Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia.

This paper describes a mechanism for degradation of low-density lipoprotein (LDL) in fibroblasts unable to synthesize the LDL receptor. In this cell line, long-chain free fatty acids (FFA) activated 125I-LDL uptake; unsaturated FFA were the most efficient. The first step of this pathway was the binding of LDL apoB to a single class of sites on the plasma membrane and was reversible in the presence of greater than or equal to 10 mM suramin. Binding equilibrium was achieved after a 60-90-min incubation at 37 degrees C with 1 mM oleate; under these conditions, the apparent Kd for 125I-LDL binding was 12.3 micrograms/mL. Both cholesterol-rich (LDL and beta-VLDL) and triglyceride-rich (VLDL) lipoproteins, but not apoE-free HDL, efficiently competed with 125I-LDL for this FFA-induced binding site. After LDL bound to the cell surface, they were internalized and delivered to lysosomes; chloroquine inhibited subsequent proteolysis of LDL and thereby increased the cellular content of the particles. A physiological oleate to albumin molar ratio, i.e., 1:1 (25 microM oleate and 2 mg/mL albumin), was sufficient to significantly (p less than 0.01) activate all three steps of this alternate pathway: for example, 644 +/- 217 (25 microM oleate) versus 33 +/- 57 (no oleate) ng of LDL/mg of cell protein was degraded after incubation (2 h, 37 degrees C) with 50 micrograms/mL 125I-LDL. We speculate that this pathway could contribute to the clearance of both chylomicron remnants and LDL.     

Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.

125I-labeled and ferritin-labeled low density lipoprotein (LDL) were used as visual probes to study the surface distribution of LDL receptors and to examine the mechanism of the endocytosis of this lipoprotein in cultured human fibrobasts. Light microscopic autoradiograms of whole cells incubated with 125I-LDL at 4 degrees C showed that LDL receptors were widely but unevenly distributed over the cell surface. With the electron microscope, we determined that 60-70% of the ferritin-labeled LDL that bound to cells at 4 degrees C was localized over short coated segments of the plasma membrane that accounted for no more than 2% of the total surface area. To study the internalization process, cells were first allowed to bind ferritin-labeled LDL at 4 degrees C and were then warmed to 37 degrees C. Within 10 min, nearly all the surface-bound LDL-ferritin was incorporated into coated endocytic vesicles that were formed by the invagination and pinching-off of the coated membrane regions that contained the receptor-bound LDL. With increasing time at 37 degrees C, these coated vesicles were observed sequentially to migrate through the cytoplasm (1 min), to lose their cytoplasmic coat (2 min), and to fuse with either primary or secondary lysosomes (6 min). The current data indicate that the coated regions of plasma membrane are specialized structures of rapid turnover that function to carry receptor-bound LDL, and perhaps other receptor-bound molecules, into the cell.     

Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.     

Specific, saturable, and high affinity binding of 125I-low density lipoprotein to glass beads.

¹²⁵I-Low density lipoprotein (¹²⁵I-LDL)¹ binds tightly to glass beads at physiologic pH and ionic strength. This binding shows saturability, high affinity (half maximal binding achieved at 10–15 μg protein/ml), and specificity (unlabeled LDL but not HDL or albumin competes with ¹²⁵I-LDL for binding to the glass beads). In contrast to the binding of ¹²⁵I-LDL to the physiologic LDL receptor on the surface of human fibroblasts and lymphocytes, the binding of ¹²⁵I-LDL to bind to inert substances such as glass must be considered in the interpretation of studies in which ¹²⁵I-LDL binding to membrane receptors is measured. The data emphasize the importance of correlating observed ¹²⁵I-LDL binding with a physiologic action of the lipoprotein.     

Subcellular structures involved in internalization and degradation of epidermal growth factor

