Internalization of low-density-lipoprotein-specific receptors in leukemic guinea pig lymphocytes

Leukemic guinea pig lymphocytes (L2C) have ten times as many low-density lipoprotein (LDL) receptors as healthy lymphocytes, but LDL accounts for only 38% of the cholesterol in L2C cells, compared to more than 95% in normal cells. Our data show that LDL fails to regulate cholesterol biosynthesis and that there is a defect in LDL internalization and receptor turnover in L2C cells. We also demonstrate that the degradation of LDL is not a limiting process. By discriminating between binding and internalization, we show that internalization in L2C is much slower than in normal cells and that the decrease in metabolism is related to the slow turnover of the LDL receptors.     

Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome

To study the fusion and separation of endocytic compartments, we have used digital image analysis to quantify the accumulation of fluorescent ligands in endosomes during continuous endocytosis for periods of 1-20 min. Fluorescently labeled transferrin (Tf) and low density lipoproteins (LDL) were used as markers of recycling receptors and lysosomally directed ligands respectively. By measuring the intensity of individual endosomes, we found that the amount of LDL per endosome increases 30-40-fold between 1 and 10 min and then plateaus. In contrast, the amount of Tf per endosome reaches a steady state within 2 min at a level that is only three to four times that at 1 min. We used pulse-chase double label methods to demonstrate that Tf cycles through the compartment in which the LDL accumulates. When both Tf and LDL are added to cells simultaneously for 2 min, nearly all endosomes contain both labels. With 2-4 min further incubation in the absence of external ligands, LDL-containing compartments become depleted of Tf as Tf is directed to para-Golgi recycling endosomes. However, if Tf is added to the medium 2-4 min after a pulse with LDL, most of the LDL-containing endosomes become labeled with Tf. The data indicate that at least 30-40 endocytic vesicles containing both Tf and LDL fuse with an endosomal compartment over a period of 5-10 min. LDL accumulates within this compartment and Tf is simultaneously removed. Simple mathematical models suggest that this type of iterative fractionation can lead to very high efficiency sorting.     

LDL-mediated targeting of liposomes to leukemic lymphocytes in vitro.

We describe a method for the covalent coupling of low-density lipoproteins (LDL) to the surface of small unilamellar vesicles, and the delivery of the liposome content to leukemic L2C lymphocytes in vitro. We demonstrate the stability of the linkage between LDL and liposomes, the preservation of vesicle integrity and the affinity of the LDL for their specific receptors after the coupling reaction. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted liposomes, and delivered into the cytoplasm of leukemic L2C lymphocytes by the LDL pathway, as demonstrated by the lethal effect on cells measured by 51chromium-release assay.     

Thiolation of low-density lipoproteins and their interaction with L2C leukemic lymphocytes

We present here, a new method for coupling sulfhydryl groups (SH) to low-density lipoprotein (LDL) surface. This method uses homocysteine thiolactone (HCTL) which reacts with lysine residues in a very mild manner, and permits the selection of the number of SH bound per LDL. Under our experimental conditions (8 SH/LDL), the affinity of thiolated LDL for the specific receptors and their further internalization by L2C lymphocytes are preserved.     

Characterization of Low Density Lipoprotein Receptors in Freshly Isolated Leukemic Guinea Pig Lymphocytes (L2C)

Abstract

The present study shows that L₂C leukemic guinea pig lymphocytes have 10 times as many low density lipoprotein (LDL) receptors per cell as normal lymphocytes. The affinity of these receptors is higher for guinea pig LDL than for human LDL. In contrast to normal cells, in which the degradation of the receptor-bound LDL is quite efficient, the leukemic cells only degraded a small fraction of these same receptor-bound LDL. Thus, the internalization index was nearly 4 times higher in the normal cells than in the leukemic cells.

