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.     

alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.     

Primary amines do not prevent the endocytosis of epidermal growth factor into 3T3 fibroblasts

Various amines block the degradation of endocytosed epidermal growth factor (EGF) without affecting the binding of the hormone to its surface receptors. However, studies based on fluorescence microscopy demonstrate that amines block the internalization of alpha 2-macroglobulin and EGF by preventing it from clustering in clathrin coated pits. In order to resolve this controversy we have studied in detail the effect of various amines on the localization and processing of fluorescent and radiolabelled EGF. We have explored the effect of amines on EGF binding and localization, receptor mobility, membrane fluidity, receptor down regulation, hormone degradation and release of degradative products as a function of time and temperature. Our conclusions are as follows. 1. Primary amines prevent the formation of visible patches of fluorescent EGF and alpha 2-macroglobulin on the cell surface at least for 15 min, thus increasing the diffusion coefficients and the mobile fraction of EGF-receptor complexes on the cell surface. 2. Amines do not block the endocytosis of EGF and alpha 2-macroglobulin. On most cells fluorescent EGF and alpha 2-macroglobulin are clustered and endocytosed within 30-45 min at 37 degrees C. 3. Amines do not effect the internalization of 125I-labelled-EGF and the down regulation of EGF receptors. 4. Amines block the degradation of the endocytosed EGF.     

Binding and internalization of alpha 2-microglobulin by cultured fibroblasts. Effects of monovalent ionophores

Receptor-bound alpha 2-macroglobulin (alpha 2M) undergoes a two-step process in its internalization by cultured fibroblasts. First, the receptor- alpha 2M complexes concentrate in coated pits on the cell surface. Second, the alpha 2M is internalized into endocytic vesicles we have termed receptosomes. Using a variety of monovalent ionophores and inhibitors of ATP synthesis, the present report provides data that discriminates between these two steps. Appearance of alpha 2M-receptor complexes in coated pits occurs at 4 degrees C and is inhibited by primary amines as well as some other drugs and chemical reagents [1, 2]. Internalization of alpha 2M-receptor complexes into receptosomes is inhibited by monovalent ionophores that disrupt proton gradients (monensin, nigericin, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and 3,3',4',5-tetrachlorosalicyanilide), but not the Na+ specific ionophore antamanide or the K+ specific ionophore valinomycin. Using electron microscopy, the proton ionophores appear to interfere with the transfer of alpha 2M from coated pits to receptosomes. Prolonged incubation with monensin in the presence of alpha 2M also decreases the number of alpha 2M receptors on the cell surface, but this did not appear sufficient to account for the extensive inhibition of internalization. Monensin also inhibited the internalization of vesicular stomatitis virus and epidermal growth factor (EGF). Our data suggest that a proton gradient may be necessary for receptor-mediated endocytosis of alpha 2M and some other ligands.     

Receptor-mediated endocytosis of alpha 2-macroglobulin and transferrin in rat caput epididymal epithelial cells in vitro

The endocytic activity of epithelial cells from the rat epididymis in vitro has been examined by following the uptake of tracer compounds conjugated to proteins. Transferrin-gold and alpha 2-macroglobulin-gold were taken up initially in coated pits, internalized and sequestered into tubular-vesicular structures, multivesicular bodies and, in the case of alpha 2-macroglobulin, into lysosomes. Uptake could be prevented by an excess of unlabeled protein. Studies using 125I-alpha 2-macroglobulin and 125I-transferrin also showed that the uptake of these proteins was specific and could be displaced with increasing amounts of unlabeled protein. In addition, binding of 125I-transferrin to cells was saturable at 4 degrees C. These studies indicate that transferrin and alpha 2-macroglobulin are taken up by receptor-mediated endocytosis. In contrast, a fluid phase marker, bovine serum albumin-gold (BSA-gold), was initially taken up predominantly in uncoated caveolae rather than coated pits, and could not be displaced with excess BSA. By virtue of their charge, polycationized ferritin and unlabeled colloidal gold were taken up and internalized by adsorptive endocytosis, a pathway which is similar to fluid phase endocytosis. The uptake and internalization of alpha 2-macroglobulin and transferrin differed in a number of respects. Uptake and internalization of alpha 2-macroglobulin but not of transferrin was dependent on extracellular calcium. Only alpha 2-macroglobulin was transferred into lysosomes, whereas transferrin was recycled to the cell surface. Although the proton ionophore, monensin, and the transglutaminase inhibitor, dansylcadaverine, did not stop uptake and internalization of either alpha 2-macroglobulin or transferrin, they did prevent the transfer of alpha 2-macroglobulin to lysosomes.     

Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.     

Loss of alpha 2-macroglobulin and epidermal growth factor surface binding induced by phenothiazines and naphthalene sulfonamides

We have found that certain naphthalenesulfonamides [e.g., N-6(-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)] and phenothiazines [e.g., trifluoperazine (TFP)] induce a loss of cell-surface receptors for alpha 2-macroglobulin, and epidermal growth factor (EGF) in fibroblasts. The loss of alpha 2-macroglobulin receptors is independent of receptor occupancy and is rapidly reversed upon removal of these agents from the culture medium. The extent of EGF receptor loss is less than for alpha 2-macroglobulin, and the EGF receptors do not reappear at the surface when W-7 is removed. Receptor loss was measured as a change in the capacity for binding iodinated ligands; no change in affinity of binding was observed. This receptor loss could reflect inactivation of receptors or internalization. W-7 did not induce a loss of cell surface beta 2-microglobulin, a membrane protein which is excluded from coated pits and which is not internalized, indicating that the effect of W-7 was specific for membrane receptors and not a result of bulk depletion of plasma membrane. The loss of alpha 2-macroglobulin and EGF receptors occurs at concentrations which do not cause an increase in the pH of endocytic vesicles or the cytoplasm, indicating that these agents act by a mechanism distinct from the effect of other weak bases. Since both TFP and W-7 are potent inhibitors of calmodulin, we investigated the possibility that inhibition of calmodulin was responsible for the loss of receptors. Three lines of evidence suggest that calmodulin inhibition is not responsible for the inhibition of binding and endocytosis: 1) Promethazine, a phenothiazine that is a poor inhibitor of calmodulin, is nearly as effective as TFP at inhibiting endocytosis; calmidazolium, a potent inhibitor of several calmodulin functions, did not cause a loss of binding; 2) the microinjection of calmodulin into cells did not reverse the effects of W-7; using pressure microinjection, we introduced up to a 100-fold excess of calmodulin over native levels into individual gerbil fibroma cells; using rhodamine-labeled alpha 2-macroglobulin, we saw that the W-7 induced inhibition of receptor-mediated endocytosis was the same in injected and uninjected cells; 3) we injected calcineurin, a calmodulin-binding protein, into cells (1-3 pg/cell) and observed no effect on the receptor-mediated endocytosis of rhodamine-labeled alpha 2-macroglobulin. These data indicated that cell surface receptor numbers can be regulated by a cellular component that is not cytoplasmic calmodulin but that shares some drug sensitivities with calmodulin.     

Lactate, alanine and glutamine metabolism in isolated canine pup liver cells

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.     

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.     

Direct demonstration of rapid insulin-like growth factor II Receptor internalization and recycling in rat adipocytes. Insulin stimulates 125I-insulin-like growth factor II degradation by modulating the IGF-II receptor recycling process