Epidermal Growth Factor (EGF), a small polypeptide which acts as a mitogen for many cell types, has previously been shown to bind to a specific plasma membrane receptor on 3T3 cells. If ¹²⁵I-EGF is bound to 3T3 cells for one hour at 4°C, it remains predominantly associated with the plasma membrane-containing fractions obtained by subjecting cell supernatants to equilibrium sedimentation on sucrose gradients. When binding is followed by a 10-minute incubation at 37°C, over 50% of the ¹²⁵I-EGF is associated with two internal membrane-containing peaks having higher densities than the plasma membrane. After one hour at 37°C, over 80% of the ¹²⁵I-EGF is degraded and removed from the cells. The most rapidly labeled internal peak corresponds in density to brain-coated vesicles (CVs). Antiserum prepared against coated vehicles from brain precipitates the ¹²⁵I-EGF in this peak. In addition, CVs containing ¹²⁵I-EGF can be co-purified from 3T3 cells exposed to ¹²⁵I-EGF, using brain as a carrier. Several lines of evidence suggest that the other ¹²⁵I-EGF-labeled intracellular peak is ¹²⁵I-EGF in lysosomes. These results provide kinetic and biochemical evidence for a unidirectional pathway for EGF catabolism by 3T3 cells. EGF first binds to the plasma membrane bound receptors, is then moved to the cytoplasm in CVs, and finally appears in lysosomes, where it is degraded and released from the cells. Ten-millimolar NH₄Cl blocks lysosomal hydrolysis of EGF almost completely. Subsequently, EGF internalization is inhibited. This finding suggests that the pathway for EGF internalization and degradation is tightly coupled.     

Apolipoprotein E and Low-Density Lipoprotein Binding and Internalization in Primary Cultures of Rat Astrocytes: Isoform-Specific Alterations

Abstract: Apolipoprotein (apo) E is likely involved in redistributing cholesterol and phospholipids during compensatory synaptogenesis in the injured CNS. Three common isoforms of apoE exist in human (E2, E3, and E4). The apoE4 allele frequency is markedly increased in both late-onset sporadic and familial Alzheimer's disease (AD). ApoE concentration in the brain of AD subjects follows a gradient: ApoE levels decrease as a function of E2 > E3 ? E4. It has been proposed that the poor reinnervation capacity reported in AD may be caused by impairment of the apoE/low-density lipoprotein (LDL) receptor activity. To understand further the role of this particular axis in lipid homeostasis in the CNS, we have characterized binding, internalization, and degradation of human ¹²⁵I-LDL to primary cultures of rat astrocytes. Specific binding was saturable, with a K_D of 1.8 nM and a B_max of 0.14 pmol/mg of proteins. Excess unlabeled human LDL or very LDL (VLDL) displaced 70% of total binding. Studies at 37°C confirmed that astrocytes bind, internalize, and degrade ¹²⁵I-LDL by a specific, saturable mechanism. Reconstituted apoE (E2, E3, and E4)-liposomes were labeled with ¹²⁵I and incubated with primary cultures of rat astrocytes and hippocampal neurons to examine specific binding. Human LDL and VLDL displaced binding and internalization of all apoE isoforms similarly in both astrocytes and neurons. ¹²⁵I-ApoE2 binding was significantly lower than that of the other ¹²⁵I-apoE isoforms in both cell types. ¹²⁵I-ApoE4 binding was similar to that of ¹²⁵I-apoE3 in both astrocytes and neurons. On the other hand, ¹²⁵I-apoE3 binding was significantly higher in neurons than in astrocytes. These isoform-specific alterations in apoE-lipoprotein pathway could explain some of the differences reported in the pathophysiology of AD subjects carrying different apoE alleles.     

Characterization of the parathyrin receptor in renal plasma membranes by labelled hormones and labelled antibody binding techniques

The parathyrin receptor in renal cortex has been investigated by studying the binding of ¹²⁵I-labelled parathyrin, or of unlabelled parathyrin detected with ¹²⁵I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 °C but this phenomenon was not detectable at 37 °C. Specific displacement of lactoperoxidase labelled ¹²⁵I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was $\text{2.3 · 10}{}^8\text{M}{}^{\mathrm{?1}}$ and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0–7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1–34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glueagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.     

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of ¹²⁵I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of ¹²⁵I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10⁸ cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of ¹²⁵I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.     

Binding and degradation of 125I-labeled insulin by a clonal line of rat pituitary tumor cells

Receptor sites for insulin on GH₃ cells were characterized. Uptake of ¹²⁵I-labeled insulin by the cells was dependent upon time and temperature, with apparent steady-states reached by 120, 20 and 10 min at 4, 23 and 37°C, respectively. The binding sites were sensitive to trypsin, suggesting that the receptors contain protein. Insulin competed with ¹²⁵I-labeled insulin for binding sites, with half-maximal competition observed at 5 nM insulin. Neither adrenocorticotropic hormone nor growth hormone competed for ¹²⁵I-labeled insulin binding sites. ¹²⁵I-labeled insulin binding was reversible, and saturable with respect to hormone concentration. ¹²⁵I-labeled insulin was degraded at both 4 and 37°C by GH₃ cells, but not by medium conditioned by these cells. After a 5 min incubation at 37°C, products of ¹²⁵I-labeled insulin degradation could be recovered from the cells but were not detected extracellularly. Extending the time of incubation resulted in the recovery of fragments of ¹²⁵I-labeled insulin from both cells and the medium. Native insulin inhibited most of the degradation of ¹²⁵I-labeled insulin suggesting that degradation resulted, in part, from a saturable process. At steady-state, degradation products of ¹²⁵I-labeled insulin, as well as intact hormone, were recovered from GH₃ cells. After 30 min incubation at 37°C, 80% of the cell-bound radioactivity was not extractable from GH₃ cells with acetic acid.     

