Three types of low density lipoprotein receptor-deficient mutant have pleiotropic defects in the synthesis of N-linked, O-linked, and lipid- linked carbohydrate chains

Biochemical, immunological, and genetic techniques were used to investigate the genetic defects in three types of low density lipoprotein (LDL) receptor-deficient hamster cells. The previously isolated ldlB, ldlC, and ldlD mutants all synthesized essentially normal amounts of a 125,000-D precursor form of the LDL receptor, but were unable to process this receptor to the mature form of 155,000 D. Instead, these mutants produced abnormally small, heterogeneous receptors that reached the cell surface but were rapidly degraded thereafter. The abnormal sizes of the LDL receptors in these cells were due to defective processing of the LDL receptor's N- and O-linked carbohydrate chains. Processing defects in these cells appeared to be general since the ldlB, ldlC, and ldlD mutants also showed defective glycosylation of a viral glycoprotein, alterations in glycolipid synthesis, and changes in resistance to several toxic lectins. Preliminary structural studies suggested that these cells had defects in multiple stages of the Golgi-associated processing reactions responsible for synthesis of glycolipids and in the N-linked and O-linked carbohydrate chains of glycoproteins. Comparisons between the ldl mutants and a large number of previously isolated CHO glycosylation defective mutants showed that the genetic defects in ldlB, ldlC, and ldlD cells were unique and that only very specific types of carbohydrate alteration could dramatically affect LDL receptor function.     

Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild- type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.     

LDLC encodes a brefeldin A-sensitive,peripheral Golgi protein required for normal Golgi function

Two genetically distinct classes of low density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants, ldlB and ldlC, exhibit nearly identical pleiotropic defects in multiple medial and trans Golgi-associated processes (Kingsley, D., K. F. Kozarsky, M. Segal, and M. Krieger. 1986. J. Cell Biol. 102:1576-1585). In these mutants, the synthesis of virtually all N- and O-linked glycoproteins and of the major lipid-linked oligosaccharides is abnormal. The abnormal glycosylation of LDL receptors in ldlB and ldlC cells results in their dramatically reduced stability and thus very low LDL receptor activity. We have cloned and sequenced a human cDNA (LDLC) which corrects the mutant phenotypes of ldlC, but not ldlB, cells. Unlike wild-type CHO or ldlB cells, ldlC cells had virtually no detectable endogenous LDLC mRNA, indicating that LDLC is likely to be the normal human homologue of the defective gene in ldlC cells. The predicted sequence of the human LDLC protein (ldlCp, approximately 83 kD) is not similar to that of any known proteins, and contains no major common structural motifs such as transmembrane domains or an ER translocation signal sequence. We have also determined the sequence of the Caenorhabditis elegans ldlCp by cDNA cloning and sequencing. Its similarity to that of human ldlCp suggests that ldlCp mediates a well- conserved cellular function. Immunofluorescence studies with anti-ldlCp antibodies in mammalian cells established that ldlCp is a peripheral Golgi protein whose association with the Golgi is brefeldin A sensitive. In ldlB cells, ldlCp was expressed at normal levels; however, it was not associated with the Golgi. Thus, a combination of somatic cell and molecular genetics has identified a previously unrecognized protein, ldlCp, which is required for multiple Golgi functions and whose peripheral association with the Golgi is both LDLB dependent and brefeldin A sensitive.     

Reversible defects in O-linked glycosylation and LDL receptor expression in a UDP-Gal/UDP-GalNAc 4-epimerase deficient mutant

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.     

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

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

Heterozygous familial hypercholesterolemia: failure of normal allele to compensate for mutant allele at a regulated genetic locus.

