Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca(2+)-dependent and Ca(2+)-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[(14)C]glutamate and [(3)H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 microM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 microM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca(2+)-stimulated l-[(14)C]glutamate and [(3)H]GABA release and increase the Ca(2+)-independent release of l-[(14)C]glutamate, but not of [(3)H]GABA. Disulfide-reducing reagents (dithiothreitol and beta-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca(2+)-stimulated neurotransmitter release.     

The surface content of asialoglycoprotein receptors on isolated hepatocytes is reversibly modulated by changes in temperature

We have investigated the effect of temperature on the content of surface asialoglycoprotein receptors on isolated rat hepatocytes. Receptor was determined by measuring the specific binding of 125I- or [3H] asialo-orosomucoid at 0 degrees C. As reported previously, the receptor number/cell increases 2-3-fold within 30-60 min when freshly isolated cells are warmed from 0-37 degrees C (Weigel, P. H. (1980) J. Biol. Chem. 255, 6111-6120). This increase in receptor number is not inhibited by cycloheximide and also occurs on cells which have first been treated with EDTA to expose a population of cryptic receptors on the cell surface. The rate and extent of the receptor number increase on the cell surface are proportional to the temperature above about 17 degrees C. If cells are first equilibrated at 37 degrees C and then transferred to a lower temperature, the surface receptor number decreases at a rate and to an extent dependent on the temperature. The surface receptor number can be modulated up and down by successive temperature change cycles between 25 and 37 degrees C. In this temperature range, the number of surface receptors/cell is dependent on the final temperature but independent of the pathway to that temperature and is, therefore, a function of state with respect to temperature. The results demonstrate that temperature changes reversibly modulate the number of receptors on the hepatocyte surface. We conclude that, in the absence of ligand, surface receptors can either recycle or can be reversibly internalized or sequestered to prevent access to ligand. The results may also explain why different laboratories have reported a wide range of values for the number of receptors per hepatocyte.     

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.     

Tyrosine phosphorylation of the asialoglycoprotein receptor

The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with [gamma-32P]ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.     

Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.     

Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.     

Effect of hypo-osmotic incubation on membrane recycling

Incubation of alveolar macrophages in hypo-osmotic media causes a time-and temperature-dependent increase in the number of surface receptors for three different ligands. Exposure of cells to solutions of 210 mOsM or less, at 37 degrees C but not at 0 degree C, resulted in an increase in the number of surface receptors for diferric transferrin, alpha-macroglobulin-protease complexes, and mannose-terminated glycoproteins. Upon media dilution at 37 degrees C, surface receptor number reached a maximum within 5 min and returned to near-normal values by 30 min. The increase in surface receptor number was the result of a decrease in the rate of internalization of receptors, either occupied or unoccupied. The rate of receptor exteriorization was unaltered by hypo-osmotic incubation of cells. The rate of fluid-phase pinocytosis was also inhibited upon incubation in hypo-osmotic solution. In experiments in which both receptor-mediated endocytosis and fluid phase pinocytosis were measured on the same samples, inhibition of both processes occurred with the same kinetics and to a similar extent. The rate of receptor-mediated endocytosis recovered to normal rates after 60 min in hypo-osmotic solutions, whereas the rate of fluid phase pinocytosis did not recover to the same extent.     

Low temperature-induced cell surface membrane vesicle shedding is associated with DNA fragmentation

Temperature shift conditions of 0 degree to 22 degrees C or 0 degree to 37 degrees C induce the formation and shedding of membrane vesicles (MV) from P815 tumor cell surfaces. When the MV shedding process takes place at 22 degrees C it occurs without changes in cell surface membrane permeability, whereas at 37 degrees C, changes in permeability to 51Cr and trypan blue do occur, thus mimicking the lymphocyte-mediated lytic process of tumor cells [1]. The present studies demonstrate that nuclear DNA fragmentation also occurs in both 0 degree to 22 degrees C and 0 degree to 37 degrees C temperature shifts but cell surface membrane permeability to DNA fragments occurs only in the latter condition, i.e., 0 degree to 37 degrees C. The microtubule-stabilizing agent deuterium oxide (D2O) inhibited the MV shedding process, the changes in membrane permeability, and DNA fragmentation. When P815 cells which had been induced to shed MV by the 0 degree to 22 degrees C temperature shift were labeled with 51Cr and used as targets for alloimmune lymphocytes, they were found to be as susceptible to T-cell lysis as control P815 cells. This result indicates that the lytic effect of alloimmune T lymphocytes can be exerted at the target cell surface membrane level independently of nuclear DNA fragmentation.     

