Effects of lectins on cAMP production and steroidogenesis in cultured adrenal tumor cells

The plant lectins, concanavalin A (conA), wheat germ agglutinin (WGA), and phytohemagglutinin (PHA) stimulate steroidogenesis in cultured adrenal tumor cells. ConA maximally stimulated steroidogenesis at 100 μg/ml following an approximate 4 h lag phase. ConA stimulation was completely inhibited by α-methyl-d-mannopyranoside and the WGA effect was prevented by N-acetyl-d-glucosamine. It was also found that conA alone did not cause a measurable increase in either intra- or extracellular cyclic adenosine 3′5′-monophosphate (cAMP) production. In addition, conA when added simultaneously with adrenocorticotropin (ACTH) doubled the intra- and extracellular cAMP production over controls treated with ACTH alone. This enhancement effect was dose dependent. When Y-1 cells were preincubated with conA and then treated with either ACTH or cholera enterotoxin (CT) there was a dose- and time-dependent inhibition of induced cAMP production. In the case of CT, the inhibitory effect occurred even with simultaneous addition of conA and CT. This effect was reversed by addition of both α-methyl-d-mannopyranoside and washing with Eagle's minimal essential medium (MEM) 1 h after CT had bound to its receptor. This reversal was not apparent for the inhibitory effect of conA on ACTH-induced cAMP production which occurred after 2 h of preincubation with conA. These results demonstrate that conA, as well as the other plant lectins, interact with specific membrane receptors to reversibly stimulate steroid production as well as enhancing or inhibiting ligand-induced cAMP production in cultured adrenal tumor cells.     

In vivo quantification of receptor-mediated uptake of asialoglycoproteins by rat liver

The in vivo kinetics of hepatic clearance of 125I-asialo-orosomucoid and 125I-asialofetuin was determined with a portal vein injection technique in barbiturate-anesthetized rats. Nonlinear regression analyses of saturation data gave the following parameters for asialo-orosomucoid, Km = 0.26 +/- 0.06 mg/ml, Vmax = 320 +/- 70 micrograms/min/g, and for asialofetuin, Km = 0.32 +/- 0.07 mg/ml, Vmax = 240 +/- 40 micrograms/min/g. Unlabeled asialofetuin inhibited the clearance of 125I-asialo-orosomucoid with a Ki = 0.25 +/- 0.04 mg/ml. Based on a model assuming that in vivo receptor concentration much greater than receptor KD, then the maximal binding capacity of the external surface of liver cells in vivo for asialo-orosomucoid is 2Km or 520 micrograms/ml or 52 micrograms/g of liver, assuming the liver interstitial space is 0.1 ml/g. Our estimate of in vivo binding capacity approximates in vitro estimates of total hepatic binding capacity, but is 10-fold greater than in vitro estimates of binding capacity on the external surface of liver cells. These results suggest the large majority of asialoglycoprotein receptors are located on the external surface of liver cells. The saturability of 125I-asialo-orosomucoid clearance was also demonstrated with a portal vein double bolus technique, wherein the portal injection of 20-1000 micrograms of unlabeled asialo-orosomucoid was followed 30 s later by the portal injection of tracer. Maximal inhibition of uptake was obtained with a portal vein injection of greater than or equal to 500 micrograms of asialo-orosomucoid. The specific extraction of the 125I-asialo-orosomucoid, which was near zero shortly after a 400-micrograms loading dose, gradually increased toward normal levels with a t1/2 of 21 min. This t1/2 may represent the in vivo rate of receptor recycling, since the gradual increase in unoccupied receptor sites is consistent with the model of receptor binding, internalization, and recycling.     

Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin

Freshly prepared plasma membranes from rat corpora lutea were examined for the presence of low density lipoprotein (LDL) and high density lipoprotein (HDL) receptors by determining the specific binding of 125I-LDL and 125I-HDL. These membranes have two types of binding site for 125I-LDL, one with high affinity (Kd = 7.7 micrograms of LDL protein/ml), the other with low affinity (Kd = 213 micrograms of LDL protein/ml) and one type of binding site for 125I-HDL with Kd = 17.8 micrograms of HDL protein/ml. LDL receptor is sensitive to pronase and trypsin; HDL receptor, however, is resistant. The binding reaction was further characterized with respect to effect of time and temperature of incubation, requirement of divalent metal ion, influence of ionic strength, and binding specificity. In vivo pretreatment of rats with human choriogonadotropin (hCG) resulted in induction of both LDL and HDL receptors in a dose- and time-dependent manner when compared with saline-injected controls. The induction of lipoprotein receptors by hCG treatment is target organ-specific since the increase was seen only in the ovarian tissue. Membranes prepared from liver, kidney, and heart did not show an increase in lipoprotein receptors after hCG injection. An examination of the equilibrium dissociation constants for 125I-LDL and 125I-HDL binding after hCG administration revealed that the increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, rat corpus luteum possesses specific receptors for both LDL and HDL and these receptors are regulated by gonadotropins.     

Regulation of atrial natriuretic peptide receptors in cultured vascular smooth muscle cells of rat

To elucidate the regulation of vascular receptors for atrial natriuretic peptide (ANP), we have studied the binding capacity of 125I-labeled rat (r) ANP using cultured vascular smooth muscle cells from rat aorta. After preincubation with 3.2 X 10(-8) M rANP at 37 degrees C, the binding capacity decreased as a function of time; the maximal receptor loss (70-75%) occurred after 4 hrs and persisted for 24 hrs. Pretreatment with cycloheximide (20 micrograms/ml) and actinomycin D (2 micrograms/ml) similarly caused a dramatic reduction (approximately 80%) of the binding capacity after 24 hrs; the half-life (t1/2) of the receptor loss was approximately 7-8 hrs. Following removal of rANP, the "down-regulated" ANP receptors fully recovered in the presence of 10% fetal calf serum, but not in combination with either actinomycin D or cycloheximide. Concanavalin A dose-dependently inhibited the binding. The binding capacity also decreased with time in the presence of tunicamycin (1 microgram/ml) with t1/2 of approximately 30 hrs. These data indicate that protein and carbohydrate moieties are essential for the functional integrity of the vascular receptor binding sites for ANP, and suggest that the recovery of the receptor loss by "down-regulation" requires concomitant RNA and protein synthesis.     

Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.     

Type I insulin-like growth factor receptors in human ovarian stroma

Hyperandrogenism observed in a variety of hyperinsulinemic states is thought to be due to an effect of insulin mediated through the type I insulin-like growth factor (IGF) receptors. These receptors, however, have not yet been demonstrated in normal human ovarian cells capable of androgen production. We now report the presence of type I IGF receptors in membrane preparations of human ovarian stroma. The ovarian stromal tissue was obtained from women undergoing indicated oophorectomy. Stromal plasma membranes were prepared. Specific 125I-IGF-I binding was 6.6 +/- 0.2%/100 micrograms protein. The affinity constant estimated by Scatchard analysis was 4.6 X 10(-9) M. 50% inhibition of 125I-IGF-1 binding was observed at 5 ng/ml of IGF-1. Specificity of the 125I-IGF-I-binding sites was confirmed by analogue specificity studies and in experiments utilizing monoclonal antibody to the IGF-I receptor, alpha-IR-3. IGF-II and insulin competed with 125I-IGF-I for the binding sites, but with an affinity significantly lower than that of IGF-I: 50% inhibition was observed at approximately 60 ng/ml of IGF-II or insulin. alpha-IR-3, a monoclonal antibody with high specificity for the type I IGF receptor, effectively inhibited 125I-IGF-I binding in a dose-dependent manner, confirming that the 125I-IGF-I binding was indeed to the type I IGF receptor. We conclude that type I IGF receptors are present in human ovarian stroma. These receptors may mediate effects of insulin on the ovary in hyperinsulinemic insulin-resistant states.     

