Receptor-bound thrombin is not internalized through coated pits in mouse embryo cells

The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity-purified antithrombin rabbit antibody and colloidal gold labeled anti-rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor-bound thrombin was internalized by receptor-mediated endocytosis, ME cells were incubated with 125I-thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I-thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4 degrees C and the temperature increased to 37 degrees C and where initial incubation was at 37 degrees C, the receptor-directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor-mediated endocytosis.     

Role of specific cell surface receptors in thrombin-stimulated cell division

Previous studies have shown that thrombin action at the cell surface is sufficient to bring about division of cultured fibroblast-like cells (Carney and Cunningham, 1978). This prompted the present binding experiments with ¹²⁵I-thrombin which led to the Identification of a thrombin receptor on the surface of mouse embryo cells. Scatchard plots of binding data at 4, 22 and 37°C were linear over a broad range of thrombin concentrations, indicating a single affinity class of receptors. The association constant was about 1 × 10⁹ M^?1 and there were approximately 2 × 10⁵ receptors per cell. Neither insulin, epidermal growth factor nor prothrombin competed for thrombin binding to its receptor, indicating that It was unique for thrombin. Comparisons of thrombin binding and the amount of cell division produced by various concentrations of thrombin indicated that there was a relationship between receptor occupancy and increase in cell number. Low concentrations of serum (0.1%) inhibited both the mitogenic action of thrombin and the specific binding of thrombin to its receptor. It did not, however, inhibit nonspecific association of ¹²⁵I-thrombin with the cells. Experiments showed that this inhibition by serum resulted from a masking of thrombin receptors on the cells and not from binding of thrombin by serum factors. Together these studies suggest that thrombin must bind to Its surface receptor to stimulate cell division.     

Thrombin interaction with platelets. Influence of a platelet protease nexin

A fraction of the 125I-thrombin that binds to human platelets is taken into a sodium dodecyl sulfate-resistant 77 kDa complex with a platelet factor (Bennett, W. F., and Glenn, K. C. (1980) Cell 22, 621-627). Here we show that this platelet factor is in several respects similar to protease nexin I (PNI), a fibroblast thrombin inhibitor. The complexes are of the appropriate size, bind to Sepharose that has been derivatized with anti-PNI antibody, do not form when the thrombin active site has been blocked with diisopropylphosphofluoridate, and do not appear on platelets when heparin is present. However, the platelet factor does not bind urokinase, indicating that this "platelet PN" may be distinct from PNI. Following brief incubation with 125I-thrombin, platelet PN X 125I X thrombin complexes are found both associated with the platelets and free in the binding medium. 125I-Thrombin has a higher affinity for platelet PN than for platelet receptors. In 30-s binding incubations carried out with thrombin at concentrations below 0.3 nM, formation of the 77-kDa complex accounts for most of the platelet specific binding of 125I-thrombin. Subtracting this large contribution to 125I-thrombin-specific binding reveals that the reversible binding of 125I-thrombin to platelet receptors exhibits sigmoidal thrombin dose-dependence. Thrombin stimulation of platelet [14C]serotonin release exhibits similar thrombin dose dependence. These results indicate that platelets may possess a mechanism for suppressing their interaction with active thrombin at thrombin doses below 0.3 nM. It is possible that platelet PN carries out this function by capturing thrombin before thrombin binds to its signal-transmitting receptors.     

Cell surface action of thrombin is sufficient to initiate division of chick cells