Effects of epidermal growth factor on transferrin receptor phosphorylation and surface expression in malignant epithelial cells

The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.     

Effect of phorbol esters on down-regulation and redistribution of cell surface receptors for tumor necrosis factor-alpha

The effects of various phorbol esters on the interaction of human cells with recombinant human tumor necrosis factor-alpha (rTNF-alpha) was investigated. Preexposure of several different types of cells with only biologically active tumor promoter, i.e. 4 beta-phorbol 12-myristate 13-acetate (PMA), inhibited the specific binding of rTNF-alpha to its receptor. The reduction in specific binding of TNF-alpha was observed only by PMA but not with several other phorbol esters tested. 1-oleoyl-2-acetylglycerol, which is an analogue of the natural protein kinase C activator, diacylglycerol, was active in down-regulating TNF-alpha receptors but only at 1000 times concentration than PMA. Scatchard analysis of the binding data on U-937 cells revealed that PMA caused a decrease in high affinity cell surface receptor number (approximately 8300 versus approximately 2500 binding sites/cell) without any significant change in the dissociation constant (0.38 nM versus 0.32 nM). This decrease in receptor number is dependent on temperature, the time of exposure, and dose of PMA. Greater than 95% of the specific binding of 125I-TNF-alpha could be abolished within 10 min by preexposure of cells to 10 nM PMA at 37 degrees C. The down-regulation of receptors by PMA occurred only at 37 degrees C but not at 4 degrees C, suggesting a probable internalization of the receptors. The specific binding of TNF-alpha to detergent-solubilized cell extracts remained unchanged after exposure of cells to PMA. The rates of dissociation of TNF-alpha from the cell surface and the rate of internalization was not significantly affected by PMA, but the rate of disappearance from cell interior and its appearance into the medium was slightly enhanced by PMA. PMA did not alter the rate of degradation of the TNF-alpha nor cause the shedding of receptors into the medium. Approximately 70% of TNF-alpha cell surface receptors could be regenerated within 16 h after PMA removal. These results suggest the involvement of PMA-activated protein kinase C in down-regulation and redistribution of TNF-alpha receptors.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

A rapid redistribution of the transferrin receptor to the cell surface of HL-60 cells and K562 cells upon treatment with dimethyl sulfoxide due to slowing of endocytosis

Treatment of two human leukemia cell lines with 1.25% dimethyl sulfoxide at 37 degrees C results in a rapid increase in the number of transferrin receptors on the cell surface detected by fluorescein-labeled anti-transferrin receptor antibodies. Both HL-60 cells, a human myeloid cell line, and K562 cells, a human erythroid-myeloid cell line, showed a 25-65% increase in cell surface transferrin binding in parallel experiments. Scatchard plot analysis of the data indicates that the number of receptors increases while the affinity of transferrin for the receptor remains the same. This rapid increase in the number of receptors at the cell surface appears to be due to a slowing of endocytosis rather than an increase in externalization of the receptor.     

Protein kinase C-linked inactivation of the interleukin-1 receptor in a human transformed B-cell line

The effect of tumor-promoting phorbol ester treatment on the binding of interleukin-1 beta (IL-1 beta) to specific cell surface receptors was investigated. A 1 h exposure of Raji human B lymphoma cells with the protein kinase C-activating phorbol ester, phorbol dibutyrate (PDBu), reduced IL-1 beta binding by up to 90% of control cells. This effect was dose-dependent and was not observed with 4-alpha-phorbol, an inactive tumor promoter. Analysis of 125I-labeled IL-1 beta binding to intact cells revealed that PDBu caused a 91% decrease in high-affinity cell-surface receptor number without an effect on receptor affinity. The phorbol ester response was rapid (30 min), observed both at 4 and 37 degrees C, and was preceded by the rapid translocation (t much less than 6 min) of protein kinase C (PKC) from the cytosol to the cell membrane. The PDBu-induced decrease in IL-1 beta receptor number was inhibited by prior incubation of cells for 30 min with the PKC inhibitor 1-(5-Isoquinoline sulfonyl)-2-methylpiperazine (H7). The decrease in receptor binding was not due to enhanced IL-1 beta receptor internalization or shedding into the extracellular medium, since a similar effect was observed with solubilized IL-1 beta receptor. The most likely explanation for the phorbol ester effect appears to be cell surface inactivation of IL-1 receptors. These data suggest that modulation of PKC activity could play a role in the regulation of the IL-1 beta receptor.     