The photoactive insulin-like growth factor (IGF)-II analogue 4-azidobenzoyl-125I-IGF-II was synthesized and used to label specifically and covalently the Mr = 250,000 Type II IGF receptor. When rat adipocytes are irradiated after a 10-min incubation with 4-azidobenzoyl-125I-IGF-II at 10 degrees C and immediately homogenized, most of the labeled IGF-II receptors are associated with the plasma membrane fraction, indicating that receptors accessible to the labeling reagent at low temperature are on the cell surface. However, when the photolabeled cells are incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors are rapidly internalized with a half-time of 3.5 min as evidenced by a loss from the plasma membrane fraction and a concomitant appearance in the low density microsome fraction. The low density microsomes were previously shown to contain intracellular membranes (Oka, Y., and Czech, M.P. (1984) J. Biol. Chem. 259, 8125-8133). The steady state level of cell surface IGF-II receptors in the presence or absence of IGF-II, measured by the binding of anti-IGF-II receptor antibody to cells, remains constant under these conditions, demonstrating that IGF-II receptors rapidly recycle back to the cell surface at the same rate as receptor internalization. Using the above methodology, it is shown that acute insulin action: 1) increases the steady state number of cell surface IGF-II receptors; 2) increases the number of ligand-bound IGF-II receptors that are internalized per unit of time, as evidenced by a large increase in the photolabeling of intracellular membrane IGF-II receptors when cells are incubated at 37 degrees C with insulin and 4-azidobenzoyl-125I-IGF-II prior to photoactivation; and 3) increases the rate of cellular 125I-IGF-II degradation by a process that is blocked by anti-IGF-II receptor antibody. The results indicate that the action of insulin to elevate the steady state number of cell surface IGF-II receptors leads to an increased internalization flux of IGF-II-bound receptors, mediating increased IGF-II uptake and degradation.     

Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts

Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.     

Binding, surface mobility, internalization, and degradation of rhodamine-labeled alpha 2-macroglobulin

We have used quantitative fluorescence methods to examine the fate of rhodamine-labeled alpha 2-macroglobulin (R-alpha 2 M) after binding to cell-surface receptors on NRK and Swiss 3T3 cells. From measurements of fluorescence intensities in NRK cells fixed after incubation with R-alpha 2M, we found that uptake was saturable and that half-maximal uptake occurred at 130 nM R-alpha 2M. Fluorescence measurements on cell extracts of NRK and Swiss 3T3 cells also showed a half-maximal uptake of R-alpha 2M near 130 nM. We estimate that NRK cells can take up 10(6) molecules of R-alpha 2M per hour via receptor-mediated endocytosis. The mobility of alpha 2-macroglobulin receptors on the surface of Swiss 3T3 cells was measured by using fluorescence photobleaching recovery. The two-dimensional effective diffusion coefficient of R-alpha 2M receptors was approximately 8 X 10(-10) cm2 s-1, a value close to that previously obtained for insulin and epidermal growth factor receptors. Degradation of R-alpha 2M by the cells was followed by using the loss of fluorescence from the 185000-dalton band in sodium dodecyl sulfate--polyacrylamide gels. Rhodamine fluorescence was detected in the gels by using a microscope fluorescence spectrophotometer. NRK cells degraded alpha 2M to low molecular weight fragments with a t 1/2 of 15 min. Swiss 3T3 cells degraded about 75% of the alpha 2M with a t 1/2 of 1 h. The remaining 25% remained as the intact 185000-dalton peptide after 24 h. No significant accumulation of large breakdown products was observed in Swiss 3T3 or NRK cells.     

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.     

Binding and degradation of alpha 2-macroglobulin by cultured fibroblasts

We studied the interactions of alpha 2-macroglobulin, a major protease inhibitor of plasma and of serum-containing culture medium, with cultured fibroblasts. Iodinated human alpha 2-macroglobulin bound specifically to washed cell layers of cultured human fibroblasts. At 0--4 degrees C, binding was saturated at a concentration of 10--20 micrograms/ml. At 37 degrees C, radiolabel appeared in the medium in a form soluble in 10% trichloroacetic acid. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that ingested iodinated alpha 2-macroglobulin transiently forms a complex with a trypsin-like protease. Indirect immunofluorescence demonstrated alpha 2-macroglobulin in vacuoles of fibroblasts grown in 10% human serum or incubated with purified alpha 2-macroglobulin. Fibroblasts transformed by SV-40 (VA-13 cells) bound and degraded less 125I-labeled alpha 2-macroglobulin than non-transformed fibroblasts and had fewer vacuoles containing alpha 2-macroglobulin. These observations indicate that cultured fibroblasts bind, take up by endocytosis, and degrade alpha 2-macroglobulin. Binding and endocytosis of alpha 2-macroglobulin by a cell may be a means of modulating proteases in the microenvironment of the cell and during endocytosis.     