Thrombin covalently linked to carboxylate-modified polystyrene beads initiated division of quiescent chick embryo (CE) cells either in medium containing low levels of serum or in serum-free medium. Release of thrombin was monitored by measuring acid-precipitable radioactivity released from 125I-thrombin beads into the medium during incubation with cells. Even if all of the acid-precipitable material released from the beads were active thrombin, it was not sufficient to account for any of the observed cell division, and was 10-30 fold less than the amount necessary to produce the increase in cell number caused by the thrombin beads. Two other kinds of experiments also showed that material released into the medium did not account for the observed initiation of cell division. First, medium taken from cultures incubated with thrombin beads did not initiate cell division when added to new quiescent cultures. Second, in coverslip experiments where populations of cells with an without thrombin feads shared the same medium, only bead-contacted cells divided. Several results suggested that the material which was released from the thrombin beads resulted from cell-associated proteolysis rather than from "leakage" of intact thrombin from the beads. For example, after incubating 125I-thrombin beads with or without CE cells, we were unable to detect any intact thrombin released into the medium. In addition, most of the material released from the beads was acid-soluble and was only released in the presence of CE cells. A few thrombin beads were endocytosed by CE cells, but they were surrounded by an intact plasma membrane. Thus they did not directly interact with the cytoplasm. The close association of many of the beads with the cell surface and the presence of a few beads in endocytic vesicles made it important to consider the possibility that thrombin might be released from the beads directly into the cells. This possibility was explored using ultrastructural (EM) autoradiography. With this technique (where one grain represented 700--900 thrombin molecules), we found that beads inside the cells had approximately the same number of grains as beads not in contact with cells. This suggested that little, if any, additional radioactive material had been released from the beads which were in contact with the cells. In addition, we were unable to detect any grains in the cytoplasm which could be attributed to released thrombin, even using an amount of 125I-thrombin beads which was 8 fold greater than the amount which produced maximal cell division. Taken together, these results provide direct evidence that thrombin action at the cell surface is sufficient to initiate division of CE cells.     

Characterization of the receptor for protease nexin-I:protease complexes on human fibroblasts

Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin-I, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin-I and its target protease via a cell-surface, high-affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4 degrees C binding of PN-I:protease complexes was competed by heparin. In addition, binding was independent of the particular protease bound to the PN-I; purified complexes of PN-I with thrombin or urokinase competed equipotently for [125]I-thrombin:PN-I binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes. At 37 degrees C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate constant) of 1.0 min-1. At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields a receptor number of 250,000 per cell and a Kd of 1 nM.     

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I- thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.     

Visualization of thrombin receptors on mouse embryo fibroblasts using fluorescein-amine conjugated human α-thrombin

The localization of thrombin receptors on mouse embryo (ME) cells has been examined by direct fluorescence microscopy using a fluorescein aminelabeled thrombin. Two fluorescein amines, 4-(N-6-aminoethyl thioureal)-fluorescein and 4-(N-6-aminohexyl thioureal)-fluorescein, were synthesized and attached to the carbohydrate moiety of highly purified human α-thrombin by periodate oxidation of the carbohydrate and selective reduction of the Schiff's base using sodium cyanoborohydride. Preparations of fluorescent thrombin with from 1 to 4 fluoresceins per molecule of thrombin retained their ability to proteolytically cleave fibrinogin to form fibrin clots, to bind to thrombin receptors on ME cells, and to initiate cell division. After incubating mitogenic concentrations of the fluorescein amine labeled thrombin with ME cells at 4°C, a diffuse fluorescent pattern was observed over the surface of the ME cells. This diffuse pattern was specific: it was not observed on cells from parallel cultures incubated with fluorescent thrombin plus a 20-fold excess of unlabeled thrombin. Thus, thrombin receptors appear to be distributed randomly over the surface of ME cells prior to interaction with thrombin. Increasing the temperature to 37°C following binding at 4° C resulted in a rapid dissociation of the fluorescent pattern from the cells leaving only the autofluorescent vesicles. This result may reflect the unique ability of thrombin to proteolytically cleave its own receptor.     

The effects of cell-released protease nexin on the measurement of thrombin binding to mouse cells

We previously showed that fibroblast-like cells release protease nexin into their growth medium. Protease nexin links to thrombin and mediates the cellular binding of thrombin via the protease nexin part of the complex to a site different from that for unlinked thrombin (1,2). To determine the effect that cell-released protease nexin had on the measurement of total cell-bound thrombin, we separately measured the cellular binding of both ¹²⁵I-thrombin and ¹²⁵I-thrombin-protease nexin complexes. Scatchard analysis of our binding data indicates that the cellular binding affinity of linked ¹²⁵I-thrombin is about 19-fold higher than that of unlinked ¹²⁵I-thrombin. We show that protease nexin acts to increase the apparent affinity of ¹²⁵I-thrombin for cellular binding sites.     