Mechanistic insight into pertussis toxin and lectin signaling using T cells engineered to express a CD8α/CD3ζ chimeric receptor

Mammalian cell-surface receptors typically display N- or O-linked glycans added post-translationally. Plant lectins such as phytohemagluttinin (PHA) can activate the T cell receptor (TCR) and other cell-surface receptors by binding to glycans and initiating receptor cross-linking. Pathogenic microorganisms such as Bordetella pertussis also express proteins with lectin-like activities. Similar to plant lectins, pertussis toxin (PTx) can activate the TCR and bind to a variety of glycans. However, whether the lectin-like activity of PTx is responsible for its ability to activate TCR signaling has not been formally proven. Here we examined the ability of PTx and a panel of lectins to activate the TCR or a CD8α/CD3ζ chimeric receptor (termed CD8ζ). We demonstrate that CD8ζ rescues PTx-induced signaling events lacking in TCR null cells. This result indicates that CD8ζ can substitute for TCR and supports the hypothesis that PTxB (functioning as a lectin) stimulates signaling via receptor cross-linking rather than by binding to a specific epitope on the TCR. Moreover, PTx is able to activate signaling by binding either N-linked or O-linked glycan-modified receptors as the TCR displays N-linked glycans while CD8ζ displays O-linked glycans. Finally, studies with a diverse panel of lectins indicate that the signaling activity of the lectins does not always correlate with the biochemical reports of ligand preferences. Comparison of lectin signaling through TCR or CD8ζ allows us to better define the structural and functional properties of lectin-glycan interactions using a biologically based signaling readout.     

Alterations in glycosylation of plasma membrane proteins during myogenesis

Highly purified plasma membranes were obtained from cells of the L6 line at three characteristic stages of myogenesis: Actively proliferating cells; post-mitotic, confluent myoblasts which have already aligned; and fused myotubes. Differential glycosylation of the plasma membrane proteins of these cells was detected by staining polyacrylamide gels of the separated components with three lectins of different specificity: Concanavalin A (conA), wheat germ agglutinin (WGA) and phytohemagglutinin (PHA) Els. Four kinds of developmentally regulated changes could be identified. 1. Those which took place only at confluency (160, 150, 90, 85, 60, 43 and 40 kD for conA binding, 190 kD for WGA binding, 190 and 110 kD for PHA Els binding. 2. Those which took place only at fusion (135, 51.5 and 38 kD for conA, 160 and 150 kD for WGA and 150 kD for PHA Els binding). 3. Those where the phenomena initiated at confluency continue during fusion (66.5 and 32 kD for conA and 120 kD for PHA binding). 4. Those where opposite changes take place at confluency and at fusion (48 kD for conA, 180, 98 and 85 kD for PHA binding). These results suggest that most developmentally regulated changes in glycosylation take place during the first cell-cell contact step of myogenesis. Metabolic labelling experiments showed that, on the contrary, only few alterations in the accumulation of plasma membrane proteins take place prior to the main burst of fusion.     

Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells

The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membrane-associated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The colocalization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular-located filaments. Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X-100 insoluble cytoskeleton.     

Characterization of wild type and mutant glucocorticoid receptors from rat hepatoma and mouse lymphoma cells

Using a combination of immunological blotting techniques and hormone affinity labeling, we have characterized the glucocorticoid receptors present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Mutant HTC and WEHI7 cells of the receptorless phenotype, which contain greatly reduced amounts of glucocorticoid hormone binding activity, show parallel decreases in immunoreactive material using a monoclonal antibody raised against the rat liver glucocorticoid receptor. This indicates that these receptorless mutant cells harbor defects in either the production or accumulation of receptor protein. Quantitation of immunoreactivity and hormone binding activity present in wild type and mutant S49 cells indicates that these cells contain significantly more immunoreactive material than hormone binding activity. We conclude that S49 cells produce, in addition to their well characterized wild type or mutant receptors, a mutant receptor from a second allele which is of wild type size, is immunologically reactive, but is unable to bind hormone. The S49 mutant cell line nti (nuclear transfer increase) contains a glucocorticoid receptor which has a molecular weight of 40,000, while the wild type receptor has a molecular weight of 94,000. Affinity labeling of glucocorticoid receptors in nti cells with [3H]dexamethasone mesylate indicates that nti cells do not contain wild type sized precursor molecules which bind hormone, nor do they contain immunoreactive fragments of a molecular mass smaller than 94 kDa. It is proposed that the 40-kDa nti receptor is produced as a truncated protein most likely resulting from a nonsense mutation or from a truncated messenger RNA.     

