Intracellular pathway followed by the insulin receptor covalently coupled to 125I-photoreactive insulin during internalization and recycling.

After it interacts with a specific receptor on the cell surface, insulin is internalized in its target cell by an adsorptive endocytotic process and eventually degraded in lysosomes. It was also recently shown that the initial surface interaction between the hormone and its receptor is followed by an internalization of the receptor, which later is recycled back to the cell surface. In the present study the insulin receptor was tagged with a 125I-photoreactive insulin analogue that can be covalently coupled to the insulin receptor by ultraviolet irradiation. Using this tool we could trace by quantitative electron microscope autoradiography the intracellular pathway followed by this labeled receptor. The quantitative analysis of the intracellular distribution of the labeled material as a function of incubation time at 37 degrees C supports the following sequence of events: association first with clear vesicles, second with multivesicular bodies, third with dense bodies, and fourth, a return to the cell surface via clear vesicles. This insulin receptor recycling process is inhibited by monensin but unaffected by cycloheximide.

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes.

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.

Plasma membrane vesicles from isolated hepatocytes retain the increase of amino acid transport induced by dibutyryl cyclic AMP in intact cells.

We have investigated the effect of cyclic AMP on hepatic amino acid transport by measuring the uptake of L-alanine in plasma membrane vesicles purified from hepatocytes incubated without or with dibutyryl cyclic AMP. The application of an Na+ gradient to vesicles from hepatocytes exposed to dibutyryl cyclic AMP, compared to control, resulted in a greater transient accumulation of L-alanine, whereas in the presence of a K+ gradient a similar slow equilibration of L-alanine was observed. Kinetic analysis of L-alanine influx revealed that the increased uptake resulted from an increased capacity (Vmax) with no change in the affinity (Km) of the carriers for L-alanine. These results strongly suggest that dibutyryl cyclic AMP induces stable changes at the membrane level probably by increasing the number of amino acid carrier molecules.

Free and membrane-bound ribosomes. II. Two-dimensional gel electrophoresis of proteins from free and membrane-bound rabbit reticulocyte ribosomes.

Free and membrane-bound polysomes were isolated from rabbit reticulocytes. The membrane-bound polysomes were liberated form the membrane with deoxycholate. Monosomes were prepared from the two types of polysomes by incubation with puromycin. The ribosomal proteins were extracted and analyzed by two-dimensional gel electrophoresis. Two proteins of the large subunit, L11 and L17 present in the free monosomes were not found in the membrane-bound monosomes. On the other hand, four additional spots were found in the protein pattern of the membrane-bound monosomes.

Free and membrane-bound ribosomes. VI. Distribution of free sulphydryl groups in rabbit reticulocyte ribosomes.

When free and membrane-bound ribosomes were titrated with N-ethyl[14C]-maleimide or 5,5'-dithio-bis(2-nitrobenzoic acid), 25 free SH groups were detected in polysomes, 10 in the 60 S subunit and 14 in the 40 S subunits. When individual ribosomal proteins were analysed, one large subunit protein L14 was found to be strongly labelled compared to the other ribosomal proteins, in polysomes, monosomes and subunits. However, two additional protein spots from the small subunit were more radioactive in monosomes and subunits than inpolysomes. In all these studies, it was found that the distribution of free SH groups in free and membrane-bound ribosomes was identical except for the few proteins which were reported before to be specific for free or membrane-bound ribosomes (Fehlmann, ., Bellemare, G. and Godin, C. (1975) Biochim. Biophys. Acta 378, 119-124 and Fehlmann, M., Bellemare, G. and Godin, C. (1975) FEBS Lett. 59, 8-12).

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes.

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.

Amino acid transport in isolated hepatocytes from streptozotocin-diabetic rats.