Insulin increases the cell surface concentration of alpha 2-macroglobulin receptors in 3T3-L1 adipocytes. Altered transit of the receptor among intracellular endocytic compartments

The present study shows that insulin causes an increase in the binding of alpha 2-macroglobulin (alpha 2M) to 3T3-L1 adipocytes. Scatchard analysis of the binding at 4 degrees C indicated an approximate 2-fold increase in the number of alpha 2M binding sites, with no change in the apparent affinity of the receptor. In addition, a 2-3-fold increase in the binding of monoclonal antibody 2C6, which recognizes a component of the alpha 2M receptor, was found in cells treated at 37 degrees C with insulin and then KCN to inhibit receptor endocytosis. An increased cellular accumulation of alpha 2M was also observed in response to insulin. Interestingly, the increase in the rate of accumulation of alpha 2M was significantly smaller than the increase in the number of alpha 2M receptors on the cell surface, suggesting that the rate of ligand internalization or subsequent processing is altered in response to insulin. Ultrastructural analysis of the internalization pathway of the alpha 2M receptor was performed using colloidal gold-coupled 2C6 monoclonal antibody. Control cells incubated for 20 min at 37 degrees C with the gold-conjugated antibody displayed 40% of cellular gold particles on the cell surface and 60% within intracellular structures. In insulin-treated cells this proportion was reversed, with 64% of the particles being found on the cell surface, and only 36% within intracellular structures. Significant differences in the distribution of gold particles among intracellular structures were detected between control and insulin-treated cells. Whereas in control cells, 18% of the total cellular gold particles internalized into tubulovesicles and multivesicular bodies, in insulin-treated cells only 3% of the gold particles were found within these structures. These data indicate that the movement of this receptor between endocytic compartments is altered in response to insulin, and suggest that the effect of insulin to increase the cell surface concentration of alpha 2M receptors and the accumulation of alpha 2M is due, at least in part, to alterations in the endocytic portion of the receptor recycling pathway.     

Reversible blockage of membrane retrieval and endocytosis in the garland cell of the temperature-sensitive mutant of Drosophila melanogaster, shibirets1

Temperature-induced structural changes in the cortical region of the garland cell, which is considered to be active in endocytosis, were investigated in a temperature-sensitive, single gene mutant of Drosophila melanogaster, shibirets1 (shi) and wild-type (Oregon-R). At 19 degrees C, both shi and wild type showed similar structural features: an irregularly extended network of labyrinthine channels, coated pits and vesicles, tubular elements and alpha vacuoles. Tannic acid (TA) impregnation showed that coated pits comprised approximately 20-25% of the total coated profiles at 19 degrees C in both shi and wild-type. When flies were incubated in a horseradish peroxidase (HRP) solution for 5 min, organelles such as coated profiles, tubular elements, and alpha vacuoles were labeled. In wild-type at 30 degrees C, minor changes were observed--mainly a decrease in the distribution of the labyrinthine channels and an increase in HRP uptake. On the other hand, in shi at 30 degrees C, the labyrinthine channels were much elongated and their network became far more complex, indicating the expansion of the surface area of the cell. Also, the coated profiles were increased in number while the number of tubular elements was decreased considerably. The TA method showed that almost all of the coated profiles were coated pits, coated vesicles being almost completely absent at 30 degrees C in shi. Furthermore, HRP uptake activity was considerably decreased at 30 degrees C. These structural changes, as well as the reduced HRP uptake activity, were reversible when the temperature was lowered to 19 degrees C. The observations suggest that in the garland cell of shi the conversion of coated pits to coated vesicles, that is, membrane pinch-off, is blocked at high temperature.     

The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process

The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.     