Characterization of the galactose-specific binding activity of a purified soluble Entamoeba histolytica adherence lectin.

We studied galactose (Gal)-specific binding of the soluble purified 260-kDa Entamoeba histolytica adherence protein to glycosylation deficient Chinese hamster ovary (CHO) cell mutants. Our goal was to further define the lectin's functional activity and carbohydrate receptor specificity. The adherence protein was purified by acid elution from an immunoaffinity column; however, exposure of the surface membrane lectin on viable trophozoites to identical acid pH conditions had no effect on carbohydrate binding activity. Saturable Gal-specific binding of soluble lectin to parental CHO cells was demonstrated at 4 degrees C by radioimmunoassay; the dissociation coefficient (Kd) was 2.39 x 10(-8) M-1 with 5.97 x 10(4) lectin receptors present per CHO cell. Gal-specific binding of lectin to Lec2 CHO cell mutants, which have increased N- and O-linked terminal Gal residues on cell surface carbohydrates, was increased due to an enhanced number of receptors (2.41 x 10(5)/cell) rather than a significantly reduced dissociation constant (4.93 x 10(-8) M-1). At 4 degrees C, there was no measurable Gal-specific binding of the adherence protein to the Lec1 and 1dlD.Lec1 CHO mutants, which contain surface carbohydrates deficient in terminal Gal residues. Binding of lectin (20 micrograms/ml) to CHO cells was equivalent at 4 degrees C and 37 degrees C and unaltered by adding the microfilament inhibitor, Cytochalasin D (10 micrograms/ml). Gal-specific binding of the lectin at 4 degrees C was calcium independent and reduced by 81% following adsorption of only 0.2% of the lectin to CHO cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Laminin increases the release of type IV collagenase from malignant cells

We have studied the effect of laminin on type IV collagenolytic activity elaborated by malignant cells in culture. Laminin (at concentrations of 4-8 micrograms/ml) added to serum-free culture supernatants of subconfluent A2058 human melanoma cells significantly increased the release of the type IV collagenolytic activity (200-300%). The induction of type IV collagenase was more pronounced (580%) using a fragment of laminin which binds to the cell surface laminin receptor. A monoclonal antibody against the human laminin receptor blocked the effect of laminin on type IV collagenase, suggesting that occupation of the laminin receptor may be necessary for the effect. Increase in the type IV collagenolytic activity mediated by laminin was also demonstrated in two other malignant cell lines, HT fibrosarcoma (168%) and mouse melanoma (B16-F10) (271%). The increase in type IV collagenase was found to be specific for laminin because another cell-binding matrix protein, fibronectin, did not have any effect, and epidermal growth factor and transferrin actually decreased the type IV collagenase in human melanoma culture medium (epidermal growth factor, 50% at 20 ng/ml; and transferrin, 20% at 10 micrograms/ml). These studies suggest that tumor cell binding to laminin, which comprises the first step of basement membrane invasion, will induce the second step, namely the collagenolytic dissolution of the basement membrane.     

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.     