The enhanced ability of liver to extract glucogenic precursors from plasma in insulinopenic diabetes was investigated at the cellular level by measuring amino acid transport in isolated hepatocytes from streptozotocin-diabetes rats. The concentrative Na+-dependent uptake of alpha-amino[1-14C]isobutyric acid (AIB), a non-metabolizable analog of alanine, was increased in hepatocytes from diabetic rats compared with controls. This increase was observed both at a low (0.1 mM) and high (30 mM) extracellular concentration of the amino acid. Analysis of the relationship between AIB influx and AIB concentration revealed that the increased uptake in diabetic rat hepatocytes was due to an enhanced capacity (i.e., a twofold increase in the Vmax) of the transport system whereas the apparent affinity (Km) for AIB was unaltered. The increased amino acid transport activity in diabetic rat hepatocytes was due mainly to an increased transport through system A (alanine-preferring) and, to a much lesser extent, through system ASC (alanine, serine, cysteine). System L (leucine-preferring) and AIB entry through simple diffusion were not affected in diabetic rat hepatocytes compared with controls. Increased AIB transport of diabetic rat hepatocytes was returned to normal by insulin treatment.

Redistribution of 125I-insulin on the surface of rat hepatocytes as a function of dissociation time.

In the present experiments, we have correlated the distribution of 125I-insulin on the surface of rat hepatocytes with the dissociation of 125I-insulin from the cell. When 125I-insulin interacts with isolated rat hepatocytes at 15 degrees C, an increasing proportion of the bound ligand becomes nondissociable under the influence of acid pH (6.0), trypsin (0.5 mg/ml), or an excess of unlabeled insulin (10(-6) M). Under these conditions, only a small percentage of the labeled material is internalized as determined by quantitative electron microscope (EM) autoradiography. This progressive nondissociability of the ligand parallels its movement from microvilli to coated pits and its progressive concentration in these later surface specializations. These data suggest that receptors in different domains of the plasma membrane may have different dissociation rates for the ligand.

Phospholipid metabolism and T cell activation: receptor triggering is associated with the inhibition of phosphatidylserine synthesis.

Activation of Jurkat T lymphocytes to produce IL2 is accompanied by a strong inhibition of phosphatidylserine (PS) synthesis. This inhibition was obtained either with the mitogenic lectin PHA, anti-CD3 monoclonal antibodies (mAb), anti-CD2 mAb or anti-Ti mAb. Bypassing membrane receptor signalling, by using a Ca2+ ionophore or a protein phosphatase inhibitor, sodium ortho-vanadate, also results in a marked inhibition of PS synthesis. Activators of phospholipid -Ca2+ dependent protein kinase C (PKC) did not significantly modify PS synthesis, suggesting that the observed changes only involve the transduction of the first activation signal. PS being a necessary cofactor for PKC, our results strongly suggest that the inhibition of PS synthesis induced by receptor triggering exerts a feed back control on PKC therefore leading to a transient activation of the enzyme upon full lymphocyte activation.

Activation of human T cells is associated with tyrosine phosphorylation of several cellular proteins.

Human T lymphocytes are activated to proliferate after triggering the T Cell Antigen Receptor Complex. CD3-Ti, with either antigen, mitogenic lectins or monoclonal antibodies against its different subunits. Stimulation of Jurkat leukemic human T cells with anti-CD3 or anti-Ti monoclonal antibodies was found to induce, within 1 min, an increase in the phosphorylation of a set of cellular proteins that can be precipitated with anti-phosphotyrosine antibodies. Seven phosphotyrosine-containing proteins were separated with respective mol. wt of 21, 25, 38, 55, 70, 80 and 110 kDa, among which the 38 kDa species is predominant. Moreover, incubation of Jurkat T cells with sodium orthovanadate, a potent inhibitor of phosphotyrosine protein-phosphatases, was found to potentiate the effects of anti-CD3 mAb on tyrosine phosphorylation. In addition vanadate also induced IL-2 secretion in Jurkat cells when associated with the phorbol ester TPA, further demonstrating the importance of these phosphorylation reactions in the process of T cell activation. Our results therefore allow us to identify several protein substrates of a tyrosine kinase activity, whose stimulation appears to be an early event in human T cell activation through the antigen receptor pathway.

A chymotryptic-type protease inhibitor decreases interleukin 2 synthesis and induces prostaglandin production in Jurkat T cells.

TPCK (N-alpha-p-tosyl-L-phenylalanine chloromethylketone), a potent inhibitor of chymotryptic-type serine proteases, was found to decrease IL2 synthesis in Jurkat T cells. Conversely, the tryptic-type protease inhibitor, TLCK (N-alpha-p-tosyl-lysine chloromethylketone), which structurally is very similar to TPCK, had no effect on IL2 synthesis. Prostaglandin synthesis, a process that is known to reduce IL2 production in T cells, was increased by TPCK but not by TLCK, suggesting that this process could be, at least in part, responsible for the inhibition of IL2 production. Our results imply that a chymotryptic-type serine protease plays an active role in the regulation of IL2 synthesis and thus in the whole process of T-lymphocyte activation.