In L₂C cells, cholesterol homeostasis derived 38% of its cholesterol input from receptor-mediated degradation of LDL and 62% from cholesterol synthesis, whereas in normal cells, these fractions were 97% and 3% respectively     

Simple method for comparing large numbers of flow cytometry histograms exemplified by analysis of the CD4 (T4) antigen and LDL receptor on human peripheral blood lymphocytes

We have developed a simple method for comparing the relative fluorescence intensity (FI) of flow cytometry histograms. It entails assessment of the FI (equivalent to the fluorescence-activated cell sorter (FACS) channel) of the 50th or 75th percentiles of either positively stained cells or the total cell population. We illustrate the method with dilution curves of 1) monoclonal antibodies against the T4 surface antigen of human peripheral blood lymphocytes and 2) fluorescent low density lipoprotein (LDL) binding to the human peripheral blood lymphocytes LDL receptor. We demonstrate the versatility of the method by characterizing the binding properties of fluorescent LDL to their receptors. Binding was shown to be specific and of high affinity, and to reach a steady state plateau at about 2 hr; the affinity of fluorescent LDL for the receptor was found to be two to three times higher than that of the unlabeled LDL.     

Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.     

Macrophage colony-stimulating factor differentially regulates low density lipoprotein and transferrin receptors

Endocytosis mediated by both LDL receptors (LDLRs) and transferrin receptors (TfRs) occurs in clathrin-coated pits and requires specific tyrosine-based internalization sequences located in the cytoplasmic domain of these receptors. Internalization of these receptors is mediated by endocytic proteins that interact with the internalization domains. We previously showed that macrophage colony-stimulating factor (M-CSF) rapidly increases LDLR-dependent uptake and metabolism of LDL. To study the mechanism by which M-CSF regulates LDL uptake, we compared the effect of M-CSF on the internalization of LDL and transferrin (Tf). Our results show that M-CSF substantially increased the rate of LDLR internalization without increasing LDLR localization on the cell surface. In contrast, M-CSF treatment of macrophages rapidly increased the localization of TfR to the cell surface but did not alter the relative rate of Tf internalization. Moreover, M-CSF regulated TfR and LDLR via the activation of distinct signaling pathways. Recruitment of TfR to the cell surface was attenuated by phosphatidylinositol 3-kinase inhibitors, whereas stimulated LDL uptake was inhibited by the serine/threonine phosphatase inhibitor okadaic acid. Taken together, our results indicate that M-CSF differentially regulates receptors that undergo endocytosis and that increased LDL uptake results from a selective increase in the rate of LDLR internalization.     

Lipolysis-induced iron release from diferric transferrin: Possible role of lipoprotein lipase in LDL oxidation.

Conditions leading to oxidation of LDL in vivo are still unknown. While the occurrence of oxidized lipoproteins and catalytic free iron in advanced atherosclerotic lesions has been demonstrated, the origin of both is unclear. In vivo, iron metabolism is tightly regulated by iron-binding proteins that ensure that virtually no free iron exists. We examined whether physiological events such as lipolysis might reduce pH, facilitate iron release from transferrin (Tf), and promote low density lipoprotein (LDL) oxidation. Lipolysis is brought about by lipoprotein lipase (LpL), a triglyceride hydrolase present on endothelial cell surfaces and in atherosclerotic lesions. LpL hydrolysis of Intralipid lowered pH from 7.40 to 7.00 in 10% human serum and from 7.40 to 6.88 in phosphate-buffered saline. Similar decreases in pH were also observed when very low density lipoproteins were hydrolyzed by LpL. Lipolysis was accompanied by a 2-fold increase in the release of 59Fe from Tf. Tf binding to subendothelial matrix (SEM), a site of key events in atherosclerosis, increased 2-fold as the pH decreased from 7.40 to 6.00. More free iron also bound to SEM as the pH decreased below 7.40. We next tested whether a reduction in pH promotes LDL oxidation. More oxidation products were found in LDL incubated at low pH for 24 h in 10% human serum. Malonaldehyde contents (nmol/mg protein), measured as TBARS, were 7.11 +/- 0.34 at pH 7.40, 7.65 +/- 0.49 at pH 7.00, 9.00 +/- 1.18 at pH 6.50, and 11. 54 +/- 0.63 at pH 6.00.Based on these results, we hypothesize that lipolysis-induced acidic conditions enhance iron release from Tf and increase formation of oxidized LDL.     

Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells

We have previously characterized the trafficking of transferrin (Tf) through HEp2 human carcinoma cells (Ghosh, R. N., D. L. Gelman, and F. R. Maxfield, 1994. J. Cell Sci. 107:2177-2189). Early endosomes in these cells are comprised of both sorting endosomes and recycling compartments, which are distinct separate compartments. Endocytosed Tf initially appears in punctate sorting endosomes that also contain recently endocytosed LDL. After short loading pulses, Tf rapidly sorts from LDL with first-order kinetics (t1/2 approximately 2.5 min), and it enters the recycling compartment before leaving the cell (t1/2 approximately 7 min). Here, we report a second, slower rate for Tf to leave sorting endosomes after HEp2 cells were labeled to steady state with fluorescein Tf instead of the brief pulse used previously. We determined this rate using digital image analysis to measure the Tf content of sorting endosomes that also contained LDL. With an 11-min chase, the Tf in sorting endosomes was 24% of steady-state value. This was in excess of the amount expected (5% of steady state) from the rate of Tf exit after short filling pulses. The excess could not be accounted for by reinternalization of recycled cell surface Tf, implying that either some Tf was retained in sorting endosomes, or that Tf was delivered back to the sorting endosomes from the recycling compartment. The former is unlikely since nearly all sorting endosomes contain detectable Tf after an 11-min chase, even though more than one third of the sorting endosomes were formed during the chase time. Furthermore, while observing living cells by confocal microscopy, we saw vesicle movements that appeared to be fluorescent Tf returning from recycling compartments to sorting endosomes. The slow rate of exit after steady-state labeling was similar to the Tf exit rate from the cell, suggesting an equilibration of Tf throughout the early endosomal system by this retrograde pathway. This retrograde traffic may be important for delivering molecules from the recycling compartment, which is a long-lived organelle, to sorting endosomes, which are transient.     

The regulation by low-density lipoproteins of the activation of oxidative enzyme-primed lymphocytes is governed by transferrin

The activation of T lymphocytes was regulated in vitro by low-density lipoproteins (LDL). Not all prereplicative events induced by the oxidative enzymatic mitogens neuraminidase and galactose oxidase (NAGO) were susceptible to inhibition by LDL. The accessory cell-independent early blastogenic response was not suppressed. LDL suppressed accessory cell-dependent responses, and the extent of LDL suppression, depended on the concentration of transferrin. A gradient of transferrin determined the point in the cell cycle at which NAGO-primed lymphocytes were suppressed by LDL. When transferrin was low (0-10 micrograms/ml) and in serum-free medium (SFM), LDL suppressed the expression of cell surface receptors for interleukin-2 (IL-2R) and transferrin (TfR), the late blastogenic response prior to DNA replication (72 hr), and DNA replication. At higher levels of transferrin, about 100 micrograms/ml, the LDL-suppressed cells were IL-2R+, TfR+ and responsive to IL-2, but did not enter S phase. LDL suppression could be ablated by IL-2 and by high levels of transferrin (250-1000 micrograms/ml). In RPMI medium containing serum (FBS), the pattern of LDL suppression was different from that in SFM: fully activated IL-2R+, TfR+ lymphocytes were unresponsive to exogenous IL-2, suggesting that they were blocked at the G1/S boundary. This block was also relieved by transferrin (greater than 100 micrograms/ml). The data suggest that the interplay between transferrin and LDL is a critical factor in the NAGO-induced stimulation of T lymphocytes. LDL and transferrin exert negative and positive control of lymphocyte activation, respectively. In SFM, LDL appear to alter transferrin utilization by accessory cells; in RPMI-FBS, by fully activated T lymphocytes.     

The effect of concanavalin A-stimulated mononuclear cells on low density lipoprotein receptor activity of cultured fibroblasts

