forms of the same mutant receptor. One of these forms bound lipoproteins with normal high affinity whereas another did not. Binding studies of transfected hamster cells expressing only the mutant human gene confirmed the single-gene origin of the different forms. The existence of functionally distinct forms of the receptor protein was supported by the observation that only lipoprotein-binding receptor molecules were trapped intracellularly and degraded following ammonium chloride treatment of cells in the presence of ligand. The lipoprotein-binding receptor population was indistinguishable from normal receptors with respect to its affinity for LDL and beta VLDL, uptake and degradation of lipoprotein, and receptor recycling. Ligand blotting versus immunoblotting of receptors revealed normal-sized mutant receptors that were not recognized by lipoprotein ligand. Despite these differences, both mutant forms of the receptor were degraded at rates similar to those of normal receptors. We propose that the single amino acid substitution in this receptor interferes with the folding and/or posttranslational processing of precursor molecules in such a way that receptors adopt alternative stable structures.     

Cells of an internalization-defective familial hypercholesterolemia mutant secrete low density lipoprotein receptors

Familial hypercholesterolemia (FH) is a congenital disorder of plasma low density lipoprotein (LDL) metabolism resulting from the defect or malfunction of LDL receptors on the cell surface. In most cases of FH, LDL binding to the cell surface is disrupted, while in some special cases LDL binding to the receptors occurs normally but the internalization of the bound LDL is inhibited (internalization-defective type). We studied the biosynthesis and transport of the LDL receptor in cultured fibroblasts obtained from one of the internalization-defective mutants by using [35S]methionine labeling and detection with anti-LDL receptor antibody. The mutant cells synthesized LDL receptors with a molecular weight slightly smaller than normal as shown in SDS-polyacrylamide gel electrophoresis. A large portion of the synthesized receptors was secreted into the medium while the other portion was associated with the cells. The apparent molecular weight of the receptors secreted into the medium was about 10 kDa smaller than that of the cell-associated receptors. The cell-associated form was converted into the secreted form following a prolonged incubation of the cells, showing the precursor-product relationship between the cell-associated and the secreted forms.     

A missense mutation in the low density lipoprotein receptor gene causes familial hypercholesterolemia in Sephardic Jews

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low density lipoprotein (LDL) receptor gene. Here, we characterize an LDL receptor mutation that is associated with a distinct haplotype and that causes FH in the Jewish Sephardic population originating from Safed, a town in northern Israel. The mutation was found in eight FH families originating from this community comprising 10% of heterozygote FH index cases screened in Israel. The mutation was not found in four additional FH heterozygotes whose hypercholesterolemia co-segregated with an identical LDL receptor gene haplotype. A guanine to cytosine substitution results in a missense mutation (asp¹⁴⁷ to his) in the fourth repeat of the binding domain encoded by exon 4 of the LDL receptor gene. The mutant receptor protein was synthesized in cultured cells as a 120kDa precursor form that failed to undergo normal processing to a mature cell surface form. Most of the receptor precursors were degraded in the endoplasmic reticulum. The small number of mutant receptors on the cell surface were unable to bind LDL or very low density lipoprotein. The abnormal behavior of the mutant receptor was reproduced by site-directed mutagenesis and expression of the mutant protein in CHO cells. The mutation can be diagnosed by allele-specific oligonucleotide hybridization of polymerase chain reaction amplified DNA from FH patients.     

Transport-deficient mutations in the low density lipoprotein receptor. Alterations in the cysteine-rich and cysteine-poor regions of the protein block intracellular transport

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.     

Mutations of the low density lipoprotein receptor in Japanese kindreds with familial hypercholesterolemia

Summary Mutations of the low density lipoprotein (LDL) receptor in 16 Japanese kindreds with homozygous familial hypercholesterolemia (FH) were studied using an anti-LDL receptor antibody. The LDL receptor mutations in Japanese FH were heterogeneous and included defects in synthesis, posttranslational processing, ligand-binding activity, and internalization of the LDL receptor. Of the 16 kindreds, 10 were receptor-negative and 5, receptor-defective types and 1 was an internalization-defective type with respect to LDL binding. The receptor-negative group was further subdivided into four groups: those with cells producing (i) no immunodetectable receptor (five kindreds); (ii) 160-kd mature receptors, which were quite scarce (two kindreds); (iii) receptors that could not be processed to the mature receptor properly (two kindreds); and (iv) receptors with an apparent molecular weight smaller than normal (one kindred). The last kindred synthesized an about 155-kd mature receptor that was rapidly degraded. This finding is compatible with the low concentration of the cell surface LDL receptors and decreased binding activity for LDL in the cells of this kindred. The receptor-defective group, which could produce a residual amount of functional receptors, exhibited a lower tendency to coronary artery disease than the receptor-negative group.     

Identification and properties of the proline664-leucine mutant LDL receptor in South Africans of Indian origin.