Sulfhydryl Reagents Alter Epidermal Growth Factor Receptor Affinity and Association with the Cytoskeleton

Abstract

Sulfhydryl (SH) reagents are known to influence the characteristics of many ligand-receptor systems. The SH reagent N-ethylmaleimide has been demonstrated to interact with EGF receptors, and to inhibit EGF receptor kinase activity. The data presented in this paper concern the effect of SH reagents on two intriguing features of the EGF receptor system, namely the presence of low and high affinity EGF binding sites, and the interaction of EGF receptors with the cytoskeleton. SH reagents were observed to induce a disappearance of high, but not low, affinity EGF receptors from the cell surface, and an increase in receptor-cytoskeleton interaction. Comparison of the effects of membrane-permeant and membrane-impermeant SH reagents on wild type and structurally modified EGF receptors suggested that sulfhydryl groups on the cytoplasmic, rather than the extracellular, receptor domain are involved. This indicates that the cytoplasmic domain of the EGF receptor plays a role in the high affinity binding of EGF, and in the interaction of EGF receptors with the cytoskeleton. Experiments with an anti-EGF receptor antibody that specifically blocks the binding of EGF to low affinity receptors indicated that EGF induces a shift in the EGF receptor from low to high affinity. SH reagents probably affect EGF binding by inhibiting this EGF-induced receptor conversion.     

Properties of enucleated cells. III. Changes in cytoplasmic architecture of enucleated BHK21 cells following trypsinization and replating.

At low concentrations of concanavalin A (conA), binding of the lectin to the erythrocytes appears to be the rate-limiting step in the agglutination of these cells. At higher concentrations of lectin the rate of agglutination is concentration-independent, indicating that the aggregation reaction is rate-determining. Only 5 to 7% of the 1.2 × 10⁵ receptor sites need be occupied by con A in order for agglutination to take place. Although trypsin-treated cells bind 30% less ¹²⁵I-conA, they agglutinate better than untreated cells. At high lectin concentrations, erythrocyte agglutination by wheat germ agglutinin (WGA) is more than 8 times faster than the conA-mediated reaction. Lowering of the temperature to 0 °C reduces the rate but not the extent of the agglutination by both lectins. Mechanical shear reduced the conA-mediated agglutination of native cells by more than 160-fold and that of trypsinized and neuraminidase-treated cells 6-fold and 4-fold, respectively.It is concluded that metabolic activity, receptor mobility (i.e. cluster or patch formation) and cytochalasin B-sensitive processes, all of which have been reported to be involved in the lectin-mediated agglutination of fibroblasts and other cells, do not play a role in erythrocyte agglutination. Lectin-mediated erythrocyte agglutination appears to be governed primarily by the rate and extent of binding of lectin to the cell surface, the cell surface charge (modifiable by enzyme treatments or polycations) and the shear forces in the suspension. Morphological studies confirm and amplify these conclusions.     

Wheat-germ-agglutinin and Ricinus communis-agglutinin-binding sites of BHK cells compared with each other and with 140 kDa fibronectin receptors.

We compared the wheat-germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) binding sites of baby-hamster kidney (BHK) cells. There were 1.01 X 10(8) WGA-binding sites per cell (Kd = 0.027 nM) and 6 X 10(6) RCA-binding sites per cell (Kd = 0.014 nM). Binding of WGA or RCA to BHK cells resulted in more than 75% of the cell-surface binding sites becoming associated with the cytoskeleton (i.e. resistant to extraction with detergent), although no more than 10% of these sites were associated with the cytoskeleton before addition of the lectins. After binding of WGA to the cells, the cell surface was cross-linked so extensively that it remained intact even after detergent extraction of the treated cells, and could be observed by electron microscopy. A similar cross-linking effect did not occur after binding of RCA to cells, which may be because there were so many more binding sites for WGA than for RCA. The composition of WGA- and RCA-binding molecules was analysed by lectin affinity chromatography of metabolically radiolabelled BHK cells. We found that in the WGA-binding-molecule preparations there were eight major polypeptides, ranging in molecular mass from 93 to 340 kDa, and that the RCA-binding molecules were a subpopulation of the WGA-binding molecules. A polyclonal antibody against the 140 kDa fibronectin (FN) receptors of Chinese-hamster ovary (CHO) cells immunoblotted a 145 kDa polypeptide component in both WGA- and RCA-binding-molecule preparations. The results indicated that the 145 kDa component was present in at least two FN-receptor complexes that differed in glycosylation, only one of which was able to bind to RCA affinity columns. The oligomeric nature of the FN-receptor complex, which contained three polypeptides with molecular masses of 120-145 kDa, was demonstrated by using anti-(CHO-cell FN receptor) antibodies to immunoprecipitate extracts prepared from radioiodinated BHK cells.     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