Direct linkage of thrombin to its cell surface receptors in different cell types

When ¹²⁵I-thrombin was incubated with foreskin fibroblasts, cervical carcinoma cells or fibrosarcoma cells of human origin, or with secondary chick embryo cells or Chinese hamster lung cells, it became directly linked to its cell surface receptors. The thrombin-receptor complex (TH-R) was derived exclusively from a pool of ¹²⁵I-thrombin that had become specifically bound to the cell surface. The linkage was probably covalent, since the complex was resistant to boiling in sodium dodecyl sulfate and 2-mercaptoethanol. Raising the pH to 12 disrupted TH-R, but did not affect a similar complex between epidermal growth factor and its receptor, suggesting that the linkage of these mitogens to their receptors was different. Mild trypsin treatment removed the ability of cells to form TH-R; however, after a 24-h incubation in serum-free medium, trypsin-treated cells recovered the capacity to form TH-R, suggesting that TH-R resulted from interaction of ¹²⁵I-thrombin with a cellular rather than a serum component. The mitogenic response of cells to thrombin was inversely related to the fraction of specifically bound ¹²⁵I-thrombin represented by TH-R. The role of TH-R in mitogenesis may be clarified in future studies by obtaining clones of Chinese hamster lung cells that vary in their capacities to form TH-R and to respond to the mitogenic action of thrombin.     

Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin

We have prepared a monoclonal antibody directed against human thrombomodulin. We used the antibody to measure thrombomodulin molecules in cultured human endothelial cells from umbilical vein and in a human lung cancer cell line (A549). Endothelial cells contain approximately 30,000-55,000 molecules of thrombomodulin/cell while the A549 cell has about 1/4 of this number. About 50-60% of thrombin binding sites on endothelial cells are thrombomodulin, while about 90% of thrombin binding sites on A549 cells are thrombomodulin. Exposure of these cells to thrombin decreased thrombomodulin on the cell surface suggesting that internalization of thrombin-thrombomodulin occurred. The internalized 125I-thrombin was degraded in the cells and thrombomodulin reappeared on the cell surface after 30 min, suggesting the recycling of thrombomodulin. The rate of protein C activation correlated with the presence of the thrombin-thrombomodulin complex on the cell surface. The binding of thrombin to cell-surface thrombomodulin accelerates protein C activation; the subsequent internalization of the thrombin-thrombomodulin complex is associated with cessation of protein C activation. Therefore, endocytosis of thrombin-thrombomodulin may serve to control protein C activation. The uptake and degradation of thrombin bound to thrombomodulin may provide a mechanism for clearance of thrombin from the circulation.     

Dispersed mammary epithelial cells. Receptors of lactogenic hormones in virgin, pregnant, and lactating rabbits.

The number and affinity of binding sites for lactogenic hormones have been determined in dispersed mammary cells from virgin, pregnant, and lactating rabbits. Dispersed epithelial cells, prepared from mammary glands by enzyme digestion, calcium chelation, and gentle shearing, were separated from nonepithelial cells by density centrifugation. 125I-labeled ovine prolactin (oPRL) and 125I-labeled human growth hormone (/GH) were used as tracers. Association and dissociation of 125I-oPRL or 125I-hGH were time- and temperature-dependent. The rate of association followed a second order reversible reaction with a rate constant of approximately 0.5 at 4 degrees C, approximately 2.0 at 23 degrees C, and approximately 9 x 10(7) M-1 min-1 at 37 degrees C. Maximum binding was achieved after 120 h at 4 degrees C, 48 h at 23 degrees C, and 2 to 4 h at 37 degrees C. Dissociation of 125I-oPRL or hGH from cells by unlabeled oPRL was complete at 4 degrees C after 160 h, following a first order reaction (5-1 = 9.9 x 10(-5) min) and incomplete at 23 degrees C and 37 degrees C even after prolonged time. Internalization of receptor-bound 125I-oPRL was studied by quantitative electron microscope autoradiography. Grain distribution over- and volume densities of cellular organelles was analyzed as a function of time and temperature. At 37 degrees C, there was a rapid and specific translocation of lactogenic hormones to intracellular organelles. Autoradiographic grains were found associated with vesicles, Golgi elements, lysosome-like structures, and the nucleus. One class of high affinity binding sites was estimated from Scatchard plot and direct kinetic analyses at 4 degrees C. Whereas the apparent affinity constant (approximately 10(10) M-1) did not change significantly throughout pregnancy and early lactation, the number of receptors extrapolated from Scatchard plots at 4 degrees C varied in an inverse relation to serum progesterone concentration. Thus, approximately 1900 sites were detected in virgin rabbits (progesterone, approximately 200 pg/ml), and midpregnancy (progesterone, approximately 15,000 pg/ml), and approximately 1800 during early lactation (progesterone, approximately 500 pg/ml). The binding properties of lactogenic hormones to dispersed cells was compared with those to Triton X-100 solubilized microsomal membrane preparations. Good correlation between the two systems was found indicating that cell dispersion did not alter binding properties. Our results indicate that dispersed mammary cells bind lactogenic hormones in a saturable and reversible process, that the number of exposed receptors varies throughout gestation and lactation, and finally that lactogenic hormones are internalized following interaction with their membrane receptors.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Insulin elicits a redistribution of transferrin receptors in 3T3-L1 adipocytes through an increase in the rate constant for receptor externalization