Hyperthermia can enhance the initiation of DNA synthesis stimulated by growth factors in Swiss mouse 3T3 cells

Confluent quiescent Swiss mouse 3T3 cells can be stimulated to initiate DNA synthesis and to divide by epidermal growth factor (EGF) and prostaglandin F2 alpha (PGF2 alpha), two mitogens of unrelated structure. Heat treatment at 46 degrees C for up to 20 min of confluent quiescent cells, which has no mitogenic effect, can enhance the stimulatory effect of suboptimal concentrations of EGF or PGF2 alpha on the initiation of DNA synthesis. Furthermore, insulin, which is not mitogenic in these cells, enhances the effect of these mitogens, but this effect is not further enhanced by heat treatment. Likewise the combination of EGF and PGF2 alpha is synergistic on DNA synthesis, and this effect is also not enhanced by the heat treatment. Incubation at 46 degrees C for longer than 20 min was inhibitory in all cases. These results suggest that heat treatment induces events which affect the regulation of the initiation of DNA synthesis in a manner depending on the duration of the heat treatment and the stimulation of the cells.     

Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.     

Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.     

Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body

We compared the internalization and intracellular sorting of epidermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinase-negative receptor was surface down-regulated only with monensin or at 20 degrees C. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGF-R recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after 10-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity.     

Distinct properties of the recognition sites for beta-very low density lipoprotein (remnant receptor) and alpha 2-macroglobulin (low density lipoprotein receptor-related protein) on rat parenchymal cells.

The properties of the recognition sites for alpha 2-macroglobulin (alpha 2-macroglobulin receptor; low density lipoprotein receptor-related protein) and beta-migrating very low density lipoprotein (beta-VLDL) (remnant receptor) on rat parenchymal cells were directly compared to analyze whether both substrates are recognized and internalized by the same receptor system. In cholesterol-fed rats, the large circulating pool of beta-VLDL is unable to diminish the liver uptake of 125I-labeled alpha 2-macroglobulin, while liver uptake of 125I-labeled beta-VLDL in these rats is reduced by 87.3% at 10 min after injection. In vitro competition studies with isolated parenchymal liver cells demonstrate that the binding of 125I-labeled alpha 2-macroglobulin to rat parenchymal cells is not effectively competed for by beta-VLDL, whether this lipoprotein is additionally enriched in apolipoprotein E or not. Binding of alpha 2-macroglobulin to parenchymal cells requires the presence of calcium, while binding of beta-VLDL does not. Incubation of parenchymal cells for 1 h with proteinase K reduced the subsequent binding of alpha 2-macroglobulin by 90.1%, while the binding of beta-VLDL was reduced by only 20.2%. In the presence of monensin, the association of alpha 2-macroglobulin to parenchymal cells at 2 h of incubation was reduced by 64.7%, while the association of beta-VLDL was not affected. Preincubation of parenchymal cells with monensin for 60 min at 37 degrees C reduced the subsequent binding of alpha 2-macroglobulin by 54.5%, while binding of beta-VLDL was only reduced by 14.6%. The results indicate that the recognition sites for alpha 2-macroglobulin and beta-VLDL on rat parenchymal cells do exert different properties and are therefore likely to reside on different molecules.     

The immunofluorescent detection of phosphorylated receptors of the epidermal growth factor during their internalization in A-431 cells]

Phosphorylated receptors of the epidermal growth factor (EGF) were localized in the human epidermoid carcinoma cells using immunofluorescent staining with antibody to phosphotyrosine. The application of EGF at 4 degrees C was seen to induce a characteristic fluorescence of the cell margins, whereas no cell staining occurs in the absence of EGF. After a 1 hour incubation of cells at 37 degrees C, within which the internalized EGF receptor complexes are accumulated in the juxtanuclear compartment near the para-Golgi region, the staining with antiphosphotyrosine antibody reveals the receptors in this region. It is concluded that the internalized EGF-receptor complexes may remain in the phosphorylated state.