Pertussis toxin-sensitive G-proteins are not involved in activation of T-lymphocytes.

Multiple effects of pertussis toxin (PT) on Jurkat T-cells can be distinguished on the basis of their dose-response and their kinetics. High concentrations of PT deliver to cells an activating signal resulting in a rapid rise in [Ca2+]i followed by IL-2 synthesis. This activation is accompanied (within 2 h) by a down-regulation of the CD3/TCR complex from the cell surface. Cells then become refractory towards stimulation by CD3 mAb or PHA. All these effects, referred to as 'mitogenic effects', present the same dose-response curves with an EC50 of 0.5 micrograms/ml. Short term effects (PT-induced Ca2+ movements, down-regulation of CD3/TCR complex and inhibition of PHA and CD3-induced Ca2+ signal) are observed under conditions where no PT-induced ADP-ribosylation can be detected. In contrast, ADP-ribosylation of the 40,000 alpha-subunit of G-proteins requires a sustained (18 h) incubation of intact cells in the presence of low concentration (EC50 = 0.3 ng/ml) of PT. Dose-response curves for PT-dependent ADP-ribosylation and mitogenic effects are separated by three orders of magnitude. Covalent modification of G-protein has no effect on CD3-induced increase in [Ca2+]i and IL-2 synthesis induced by a combination of phorbol ester and either CD3 mAb, PHA or calcium ionophore. These data indicate that transduction of the mitogenic signal does not involve a PT-sensitive G-protein. Furthermore, inhibition of mitogenic signals following PT treatment results from a PT-induced activation leading to a down-regulation of the CD3/T cell receptor complex.

Inhibition of phosphatidylserine synthesis induced by triggering CD2 or the CD3-TCR complex in a human T cell line. Relationships with G proteins and receptors modulation.

Activation of Jurkat T cells with phytohemagglutinin, CD3 or CD2 mAbs results in a marked inhibition of phosphatidylserine (PS) synthesis. Monitoring PS synthesis in T cells shows that: (i) after modulation of CD3 molecules the cells become refractory to further treatment with CD3 mAbs as well as to a further challenge with CD2 mAbs; and (ii) treatment of T cells with fluoride ions and cholera toxin, two known effectors of guanosine triphosphate-binding proteins, also resulted in a strong inhibition of the synthesis of this phospholipid. The inhibition of PS synthesis thus appears to be regulated similarly to the other activation events, suggesting that transmembrane signalling mechanisms leading to PS inhibition are the same as those previously proposed for increasing phosphatidylinositides turnover and subsequent rise in the intracellular calcium concn in lymphocytes.

Isolation and characterization of a T lymphocyte mutant defective in the protein kinase C signal transduction pathway.

The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.

Binding of antigen to Ia molecules on intact antigen presenting cells demonstrated by photoaffinity labeling.

We used a photoaffinity labeling technique to investigate whether a molecular interaction occurs between antigen and Ia molecules on antigen presenting cells (APC) in the absence of T lymphocytes. M.12.4.1 B lymphoma cells (Iad), which are able to present bovine insulin to Iad lymph node primed T cells, were given radioiodinated bovine insulin derivatized with the photoreactive group (2-nitro-4-azidophenylacetyl) at Lys 29 of the B chain of the insulin molecule. Processing of insulin was allowed by incubating the APC with antigen for increasing periods of time at 37 degrees C or 4 degrees C. The covalent coupling of the processed photoreactive antigen to any neighboring cellular protein was thereafter induced by u.v. irradiation. Immunoprecipitation of membrane proteins by monoclonal antibodies showed that under these conditions, the alpha and beta subunits of the Ia molecules were selectively photolabeled. Labeling was time- and temp-dependent as was the internalization of insulin. The apparent mol. wts of the antigen-Ia molecule complexes were not significantly different from that of native Ia molecules radioiodinated by surface labeling, indicating that only a small fragment of the antigen was covalently coupled to Ia molecules. Similar experiments performed with human B lymphoma cells (526 cells) gave similar results. These observations therefore indicate: (1) that Ia molecules expressed by intact APC are able to bind antigens in the absence of T lymphocyte antigen receptor; and (2) that this association, at least for insulin, requires uptake and a proteolytic fragmentation of the antigen by the APC.