Characterization of the spreading process in human T lymphocytes

Substratum-bound concanavalin A (conA) caused attachment and spreading of human T lymphocytes identified by monoclonal anti-T cell antibodies and sheep erythrocyte rosette formation. The simultaneous presence of conA in the medium increased the spreading, whereas preincubation of the cells with conA inhibited spreading. The tendency of conA to induce spreading was dependent on the concentration used, the higher the conA concentration the more pronounced was the spreading. For example, conA at 10 micrograms ml-1 triggered the formation of prominent substratum-attached filopodia with a length of 1-10 micron in 60-80% of T-enriched lymphocytes obtained from separate individuals. At the same conA dose the filopodia were, in 10-20% of the lymphocytes, accompanied by development of lamellipodia. With conA at 100 micrograms ml-1 the number of cells that underwent pronounced spreading was 55-90% in separate individuals. Observation of T-enriched cells fixed at different times after initiation of spreading induced by conA at 100 micrograms ml-1 indicated that filopodia formation represented the initial morphological alteration during the spreading process. This process thereafter proceeded with development of lamellipodia, extensive cytoplasmic spreading and flattening of the central cell mass. Quiescent and mitogen-activated cells exhibited the same sequence of changes during spreading. Spreading led to disappearance of the microvilli with a length of 0.1-0.7 micron present on lymphocytes in suspension, although some microvilli persisted over the cell center.     

Defective hepatic lipoprotein receptor binding of beta-very low density lipoproteins from type III hyperlipoproteinemic patients. Importance of apolipoprotein E

Apolipoprotein (apo-) E2 and beta-migrating very low density lipoproteins (beta-VLDL) (which were isolated from type III hyperlipoproteinemic subjects) both demonstrated defective binding to apo-E and apo-B,E receptors on dog liver membranes and to apo-B,E low density lipoproteins (LDL) receptors on fibroblasts. The defective binding activity of the apo-E2 and beta-VLDL varied from very poor to nearly normal. The ability of the beta-VLDL to interact with hepatic apo-E receptors was enhanced by the addition of normal apo-E3 to the beta-VLDL. Furthermore, cysteamine treatment of the apo-E2 in beta-VLDL enhanced binding of the beta-VLDL to both apo-E and apo-B,E receptors. The importance of apo-E in mediating the receptor binding of beta-VLDL to these receptors was confirmed by using monoclonal antibodies. The residual binding activity of beta-VLDL to apo-E and apo-B,E receptors was inhibited by greater than 90% with anti-apo-E, while the addition of anti-apo-B had little effect. The apo-B in the beta-VLDL was capable of binding to apo-B,E receptors after the hydrolysis of the beta-VLDL triglycerides with milk lipoprotein lipase. Lipase treatment yielded, two subfractions of beta-VLDL. One fraction (d = 1.02 to 1.03 g/ml) was enriched with apo-B100; the other fraction (d less than 1.006 g/ml) was enriched with apo-B48 and apo-E2. Significantly increased amounts of the apo-B100-enriched fraction bound to apo-B,E receptors. Inhibition of this binding caused by the addition of anti-apo-B indicated that the binding activity of this subfraction was mediated by apo-B100. The apo-B48-enriched fraction did not show a significant increase in receptor binding, suggesting that apo-B48 does not bind to these receptors. In a control experiment, it was shown that triglyceride-rich VLDL, which contain normal apo-E3 and apo-B100, bind significantly to both liver apo-E receptors and fibroblast apo-B,E receptors. This binding activity was inhibited by greater than 90% with anti-apo-E. Lipase hydrolysis of the VLDL did not further enhance their receptor-binding activity. These results demonstrate that apo-E, and not apo-B, is the major determinant mediating the receptor-binding activity of cholesterol-rich beta-VLDL and triglyceride-rich VLDL.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.