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.     

Differences in time course of internalization of receptors of insulin and insulin-like growth factor (IGF-1) in isolated rat hepatocytes

Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is a most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones at isolated rat hepatocytes, the internalization time course of ¹²⁵I-insulin and ¹²⁵I-IGF-I are traced at 37 and 12°C. There are established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37°C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. But essential differences in the internalization course of these two related peptides were obvious at the temperature of 12°C. The internalization level of insulin receptors at 12°C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocyte plasma membrane. At 12°C a slight decrease of the proportion of intracellular ¹²⁵I-IGF-I correlated with a decrease in the ¹²⁵I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12°C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar “inhibition mechanism” of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to action of cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.     

Binding, internalization, and degradation of high density lipoprotein by cultured normal human fibroblasts.

Comparative studies were made of the metabolism of plasma high density lipoprotein (HDL) and low density lipoprotein (LDL) by cultured normal human fibroblasts. On a molar basis, the surface binding of (125)I-HDL was only slightly less than that of (125)I-LDL, whereas the rates of internalization and degradation of (125)I-HDL were very low relative to those of (125)I-LDL. The relationships of internalization and degradation to binding suggested the presence of a saturable uptake mechanism for LDL functionally related to high-affinity binding. This was confirmed by the finding that the total uptake of (125)I-LDL (internalized plus degraded) at 5 micro g LDL protein/ml was 100-fold greater than that attributable to fluid or bulk pinocytosis, quantified with [(14)C]sucrose, and 10-fold greater than that attributable to the sum of fluid endocytosis and adsorptive endocytosis. In contrast, (125)I-HDL uptake could be almost completely accounted for by the uptake of medium during pinocytosis and by invagination of surface membrane (bearing bound lipoprotein) during pinocytosis. These findings imply that, at most, only a small fraction of bound HDL binds to the high-affinity LDL receptor and/or that HDL binding there is internalized very slowly. The rate of (125)I-HDL degradation by cultured fibroblasts (per unit cell mass) exceeded an estimate of the turnover rate of HDL in vivo, suggesting that peripheral tissues may contribute to HDL catabolism. In accordance with their differing rates of uptake and cholesterol content, LDL increased the cholesterol content of fibroblasts and selectively inhibited sterol biosynthesis, whereas HDL had neither effect.     

Oxidized low density lipoprotein is resistant to cathepsins and accumulates within macrophages

The rate of uptake of oxidized low density lipoprotein (LDL) by mouse peritoneal macrophages is similar to that of acetyl LDL; but only approximately 50% of the internalized oxidized LDL is ultimately degraded, in contrast to the near-complete degradation seen with acetyl LDL. The objectives of this study were to determine if this was due to increased surface binding of oxidized LDL, different uptake pathways for oxidized LDL and acetyl LDL, lysosomal dysfunction caused by oxidized LDL, or resistance of oxidized LDL to hydrolysis by lysosomal proteinases. LDL binding studies at 4 degrees C showed that the increased cell association with oxidized LDL could not be explained by differences in cell-surface binding. Immunofluorescence microscopy confirmed intracellular accumulation of apoB-immunoreactive material in macrophages incubated with oxidized LDL, but not with acetyl LDL. The scavenger receptor ligand polyinosinic acid inhibited both the cell association and degradation of oxidized LDL in macrophages by greater than 75%, suggesting a common uptake pathway for degraded LDL and nondegraded LDL. Studies in THP-1 cells also did not reveal more than one specific uptake pathway for oxidized LDL. LDL derivatized by incubation with oxidized arachidonic acid (under conditions that prevented oxidation of the LDL itself) showed inefficient degradation, similar to oxidized LDL. When macrophages were incubated with oxidized LDL together with acetyl 125I-LDL, the acetyl LDL was degraded normally, excluding lysosomal dysfunction as the explanation for the accumulation of oxidized LDL. Generation of trichloroacetic acid-soluble products from oxidized 125I-LDL by exposure to cathepsins B and D was less than that observed with native 125I-LDL. LDL modified by exposure to reactive products derived from oxidized arachidonic acid was also degraded more slowly than native 125I-LDL by cathepsins. In contrast, acetyl 125I-LDL was degraded more rapidly by cathepsins than native 125I-LDL, and aggregated LDL and malondialdehyde-modified LDL were degraded at the same rate as native 125I-LDL. It is concluded that the intracellular accumulation of oxidized LDL in macrophages can be explained at least in part by the resistance of oxidatively modified apolipoprotein B to cathepsins. This resistance to cathepsins does not appear to be due to aggregation of oxidized LDL, but may be a consequence of modification of apolipoprotein B by lipid peroxidation products.     