In normal human fibroblasts, the synthesis of a cell surface receptor for plasma low density lipoprotein (LDL) is regulated by a sensitive system of feedback suppression. The number of functional LDL receptors declines by more than 20 fold when cellular stores of esterified cholesterol are increased by incubation of cells with an exogenous source of cholesterol. Fibroblasts from patients with the heterozygous form of familial hypercholesterolemia (FH) possess one functional allele and one nonfunctional allele at the LDL receptor locus. In the current studies, we have examined the effect that this deficiency produces upon the pattern of regulation of the single functional allele at the LDL receptor locus. Under growth conditions that induced a maximal rate of LDL receptor synthesis (that is, growth in the absence of an exogenous source of cholesterol), the FH heterozygote cells produced about one half as many functional LDL receptors as did the normal cells. More importantly, when grown in the presence of increasing amounts of exogenous cholesterol, the FH heterozygote and normal cells suppressed their respective LDL receptor activities in parallel. Over a wide range of LDL receptor activities, at each level of cellular esterified cholesterol, the FH heterozygote cells expressed about one half as many receptors as did the normal cells. These data indicate that in the FH heterozygote cells, the receptor regulatory mechanism dictates that the normal allele produce only the amount of gene product that it would normally produce at a given level of cellular esterified cholesterol. The failure of the regulatory mechanism to stimulate the normal allele at the LDL receptor locus to produce twice its normal amount of gene product leaves the FH heterozygote cells with a persistent 50% deficiency in LDL receptors under all conditions of cell growth.     

Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.     

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.     

Cog2 null mutant CHO cells show defective sphingomyelin synthesis

The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ～25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C(6)-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II.     

Functional role of N-linked glycosylation on the rat melanin-concentrating hormone receptor 1

Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn13, Asn16 and Asn23) for N-linked glycosylation in its extracellular amino-terminus which may modulate its reactivity. Site-directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N-linked glycosylation sites in MCHR1 were glycosylated, and that N-linked glycosylation of Asn23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N-linked glycosylation of the MCHR1.     

Regulation of the activity of the low density lipoprotein receptor in human fibroblasts.

A specific receptor on the surface of cultured human fibroblasts binds plasma low density lipoprotein (LDL) with high affinity, and thereby initiates a cellular process by which the LDL is internalized and degraded within lysosomes and its cholesterol component is made available for cellular membrane synthesis. Current studies demonstrate that the activity of this LDL receptor is under feedback regulation. Prior incubation of fibroblast monolayers with cholesterol, 25-hydroxycholesterol, or LDL progressively reduced the ability of the cells to bind 125I-labeled LDL at the high affinity site. A series of kinetic studies indicated that this reduction in binding was due to a decrease in the number of LDL receptors. From measurements of the rate of decline in 125I-LDL binding activity after administration of cycloheximide, the LDL receptor was calculated to have a half-life of about 25 hr. LDL appeared to reduce 125I-LDL-binding activity by suppressing the synthesis of receptor molecules. Thus cultured human fibroblasts regulate their intracellular cholesterol content by regulating the activity of the LDL receptor, which in turn controls the rate of cellular entry of cholesterol derived from plasma LDL contained within the culture medium.     

N-linked glycosylation of the human bradykinin B2 receptor is required for optimal cell-surface expression and coupling

To investigate the glycosylation of the human bradykinin B2 receptor and the functional significance of this modification, we studied receptors mutated at single or multiple combinations of the three potential N-linked glycosylation sites, asparagines N3, N12 and N180, in COS-7, HEK 293 and CHO-K1 cells. Western blot experiments demonstrated that all three extracellular asparagines are glycosylated. The kinetics of bradykinin binding and receptor sequestration remained unchanged after glycosylation had been suppressed. However, the glycosylated receptors were expressed at the cell-surface to a much greater extent than the non-glycosylated receptor and coupling to phospholipase C was less efficient for receptor lacking N-terminal glycosylation. These results indicate that, for the human bradykinin B2 receptor, glycosylation is not required for optimal ligand binding, but plays an important role in cell-surface addressing and receptor function.     

Control of low density lipoprotein receptor gene expression in steroidogenic cells

Low density lipoprotein (LDL)-carried cholesterol is a primary substrate for steroid hormone synthesis by luteinized human granulosa cells. Chorionic gonadotropin and 8-bromo-cAMP both increase LDL receptor levels in granulosa cells by stimulating accumulation of the receptor mRNA. LDL and 25-hydroxycholesterol reduce LDL receptor expression, but this suppressive effect is partially overcome by 8-bromo-cAMP. Using fusion gene constructs containing the LDL receptor gene promoter transfected into JEG-3 cells, a cyclic AMP responsive enhancer could not be identified in the LDL receptor gene upstream promoter in transfection studies. We suggest that the LDL receptor gene in human steroidogenic cells is under negative control by a sterol effector, but that a cyclic AMP triggered process overcomes, to some extent, the sterol-mediated suppression. The detailed mechanisms by which sterol and cyclic AMP modulate LDL receptor gene expression remain to be elucidated.     