The incidence of familial hypercholesterolemia (FH) is high among South African Indians. The proline664-leucine low density lipoprotein (LDL)-receptor mutation was detected in four apparently unrelated Indian FH families in South Africa. This mutation was originally described in an FH subject (MM) of Indian (Gujerat province) origin (Soutar et al. 1989. Proc. Natl. Acad. Sci. 86: 4166-4170). All four South African families trace their origin to the vicinity of Surat in the Gujerat province of India. Haplotype analyses revealed that both LDL receptor genes in one of the homozygous patients are the same as those in the subject MM. The phenotype of the mutant protein was analyzed in skin fibroblasts of homozygous patients. [35S]methionine pulse-chase experiments revealed that the receptor precursors were slowly processed to mature receptors. Mature mutant receptors were degraded at faster than normal rates. This mutation, which is in the epidermal growth factor (EGF)-precursor-like domain of the LDL receptor, was previously reported to yield binding-defective receptors. Here we report that the affinity of the mutant LDL receptor for both LDL and beta-very low density lipoprotein (beta-VLDL) was normal and that the steady-state level of mutant receptors was about 20% of normal. Thus, the disease FH in these subjects is presumably due to the low steady-state level of receptor molecules that are functionally normal but exhibit accelerated turnover.     

Identification of two new LDL-receptor mutations causing homozygous familial hypercholesterolemia in a South African of Indian origin

South Africans of Indian origin have a high frequency of Familial Hypercholesterolemia (FH). Fibroblasts from a South African Indian FH homozygote, D, expressed about 30% of the normal number of LDL receptors. These receptors showed defective LDL binding. Sequence and haplotype analysis revealed that D had two different mutant LDL receptor alleles: FH Durban-1 is a point mutation [asp₆₉(GAT) to tyr(TAT)] in ligand-binding repeat 2 and FH Durban-2 is a point mutation [glu₁₁₉GAG) to lys(AAG)] in ligand-binding repeat three of the LDL receptor. Single-strand conformational polymorphism analysis, which was used in the initial detection of these mutations, was also employed for subsequent population screening assays. These mutations were not detected in amy of the South African Indian FH of hypercholesterolemic patients that were screened.     

Soluble tyrosinase is an endoplasmic reticulum (ER)-associated degradation substrate retained in the ER by calreticulin and BiP/GRP78 and not calnexin

Tyrosinase is a type I membrane protein regulating the pigmentation process in humans. Mutations of the human tyrosinase gene cause the tyrosinase negative type I oculocutaneous albinism (OCAI). Some OCAI mutations were shown to delete the transmembrane domain or to affect its hydrophobic properties, resulting in soluble tyrosinase mutants that are retained in the endoplasmic reticulum (ER). To understand the specific mechanisms involved in the ER retention of soluble tyrosinase, we have constructed a tyrosinase mutant truncated at its C-terminal end and investigated its maturation process. The mutant is retained in the ER, and it is degraded through the proteasomal pathway. We determined that the mannose trimming is required for an efficient degradation process. Moreover, this soluble ER-associated degradation substrate is stopped at the ER quality control checkpoint with no requirements for an ER-Golgi recycling pathway. Co-immmunoprecipitation experiments showed that soluble tyrosinase interacts with calreticulin and BiP/GRP78 (and not calnexin) during its ER transit. Expression of soluble tyrosinase in calreticulin-deficient cells resulted in the export of soluble tyrosinase of the ER, indicating the calreticulin role in ER retention. Taken together, these data show that OCAI soluble tyrosinase is an ER-associated degradation substrate that, unlike other albino tyrosinases, associates with calreticulin and BiP/GRP78. The lack of specificity for calnexin interaction reveals a novel role for calreticulin in OCAI albinism.     

Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones

Hps47, Grp78, have been implicated with procellagen maturation events. In particular Hps47 has been shown to blind to nascent procellagen α1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collgen has previsously been suggested to mechanism. The early association of Hps47 with procollagen and its relatively late relese suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hps47. Herein, we examined how these events occurs in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the releses of Hps47, Grp78, and Grp94 from maturing procollange is delayed. Thus, in cell experiencing metabolic stress, newly synthesized procollagen unable to property fold became stable bound to a complex of molecular chaperones. In that Hps47, Grp78, and Grp98 could be recovered with nascent procollagen and as oligomer in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.     