Processing and characterization of the low density lipoprotein receptor in the human colonic carcinoma cell subclone HT29-18: A potential pathway for delivering therapeutic drugs and genes

Low density lipoprotein (LDL) processing has been investigated in the subcloned human colonic carcinoma cell line HT29-18. LDL binding at 4°C was a saturable process in relation to time and LDL concentration. The Kd for LDL binding was 11 g/ml. ApoE-free HDL₃ or acetylated LDL did not significantly compete with¹²⁵I-LDL binding, up to 500 g/ml.¹²⁵I-LDL binding was decreased by 70% in HT29-18 cells preincubated for 24 hours in culture medium containing 100 g/ml unlabelled LDL. Ligand blotting studies performed on HT29-18 homogenates using colloidal gold labelled LDL indicated the presence of one autoradiographic band corresponding to an apparent molecular weight of 130 kDa, which is consistent with the previously reported molecular weight of the LDL receptor in human fibroblasts. At 37°C,¹²⁵I-LDL was actively internalized by HT29-18 cells and lysosomal degradation occurred as demonstrated by the inhibitory effect of chloroquine. LDL uptake and degradation by HT29-18 cells also resulted in a marked decrease in endogenous sterol synthesis. These data demonstrate that the HT29-18 human cancerous intestinal cells are able to specifically bind and internalize LDL, and that LDL processing results in down-regulation of sterol biosynthesis. Thus, intestinal epithelial cells possess specific LDL receptors that can be exploited to accomplish drug delivery and gene transfer via the receptor-mediated endocytosis pathway.Abbreviations HDL, HCL₃ high density lipoprotein - LDL low density lipoprotein     

Evidence for isomerization during binding of apolipoprotein-B100 to low density lipoprotein receptors.

To determine the kinetics of human low density lipoproteins (LDL) interacting with LDL receptors, 125I-LDL binding to cultured human fibroblasts at 4 degrees C was studied. Apparent association rate constants did not increase linearly as 125I-LDL concentrations were increased. Instead, they began to plateau which suggested that formation of initial receptor-ligand complexes is followed by slower rearrangement or isomerization to complexes with higher affinity. To test this, 125I-LDL were allowed to associate for 2, 15, or 120 min, then dissociation was followed. The dissociation was biphasic with the initial phase being 64-110-fold faster than the terminal phase. After binding for 2 min, a greater percentage of 125I-LDL dissociated rapidly (36%) than after association for 15 min (24%) or 120 min (11%). Neither the rate constants nor the relative amplitudes of the two phases were dependent on the degree of receptor occupancy. Thus, the duration of association, but not the degree of receptor occupancy affected 125I-LDL dissociation. To determine if binding by large LDL, which is predominantly via apolipoprotein (apo) E, also occurs by an isomerization mechanism, the d = 1.006-1.05 g/ml lipoproteins were fractionated by ultracentrifugation. In contrast to small LDL which bound via apoB-100 and whose dissociation was similar to that of unfractionated LDL, large LDL dissociation after 2, 15, or 120 min of binding did not show isomerization to a higher affinity. This suggests that large and small LDL bind by different mechanisms as a result of different modes of interaction of apoE and apoB-100 with LDL receptors.     

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.     

Surface interactions and intracellular localization of cationic ferritin: similarity to 125I-insulin in 3T3-L1 adipocytes

We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.     