Secretory products of freshly isolated human circulating blood cells such as platelets, monocytes, and B lymphocytes, but not T lymphocytes, have previously been shown to enhance low density lipoprotein (LDL) metabolism by arterial wall cells. This study was undertaken to evaluate how secretory factor(s) from mononuclear cells that had been stimulated by concanavalin A (Con A) alters LDL receptor activity by cultured human skin fibroblasts. Conditioned medium from Con A-stimulated mononuclear cells produced an increase of 125I-LDL degradation accompanied by increased thymidine incorporation into DNA. The effect of conditioned medium from the Con A-stimulated mononuclear cells was mediated by the LDL receptor pathway. Degradation of HDL and methylated LDL, neither of which is taken up by the classical LDL receptor pathway, was not affected. The conditioned medium from these Con A-stimulated cells also failed to stimulate fluid pinocytosis, as measured by the uptake of [14C]sucrose. Some strains of fibroblasts, deficient in LDL receptors, responded to the conditioned medium from the Con A-stimulated mononuclear cells by increasing the very small amounts of LDL degraded by these cells. Fibroblasts from other homozygous familial hypercholesterolemic cell strains were unresponsive, however. The effect on LDL receptors was characterized by an increase in LDL receptor number without a change in the affinity of LDL for its receptor. Thus stimulated mononuclear cells secrete mitogens that also stimulate LDL receptor activity in human skin fibroblasts.     

Oligomerized transferrin receptors are selectively retained by a lumenal sorting signal in a long-lived endocytic recycling compartment

Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tf10) which was prepared by chemical cross- linking of transferrin (Tf). Tf10 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tf10 was investigated using fluorescence and electron microscopy. Tf10 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tf10 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf10. The presence of Tf10 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf10 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf10 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tf10 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.     

Effect of human syncytiotrophoblast plasma membrane-soluble extracts on in vitro mitogen-induced lymphocyte proliferation. A possible inhibition mechanism involving the transferrin receptor

Transferrin (Tf) binding to lymphocytes and to syncytiotrophoblast plasma membranes (STPM) and their soluble extracts (STPM-SE) was examined, as well as the effect of the latter on mitogen-induced lymphoproliferation. Lymphocytes only express Tf receptors (Rtf) after mitogen (phytohemagglutinin or concanavalin A) stimulation, and the percentage of Tf bound by stimulated lymphocytes increased as a function of contact time with the mitogen, reaching a maximum at 72 hr. Studies of Tf binding to STPM-SE showed that the percentage of bound Tf increased proportionally to the protein concentration, but was additionally enhanced by a factor of three when STPM were pretreated with 3 M KCl. Scatchard analysis of Tf binding to lymphocytes cultured for 72 hr in the presence of mitogen, as well as to STPM and STPM-SE, revealed that this binding was specific and occurs via a single category of identical and independent receptors the numbers and affinity constants (Ka) of which have been determined. The results obtained by using STPM indicate that the Ka does not vary significantly from one preparation to another, but that the number of sites per milligram of protein increases by a factor of 10 when the STPM are pretreated with 3 M KCl (KCl-STPM). Finally, STPM-SE inhibited the mitogen-induced lymphoproliferative response whether or not they were treated with 3 M KCl. This inhibition was not due to lymphocytotoxicity, was dose dependent regardless of the preparation used, but was maximized with the KCl-STPM-SE fraction. The correlation between the inhibitory capacities of the soluble STPM extracts and the numbers of RTf sites present on their membranes leads to the hypothesis that the observed inhibition could involve the RTf. This effect may help in protecting the fetus from the maternal immune system at the time of the semi-allogenic fetal graft.     

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.     

Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay

The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.     

Lipoproteins may provide fatty acids necessary for human lymphocyte proliferation by both low density lipoprotein receptor-dependent and -independent mechanisms