A comparison of the low-density-lipoprotein receptor from bovine adrenal cortex, rabbit and rat liver and adrenal glands by lipoprotein blotting.

This paper describes the use of lipoprotein blotting to detect low-density-lipoprotein (LDL) receptors in rat and rabbit liver and adrenal glands and in bovine adrenal glands. Using this technique we show that the rabbit and rat liver LDL receptors have Mr values of 128000 and 145000 respectively. Mr values for the rabbit, rat and bovine adrenal receptors are 131000, 142000 and 132000 respectively. Differences between the bovine adrenal and rat liver receptors are not due to differences in the degree of sialylation. Lipoprotein blotting can be used to detect dietary- and drug-induced changes in the concentrations of LDL receptors. When rabbits are fed on a cholesterol-rich diet, liver LDL receptors cannot be detected, consistent with the suppression of hepatic LDL receptors by cholesterol feeding. Pharmacological doses of 17 alpha-ethinyloestradiol cause a marked increase in hepatic LDL-receptor activity in the rat. This is accompanied by a corresponding increase in the number of LDL receptors detected by lipoprotein blotting. The Mr of the induced receptor is identical with that of the receptor from control rats, which suggests that the induced receptors are produced by the same gene as LDL receptors normally present in the liver.     

Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells.

Recent evidence suggests the possibility that macrophages can influence lipoprotein metabolism. Therefore we investigated the ability of cultured macrophages to alter low density lipoprotein (LDL) uptake in a human liver cell line (HepG2). Conditioned media from phlogogenic-induced mouse peritoneal macrophages or from a human macrophage cell line stimulated with endotoxin increased HepG2 LDL uptake by as much as 60-70%. The increase was due, in part, to a significant macrophage-induced 40% increase in the number of LDL receptors per cell. Although macrophage conditioned media inhibited HepG2 cholesterol synthesis, the LDL receptor up-regulation did not appear to be due to the effects on cholesterol synthesis. The LDL receptor stimulatory activity was sensitive to proteolysis and heat. Its molecular mass was approximately 20 kDa based on gel filtration. Several macrophage secretory proteins were tested in HepG2 cultures for LDL uptake stimulation. Of these, oncostatin M (approximately 18 kDa by gel filtration) gave the strongest response. The rank order for LDL uptake stimulation was oncostatin M much greater than interleukin 6 = interleukin 1 = transforming growth factor-beta 1. A neutralizing antibody directed against oncostatin M inhibited the ability of conditioned media to up-regulate LDL receptors by 85%. Thus, our results indicate that macrophages can secrete several proteins that up-regulate LDL receptors in HepG2 cells and that most of the up-regulatory activity in macrophage conditioned media appears to be due to oncostatin M.     

Complete down-regulation of low-density lipoprotein receptor activity in human liver parenchymal cells by beta-very-low-density lipoprotein

The effect of LDL and beta-VLDL on the expression of the LDL receptor is studied in cultured human parenchymal cells. The high affinity binding of [125I]LDL to cultured human parenchymal cells was down regulated to 37.3 +/- 2.9% and 24.0 +/- 2.6% of the control value, after preincubation with LDL or beta-VLDL for 22 h, respectively. When LDL receptor synthesis was blocked at 22 h a residual receptor activity of 29% is noticed, indicating a half-life of LDL receptors in human parenchymal cells of 12 h. It is concluded that LDL receptor expression on human liver parenchymal cells is subject to complete down-regulation by beta-VLDL, which may be held responsible for the cholesterol-rich diet induced down-regulation of LDL receptors, in vivo.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).     

Regulation of glycosphingolipid glycosyltransferase by low density lipoprotein receptors in cultured human proximal tubular cells

We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis.     

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

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