The altered expression of glucose-regulated proteins 78 in different phase of streptozotocin-affected pancreatic beta-cells

Endoplasmic reticulum (ER) stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cells and contributes to the development of type 1 diabetes. The chaperone molecule, glucose-regulated proteins 78 (Grp78), is required to maintain ER function during toxic insults. In this study, we investigated the changes of Grp78 expression in different phases of streptozotocin (STZ)-affected beta-cells to explore the relationship between Grp78 and the response of beta-cells to ER stress. An insulinoma cell line (NIT-1) treated with STZ for different time periods and STZ-induced diabetic Balb/C mice at different time points were used as the model system. The level of Grp78 and C/EBP homologous protein (CHOP) mRNA were detected by real-time polymerase chain reaction and their protein by immunoblot. Apoptosis and necrosis was measured by flow cytometry. In addition, the changes of Grp78 protein in STZ-treated nondiabetic mice were also detected by immunoblot. Grp78 expression significantly increased in the early phase but decreased in the later phase of affected beta-cells, while CHOP was induced and apoptosis occurred along with the decrease of Grp78. Interestingly, the Grp78 protein of STZ-treated nondiabetic mice increased stably compared with that of the control. From the results, we can conclude that Grp78 may contribute to the response of beta-cells to ER stress, and more attention should be paid to Grp78 in the improvement of diabetes.     

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA-II adaptors (found in plasma membrane coated pits) and the HA-I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6-phosphate (M6P) receptor-a receptor which is concentrated in coated pits on both the plasma membrane and in the trans-Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA-II plasma membrane adaptor, which recognizes a 'tyrosine' signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA-I adaptors. This suggests that there is an HA-I recognition signal in another region of the M6P receptor tail, C-terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans-Golgi network, thus mediating efficient transfer of these enzymes to lysosomes.     

ANKRD13C acts as a molecular chaperone for G protein-coupled receptors

Although the mechanisms that regulate folding and maturation of newly synthesized G protein-coupled receptors are crucial for their function, they remain poorly characterized. By yeast two-hybrid screening, we have isolated ANKRD13C, a protein of unknown function, as an interacting partner for the DP receptor for prostaglandin D(2). In the present study we report the characterization of this novel protein as a regulator of DP biogenesis and trafficking in the biosynthetic pathway. Co-localization by confocal microscopy with an endoplasmic reticulum (ER) marker, subcellular fractionation experiments, and demonstration of the interaction between ANKRD13C and the cytoplasmic C terminus of DP suggest that ANKRD13C is a protein associated with the cytosolic side of ER membranes. Co-expression of ANKRD13C with DP initially increased receptor protein levels, whereas siRNA-mediated knockdown of endogenous ANKRD13C decreased them. Pulse-chase experiments indicated that ANKRD13C can promote the biogenesis of DP by inhibiting the degradation of newly synthesized receptors. However, a prolonged interaction between ANKRD13C and DP resulted in ER retention of misfolded/unassembled forms of the receptor and to their proteasome-mediated degradation. ANKRD13C also regulated the expression of other GPCRs tested (CRTH2, thromboxane A(2) (TPα), and β2-adrenergic receptor), whereas it did not affect the expression of green fluorescent protein, GRK2 (G protein-coupled receptor kinase 2), and VSVG (vesicular stomatitis virus glycoprotein), showing specificity toward G protein-coupled receptors. Altogether, these results suggest that ANKRD13C acts as a molecular chaperone for G protein-coupled receptors, regulating their biogenesis and exit from the ER.     

The low density lipoprotein receptor

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

Regulation of transport of the dopamine D1 receptor by a new membrane-associated ER protein

Many structural determinants for G protein-coupled receptor (GPCR) functions have been defined, but little is known concerning the regulation of their transport from the endoplasmic reticulum (ER) to the cell surface. Here we show that a carboxy-terminal hydrophobic motif, FxxxFxxxF, which is highly conserved among GPCRs, functions independently as an ER-export signal for the dopamine D1 receptor. A newly identified ER-membrane-associated protein, DRiP78, binds to this motif. Overexpression or sequestration of DRiP78 leads to retention of D1 receptors in the ER, reduced ligand binding, and a slowdown in the kinetics of receptor glycosylation. Our results indicate that DRiP78 may regulate the transport of a GPCR by binding to a specific ER-export signal.     

Disease-causing V(2) vasopressin receptors are retained in different compartments of the early secretory pathway

The G protein-coupled V(2) vasopressin receptor is crucially involved in water reabsorption in the renal collecting duct. Mutations in the human V(2) vasopressin receptor gene cause nephrogenic diabetes insipidus. Many of the disease-causing mutants are retained intracellularly by the quality control system of the early secretory pathway. It was previously thought that quality control system is restricted to the endoplasmic reticulum (ER). Here, we have examined the retention mechanisms of eight V(2) vasopressin receptor mutants. We show that mutants L62P, DeltaL62-R64 and S167L are trapped exclusively in the ER. In contrast, mutants R143P, Y205C, InsQ292, V226E and R337X reach the ER/Golgi intermediate compartment (ERGIC) and are rerouted to the ER. The ability of the mutant receptors to reach the ERGIC is independent of their expression levels. Instead, it is determined by their folding state. Mutant receptors in the ERGIC may be sorted into retrograde transport vesicles by an interaction of an RXR motif in the third intracellular loop with the coatomer complex I. Our data show that disease-causing mutants of a particular membrane protein may be retained in different compartments of the early secretory pathway and that the folding states of the proteins determine their retention mechanism.