Human lymphocytes respond optimally to mitogenic stimulation when cultured in serum-free medium supplemented with transferrin if fatty acids necessary for maximal proliferation are provided. Either lipoproteins or exogenous fatty acids support optimal lymphocyte responses. The current studies examined the role of cell surface receptors for low density lipoprotein (LDL) in the enhancement of lymphocyte proliferation. Support of lymphocyte growth by limiting concentrations of LDL was found to involve interaction of the lipoprotein with LDL receptors. Thus, modification of LDL by reductive methylation so as to inhibit receptor-mediated interactions markedly decreased the capacity of LDL to enhance lymphocyte proliferation. Moreover, growth of lymphocytes obtained from patients with LDL receptor-negative homozygous familial hypercholesterolemia was minimal when cultures were supplemented with low concentrations of LDL (less than 10 micrograms cholesterol/ml). LDL also enhanced lymphocyte proliferation by a receptor-independent mechanism since high concentrations (greater than or equal to 50 micrograms cholesterol/ml) supported growth of both normal and familial hypercholesterolemia lymphocytes. In contrast, support of lymphocyte proliferation by high density lipoprotein (HDL) subclass 3 was completely independent of LDL receptors. Thus, HDL3 enhanced responses of both normal and familial hypercholesterolemia lymphocytes in an equivalent concentration-dependent manner; this effect was not altered by reductive methylation of HDL3. One function of lipoproteins in this system may be the provision of fatty acids since oleic and linoleic acids enhanced DNA synthesis by both normal and familial hypercholesterolemia lymphocytes in the absence of lipoproteins. These results indicate that lipoproteins may provide fatty acids necessary for optimal proliferation of human lymphocytes by both LDL receptor-mediated and LDL receptor-independent interactions.     

Thyrotropin modulates low density lipoprotein binding activity in FRTL-5 thyroid cells

FRTL-5 cells possess high affinity low density lipoprotein (LDL) receptors which bind, internalize, and degrade LDL. When FRTL-5 cells are deprived of thyrotropin (TSH) the binding of LDL increases more than 2-fold. Upon addition of TSH, at a concentration of 1 x 10(-10) M or greater, LDL binding decreases rapidly and within 24 h reaches the level which is typical of FRTL-5 cells chronically stimulated by TSH. The data available suggest that TSH-dependent down-regulation of LDL receptor activity is exerted through a reduction of the number of active LDL receptors, with no change in affinity. It is unlikely that the synthesis of LDL receptors is impaired, since LDL receptor messenger RNA is not decreased by TSH. The effect of the hormone on LDL receptor activity can be mimicked by 8-Br-cAMP and is completely abolished by the protein synthesis inhibitor cycloheximide but not by actinomycin D. TSH regulation of LDL receptor activity is lost in v-ras Ki-transformed FRTL-5 cells (Ki Mol) which also have lost TSH dependence for adenylate cyclase activation and growth. However, 8-Br-cAMP decreases LDL binding in Ki Mol FRTL-5 cells. The reduced availability of LDL receptor in TSH-stimulated FRTL-5 cells may be related to the increased membrane fluidity (Beguinot, F., Beguinot, L., Tramontano, D., Duilio, C., Formisano, S., Bifulco, M., Ambesi-Impiombato, F. S., and Aloj, S. M. (1987) J. Biol. Chem. 262, 1575-1582) or may reflect increased degradation of LDL receptors. We propose that a lower cholesterol uptake is needed in an actively proliferating cell population, to increase the production of isoprenoids whether it be for cholesterol biosynthesis or for the synthesis of other compounds requiring isoprenoid precursors.     

The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding and internalization but not for LDL receptor clustering in coated pits

ARH is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues. Individuals lacking ARH (ARH-/-) have severe hypercholesterolemia due to impaired hepatic clearance of LDL. Immortalized lymphocytes, but not fibroblasts, from ARH-deficient subjects fail to internalize LDL. To further define the role of ARH in LDLR function, we compared the subcellular distribution of the LDLR in lymphocytes from normal and ARH-/- subjects. In normal lymphocytes LDLRs were predominantly located in intracellular compartments, whereas in ARH-/- cells the receptors were almost exclusively on the plasma membrane. Biochemical assays and quantification of LDLR by electron microscopy indicated that ARH-/- lymphocytes had >20-fold more LDLR on the cell surface and a approximately 27-fold excess of LDLR outside of coated pits. The accumulation of LDLR on the cell surface was not due to failure of receptors to localize in coated pits since the number of LDLRs in coated pits was similar in ARH-/- and normal cells. Despite the dramatic increase in cell surface receptors, LDL binding was only 2-fold higher in the ARH-/- lymphocytes. These findings indicate that ARH is required not only for internalization of the LDL.LDLR complex but also for efficient binding of LDL to the receptor and suggest that ARH stabilizes the associations of the receptor with LDL and with the invaginating portion of the budding pit, thereby increasing the efficiency of LDL internalization.     

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf. © 1993 Wiley-Liss, Inc.