Incubation of 3T3-L1 adipocytes with insulin at 37 degrees C resulted in a 2-fold increase in specific binding of transferrin to cell-surface receptors, as measured by a subsequent incubation of cells at 4 degrees C with 125I-transferrin. The insulin concentration required for half-maximal effect was 10 nM, and the half-time for insulin action was 40 s. By comparison, insulin stimulated hexose transport in 3T3-L1 adipocytes with a half-maximal effect at 8 nM and a half-time of 105 s. Scatchard analysis of 125I-transferrin binding to cells at 4 degrees C showed that the insulin-induced increase in transferrin receptor binding was due to an increase in the number of surface transferrin receptors. When cells were incubated for 2 h at 37 degrees C with 125I-transferrin to achieve steady-state binding and then exposed to insulin, there was a 1.7-fold increase in surface-bound transferrin (acid-sensitive) and a corresponding decrease in intracellularly bound transferrin (acid-insensitive). Thus, insulin elicits translocation of intracellular transferrin receptors to the plasma membrane. Concomitant with the 2-fold increase in surface receptors in response to insulin, there was a 2-fold increase in the rate of 59Fe3+ uptake from 59Fe3+-loaded transferrin. The rate of externalization of the intracellular 125I-transferrin-receptor complex at 37 degrees C was determined for basal and insulin-treated cells. Insulin increased the first-order rate constant for this process 1.7-fold. The effect of insulin on the rate of externalization is sufficient to account for the increase in surface transferrin receptors.     

Inhibitors of Urokinase and Thrombin in Cultured Neural Cells

Recent studies have suggested important roles for certain proteases and protease inhibitors in the growth and development of the CNS. In the present studies, inhibitors of urokinase or thrombin in cultured neural cells and serum-free medium from the cells were identified by screening for components that formed sodium dodecyl sulfate-stable complexes with 125I-urokinase or 125I-thrombin. Rinsed glioblastoma possessed two components that complexed 125I-urokinase. One was type 1 plasminogen activator inhibitor (PAI-1), because the 125I-urokinase-containing complexes were immunoprecipitated with anti-PAI-1 antibodies. The other component formed complexes with 125I-urokinase that were not recognized by antibodies to PAI-1 or protease nexin-1 (PN-1). Its identity is unknown. In addition to these cell-bound components, the glioblastoma cells also secreted two inhibitors that formed complexes with 125I-urokinase; one was PAI-1, and the other was PN-1. The secreted PN-1 also formed complexes with 125I-thrombin. It was the only thrombin inhibitor detected in these studies. Human neuroblastoma cells did not contain components that formed detectable complexes with either 125I-urokinase or 125I-thrombin. However, human neuroblastoma cells did contain very low levels of PN-1 mRNA and PN-1 protein. Added PN-1 bound to the surface of both glioblastoma and neuroblastoma cells. This interaction accelerated the inhibition of thrombin by PN-1 and blocked the ability of PN-1 to form complexes with 125I-urokinase. Thus, cell-bound PN-1 was a specific thrombin inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells

GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high-affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I-Tyr1]SRIF and 125I-EGF to their specific membrane receptors, these peptides are processed very differently in GH4C1 cells.     

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.     