Evidence for reutilization of surface receptors for alpha-macroglobulin.protease complexes in rabbit alveolar macrophages

Rabbit alveolar macrophages internalize α-macroglobulin ¹²⁵I-trypsin complexes subsequent to binding of complexes to high affinity surface receptors. Cells were capable of accumulating a 5–10 fold greater amount of αM · ¹²⁵I-T at 37°C than at 0°C. At 0°C cell-bound αM · ¹²⁵I-T was bound solely to surface receptors, whereas at 37°C the majority (85%) of cell-bound radioactivity was intracellular. The temperature-dependent accumulation of αM · ¹²⁵I-T did not reflect a change in surface receptor number or ligand-receptor affinity. Rather, the greater rate of uptake reflected continued internalization of αM · ¹²⁵I-T complexes. At 37°C cells took up 5–9 fmole αMT per μg cell protein per hr, whereas binding to surface receptors accounted for 0.5–0.7 fmole per μg cell protein. Once bound to surface receptors internalized αM · ¹²⁵I-T was localized in lysosomes, where it was degraded at a rate of 35–45% per hr. Following binding of αM · T to receptors at 37°C, but not at 0°C, unoccupied receptors could be found on the cell surface. Using cycloheximide to probe receptor turnover, I calculated that receptors were replenished at a rate of 15% per hr. Cells incubated in the presence of cycloheximide exhibited unaltered ligand uptake and catabolism for hours. Thus the reappearance of receptor activity during ligand uptake was not primarily due to de novo receptor synthesis. The rate of ligand uptake was a function of the number of surface receptors. Measurement of αM¹²⁵I-T binding to subcellular fractions did not reveal the presence of any intracellular reservoir of receptors. These observations are consistent with the hypothesis that continued ligand uptake reflects receptor reutilization.     

Phorbol esters inhibit the binding of low-density lipoproteins (LDL) to U-937 monocytelike cells

The present study demonstrates that U-937 monocytelike human cells possess specific LDL receptors. 125I-LDL binds at 4 degrees C on the cell surface. The bound molecules are releasable by heparin. The reaction requires Ca2+ and the binding sites are sensitive to proteolysis. Unlabeled LDL compete with 125I-LDL, whereas HDL are ineffective. At 37 degrees C, LDL are internalized and degraded by a chloroquine-sensitive pathway. Tumor-promoting phorbol esters inhibit the binding of 125I-LDL to its receptor on U-937 cells. This inhibition exhibits temperature, time, and concentration dependence. At 37 degrees C, inhibition is 50% at 5 X 10(-9) M of TPA. After removal of phorbol esters, treated cells recover their 125I-LDL-binding activity in 60 min. The inhibitory activities of various phorbol esters are proportional to their tumor-promoting activities. Inhibition appears to be due to a reduction in the number of available LDL receptors rather than a decrease in receptor affinity.     

Low-density-lipoprotein binding by mast-cell granules. Demonstration of binding of apolipoprotein B to heparin proteoglycan of exocytosed granules.

To study the interaction between low-density lipoprotein (LDL) and granules from rat serosal mast cells in vitro, mast cells were stimulated with the degranulating agent 48/80 to induce exocytosis of the secretory granules. Subsequent incubation of the exocytosed granules with 125I-LDL resulted in binding of the labelled LDL to the granules. When increasing amounts of agent 48/80 were added to mast-cell suspensions, a dose-dependent release of granules was observed and a parallel increase in the amount of 125I-LDL bound to granules resulted. 125I-LDL bound to a single class of high-affinity binding sites on the granules. At saturation, 105 ng of LDL were bound per microgram of granule protein. The lipoprotein binding to mast-cell granules was apolipoprotein(apo)-B + E-specific. Thus 125I-LDL binding to the granules was effectively compared for by LDL (apo-B) or by dimyristoyl phosphatidylcholine vesicles containing apo-E, but not by high-density lipoprotein (HDL3) containing apo-AI as their major protein component. Neutralization by acetylation of the positively charged amino groups of apo-B of LDL or presence of a high ionic strength in the incubation medium prevented LDL from binding to the granules, indicating the presence of ionic interactions between the positively charged amino acids of LDL and negatively charged groups of the granules. It could be demonstrated that LDL bound to the negatively charged heparin proteoglycan of the granules. Thus treatment of granules with heparinase resulted in loss of their ability to bind LDL, and substances known to bind to heparin, such as Toluidine Blue, avidin, lipoprotein lipase, fibronectin and protamine, all effectively competed with LDL for binding to the granules. The results show that LDL is efficiently bound to the heparin proteoglycan component of mast-cell granules once the mast cells are stimulated to release their granules into the extracellular space.