The cellular dynamics of hepatic receptors for alpha 2-macroglobulin.protease complex and for insulin are different

Making freshly isolated rat hepatocytes permeable by 0.4 g/liter digitonin doubled the number of binding sites for alpha 2-macroglobulin.trypsin complex without changing the affinity. Thus, digitonin unmasked a receptor pool, probably of intracellular origin. The total cellular binding capacity was measured in the presence of digitonin, the surface-exposed in its absence. Upon preincubation of the cells at 37 degrees C, the total cellular binding capacity for alpha 2-macroglobulin.trypsin decreased over a 2-h period to 0.26 of the initial value. By contrast, the surface-exposed binding capacity initially increased in response to a preincubation at 37 degrees C, reached after 20 min a peak value 1.74 times that at 0 time, followed by a decrease. Neither the increase in nor the loss of surface-exposed binding capacity was influenced by inhibitors of lysosomal functions, protein synthesis and glycosylation. Colchicine abolished the increase in surface-exposed binding capacity but not the disappearance. By contrast, phenylarsine oxide (inhibitor of endocytosis), N-ethylmaleimide, and phenylmethanesulphonyl fluoride inhibited the receptor loss, suggesting that the loss occurred by proteolysis. The insulin receptor concentration, studied in parallel, remained practically constant in the investigated period in the presence and absence of digitonin. Thus, the hepatic receptor for alpha 2-macroglobulin.protease complexes is regulated independently of other specialized plasma membrane proteins.     

Acute effects of insulin on surface insulin receptors in isolated hepatocytes. Evidence for a recycling pathway

Isolated rat hepatocytes were incubated for 1 h at 37 degrees C with 10 nM insulin. Following washout of insulin, cells were incubated with [125I] monoiodoinsulin at 15 degrees C to assess surface insulin binding. Preincubation with 10 nM insulin did not cause a decrease in insulin binding. Scatchard analysis confirmed that insulin receptor number remained constant. In the presence of 200 microM chloroquine or 25 microM monensin, surface insulin binding after preincubation with 10 nM insulin fell to 81.1 +/- 1.2% or 39.0 +/- 2.7% of control, respectively. It is suggested that the maintenance of insulin receptor number following acute insulin treatment in vitro is due to an insulin receptor recycling pathway, possibly involving lysosomes and/or the Golgi apparatus.     

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.     

Bradykinin binding to B2 kinin receptors and stimulation of phosphoinositide turnover and arachidonic acid release in primary cultures of cells from late pregnant rat myometrium.

Primary cultures of cells from late pregnant rat myometrium contain B2 kinin receptors through which bradykinin (BK) stimulates inositol phosphate (InsP) formation and arachidonic acid (20:4) release. Equilibrium binding at 4 degrees C revealed that [3H]BK identified a maximal number of cell surface B2 kinin receptor binding sites on rat myometrial cells of 308 +/- 78 fmol/10(6) cells with apparently a single equilibrium dissociation constant of 1.8 +/- 0.2 nM. At 37 degrees C, [3H]BK binding was associated with a time-dependent decrease in the reversibility of the binding. This decrease was due in part to formation of slowly dissociating cell surface receptor [3H]BK binding and in part to internalization of the receptor-bound [3H]BK. Exposure of labeled cells to BK resulted in dose-dependent increases in [3H]InsP3, [3H]InsP2 ([3H]Ins(1,4)P2), and [3H]InsP1 ([3H]Ins(1)P1) formation and [3H]20:4 release. Pretreatment with 100 ng/mL pertussis toxin did not perturb BK stimulation of [3H]InsP formation but partially (approximately 30%) inhibited BK stimulation of [3H]20:4 release. BK stimulation of [3H]20:4 release was directly proportional to the number of receptor sites occupied by BK. In contrast, stimulation of [3H]InsP formation required a threshold level of receptor occupancy, which decreased as a function of time of BK exposure. These results show that BK interacts with B2 kinin receptors on rat myometrial cells with apparently a single affinity through which BK stimulates [3H]InsP formation and [3H]20:4 release. BK stimulation of [3H]InsP formation requires a threshold BK concentration, which decreases with time, and we suggest that the decrease is due to a time-dependent formation of a BK receptor binding state from which BK slowly dissociates.