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H-2 major histocompatibility complex.

Insulin receptors from C57BL/6J mouse (H-2b) liver membranes were specifically labeled with 125I-photo-reactive insulin by UV irradiation. Membranes were solubilized and the capacity of various antibodies reacting with the major histocompatibility complex to immunoprecipitate insulin receptors was tested. About 5% of the labeled receptors were immunoprecipitated by a conventional mouse antiserum against H-2b histocompatibility antigens and by a monoclonal antibody against Class 1 antigens of the H-2b haplotype (Kb and Db). No immunoprecipitation was obtained with a monoclonal antibody against Class 2 antigens of I-Ab or against Class 1 antigens of the H-2k haplotype. Insulin receptors can thus be specifically immunoprecipitated by antibodies against class I histocompatibility antigens.

Progesterone action through aggregation of a receptor on the sperm plasma membrane.

Rapid steroid effects, reported in several cell types, have pointed out the possibility of non-genomic mechanisms of action, presumably on cell surface receptors. Here we analyzed the effects of antibody-mediated aggregation of a novel type of progesterone receptor on the plasma membrane of human sperm cells. We report that aggregation of hormone-receptor complexes induces Ca2+ influx and a Ca(2+)-dependent exocytotic event in this system. These data suggest a possible mechanism for rapid steroid-induced events. Further research is warranted to examined if a similar mechanism is involved in rapid steroid effects in other cell types.

Insulin receptors in rat brain: insulin stimulates phosphorylation of its receptor beta-subunit.

In rat brain cortex synaptosomes insulin stimulated the phosphorylation of its own receptor beta-subunit (94 kDa) as identified by immunoprecipitation with anti-insulin or anti-receptor antiserum. The receptor alpha-subunit (115 kDa) was characterized by specific labeling with 125I-labeled photoreactive insulin. These observations indicate that: (i) insulin receptors in brain are composed of alpha-subunits which bind insulin, and beta-subunits, the phosphorylation of which can be stimulated by insulin; (ii) the size of alpha-subunits in brain is significantly smaller than in other tissues (115 vs 130 kDa), whereas beta-subunits (94 kDa) are identical. We suggest that brain insulin receptors represent a subtype regarding their binding function, whereas their enzyme function is more conserved.

Insulin receptors in the mammalian central nervous system: binding characteristics and subunit structure.

Insulin receptors in rat and human central nervous system have been identified by binding of 125I-insulin on purified synaptic plasma membranes; affinity labelling of receptors by chemical cross-linking 125I-insulin; or phosphorylation of receptors with [gamma-32P]ATP. Brain insulin receptors showed significant differences in their binding characteristics and subunit structure when compared with receptors in other tissues like adipose and liver cells: absence of negatively cooperative interactions; a distinct binding specificity i.e. porcine proinsulin, coypu insulin and insulin-like growth factor I and II showed 2-5 times higher binding affinity in brain than in other cell types; a smaller molecular size of the brain receptor alpha-subunit than in other tissues (Mr approximately 115,000 instead of 130,000). In contrast, the size (Mr approximately 94,000) and function of the insulin receptor beta-subunit kinase was identical with that described in other cells. We conclude, that insulin receptors in mammalian brain represent a receptor subtype which may mediate growth rather than metabolic activity of insulin.

Glucose transport by horse kidney brush borders. I.--Transport properties of brush border membrane closed vesicles.

Brush border membranes isolated from horse kidney cortex as closed right-side out vesicles show selective permeability when analyzed on sucrose and dextran gradients. These vesicles can actively accumulate D-glucose. The preservation of the glucose transport system is demonstrated by the following features: (a) the uptake and release rates of D-glucose are higher in the presence of a sodium gradient, showing that D-glucose transport is a sodium-dependent process; (b) this transport, specific for the D-isomer, is inhibited by phlorizin; (c) the D-glucose transport system is saturable; (d) no inhibition of D-glucose transport is found with C-mannose; (e) the D-glucose uptake is sensitive to osmotic variations.