Recycling of the asialoglycoprotein receptor and the effect of lysosomotropic amines in hepatoma cells

Receptor-mediated uptake and degradation of 125I-asialoorosomucoid (ASOR) in human hepatoma HepG2 cells is inhibited by the lysosomotropic amines chloroquine and primaquine. In the absence of added ligand at 37 degrees C, these amines induce a rapid (t1/2 5.5-6 min) and reversible loss of cell surface 125I-ASOR binding sites as well as a rapid decrease in 125I-ASOR uptake and degradation. There is no effect of these amines on the binding of 125I-ASOR to the cell surface at 4 degrees C or on the rate of internalization of prebound 125I-ASOR. The loss of 125I-ASOR surface binding at 37 degrees C is not attributable to altered affinity of ligand-receptor binding. In the presence of added ligand at 37 degrees C, there is a more rapid (t1/2 2.5-3 min) loss of hepatoma cell surface receptors. In addition, the amines inhibit the rapid return of the internalized receptor to the cell surface. We examined the nature of this loss of 125I-ASOR surface binding sites by following the fate of receptor molecules after biosynthetic labeling and after cell surface iodination. At 37 degrees C, chloroquine and primaquine induce a loss of asialoglycoprotein receptor molecules from the hepatoma cell surface to an internal pool.     

Rapid agonist-induced decrease of 125I-pindolol binding to beta-adrenergic receptors. Relationship to desensitization of cyclic AMP accumulation in intact heart cells

Cyclic AMP accumulation in embryonic chick heart cells and binding of the beta-adrenergic antagonist 125I-pindolol to intact cells has been examined during the first 30 min of (-)-isoproterenol-induced desensitization. Myocardial beta-adrenergic receptors exist in two states which bind agonists with high (KD congruent to 10 nM) and low (KD congruent to 10 microM) affinities. Both activation and desensitization of cyclic AMP accumulation were mediated by (-)-isoproterenol binding to high affinity receptors. (-)-Isoproterenol-induced desensitization of cyclic AMP accumulation occurred with a t1/2 of 3.8 min. Desensitization was accompanied by a decrease in the number of 125I-pindolol binding sites assessed by equilibrium radioligand binding assays conducted at 4 degrees C or short (80 s) binding assays conducted at 37 degrees C. There was an excellent temporal correlation between loss of binding and loss of (-)-isoproterenol-stimulated cyclic AMP accumulation. After (-)-isoproterenol-induced desensitization, most of the remaining receptors assayed at 4 degrees C bound (-)-isoproterenol with high affinity. A rapid (-)-isoproterenol-induced decrease in the number of 125I-pindolol binding sites also occurred in adult canine heart cells and rat adipocytes. The data suggest that agonists do not cause uncoupling of surface receptors. Receptors may be uncoupled as a consequence of rapid sequestration into a hydrophobic compartment.     

Binding and receptor-mediated endocytosis of erythropoietin in Friend virus-infected erythroid cells

The binding of labeled erythropoietin (EP) to cell surface receptors and subsequent processing of the hormone within the cell was studied in erythroid cells procured from the spleens of mice infected with the anemia strain of Friend virus. These immature erythroid cells respond to EP in culture to differentiate into reticulocytes and erythrocytes. Radiolabeled EP (both iodinated and tritiated) binds to 800-1000 cell surface receptors on these cells at 4 degrees C. Using 125I-EP, we found that 300 of these cell surface receptors have a higher affinity for EP (Kd = 0.09 nM) than the remaining receptors (Kd = 0.57 nM). The number of molecules of EP bound per cell increased about 2-fold when binding was carried out at 37 degrees C. Treatment of the cell surface with pronase or removal of surface-bound EP with a low pH wash revealed that radiolabeled EP is internalized by the cells at 37 degrees C. Pulse chase experiments showed that degradation products of radiolabeled EP are released into the medium with a corresponding loss of label from the interior of the cell. Inhibitors of lysosomal function greatly reduced this degradation of 125I-EP. Since 180 of the 300 high affinity receptors and very few of the low affinity receptors are occupied at the concentration of EP which elicits the maximum biological response in these cells, we suggest that interaction of EP with the high affinity receptors are necessary for the full biological effect of the hormone. A different murine erythroleukemia cell line which does not differentiate in response to EP was found to have only the lower affinity binding sites for the hormone.