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Insulin receptor substrate (IRS) proteins are important intracellular molecules that mediate insulin receptor tyrosine kinase signaling. A decreased content of IRS proteins has been found in insulin-resistant states in animals, humans, and cultured cells under various conditions. However, the molecular mechanism that controls cellular levels of IRS proteins is unknown. We report that chronic insulin treatment induces the degradation of IRS-1, but not IRS-2, protein in cultured cells. The insulin-induced degradation of IRS-1 can be prevented by pretreatment with lactacystin, a specific inhibitor for proteasome degradation. These data demonstrate, for the first time, that insulin-induced degradation of IRS-1 is mediated by the proteasome degradation pathway. IRS-2 can escape from the insulin-induced proteasome degradation, suggesting the existence of specific structural requirements for this degradation process.

Insulin receptor substrate (IRS) proteins play a crucial role as signaling molecules in insulin action. Serine phosphorylation of IRS proteins has been hypothesized as a cause of attenuating insulin signaling. The current study investigated serine kinase activity toward IRS-1 in several models of insulin resistance. An in vitro kinase assay was developed that used partially purified cell lysates as a kinase and glutathione S-transferase fusion proteins that contained various of IRS-1 fragments as substrates. Elevated serine kinase activity was detected in Chinese hamster ovary/insulin receptor (IR)/IRS-1 cells and 3T3-L1 adipocytes chronically treated with insulin, and in liver and muscle of obese JCR:LA-cp rats. It phosphorylated the 526-859 amino acid region of IRS-1, whereas phosphorylation of the 2-516 and 900-1235 amino acid regions was not altered. Phosphopeptide mapping of the 526-859 region of IRS-1 showed three major phosphopeptides (P1, P2, and P3) with different patterns of phosphorylation depending on the source of serine kinase activity. P1 and P2 were strongly phosphorylated when the kinase activity was prepared from insulin-resistant Chinese hamster ovary/IR/IRS-1 cells, weakly phosphorylated by the kinase activity from insulin-resistant 3T3-L1 adipocytes, and barely phosphorylated when the extract was derived from insulin-resistant liver. In contrast, P3 was phosphorylated by the serine kinase activity prepared from all insulin-resistant cells and tissues of animals. P1 and P2 phosphorylation can be explained by mitogen-activated protein kinase activity based on the phosphopeptide map generated by recombinant ERK2. In contrast, mitogen-activated protein kinase failed to phosphorylate the P3 peptide, suggesting that another serine kinase regulates this modification of IRS-1 in insulin-resistant state.

On-line databases of anatomical information are being compiled for a number of genetically manipulable organisms, including the fruit fly, Drosophila melanogaster. Based on the success of the molecular databases that preceded them, they face formidable problems in data cataloguing, storage, and retrieval. The prospect for such databases, which is apparent already, is to alter permanently the approach to neuroanatomy in such species. Experience with Drosophila indicates the possibility to arbitrate controversies over and, in some cases, to redefine the borders drawn in the brain by conventional neuroanatomical methods. Two publications in this issue of Journal of Comparative Neurology by Nassif et al. and by Hartenstein et al. highlight one of the first demonstrations of a further opportunity in Drosophila. In some cases, it is already possible to suggest how individual cellular elements--neurons, tracts, and neuropil regions--might be traced from the time when they first express a precocious marker, such as the product of the fasciclin-II gene, through the metamorphic pupal stage, and into the adult. In this way, it becomes possible to identify the structures of the adult brain from the time of their first emergence in the embryo and to follow their transitional positions throughout the course of development. Critical in this process is the neuroanatomical organization of the larval brain, which contains not only the fully functional central nervous system of the larva but also the developing elements of the adult brain, because this holds the key to understanding both the cellular elements that are inherited from the embryo and the elements that are in the process of forming the adult nervous system.

Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin-peroxidase conjugate, is both faster and less labor intensive than photo-oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti-serotonin as an example.

In order to study the different mechanisms of dynorphin spinal analgesia and neurotoxicity at low and high doses, the effects of various concentrations of dynorphin A-(1-17) on the free intracellular Ca2+ concentration ([Ca2+]i) in the cultured rat spinal neurons were studied using single cell microspectrofluorimetry. While dynorphin A-(1-17) 0.1-100 microM had no significant effect on basal [Ca2+]i, dynorphin A-(1-17) 0.1 and 1 microM significantly decreased the high KCl-evoked peak [Ca2+]i by 94% and 83% respectively. Dynorphin A-(1-17) 10 and 100 microM did not affect the peak [Ca2+]i following K+ depolarization, but in all these neurons there was a sustained and irreversible rise in [Ca2+]i following high-K+ challenge. Pretreatment with the specific kappa-opioid receptor antagonist nor-binaltorphimine 10 microM, but not the competitive NMDA receptor antagonist, DL-2-amino-5-phosphonovalerate (APV) 10 microM, significantly blocked the inhibitory effect of dynorphin A-(1-17) 0.1 microM on peak [Ca2+]i. However, APV 10 microM and nor-binaltorphimine 10 microM significantly antagonized the sustained rise in [Ca2+]i induced by a high concentration of dynorphin A-(1-17) 10 microM. Furthermore, in the presence, and following the addition, of increasing concentrations of dynorphin A-(1-17) (0.1, 1, 10 and 100 microM), the high concentrations of dynorphin A-(1-17) failed to produce a sustained rise in peak [Ca2+]i. These results suggested that dynorphin exerted a dualistic modulatory effect on [Ca2+]i in cultured rat spinal neurons, inducing a sustained and irreversible intracellular Ca2+ overload via activation of both NMDA and kappa-opioid receptors at higher concentrations, but inhibiting depolarization-evoked Ca2+ influx via kappa-opioid but not NMDA receptors at lower concentrations. Serial addition of graded concentrations of dynorphin A-(1-17) prevented the effect of high concentrations of dynorphin A-(1-17) on [Ca2+]i.

The 60 kDa insulin receptor substrate in rat adipocytes that binds to the PI-3 kinase displays several functional characteristics in common with the IRS proteins; so we propose the name pp60(IRS3) to distinguish it from other tyrosine phosphorylated proteins of similar size. During insulin stimulation, p85 associated with pp60(IRS3) more rapidly than with IRS-1 or IRS-2. In mice lacking IRS-1, p85 associated more strongly with pp60(IRS3) than with IRS-2, suggesting that pp60(IRS3) provides an alternate pathway in these cells. Synthetic peptides containing two phosphorylated YMPM motifs displace pp60(IRS3) and IRS-1 from alphap85 immune complexes, suggesting that pp60(IRS3), like IRS-1, engages both SH2 domains in p85. Moreover, pp60(IRS3) binds to immobilized peptides containing a phosphorylated NPXY motif, suggesting that it contains a PTB domain with similar specificity to that in IRS-1. The cloning of pp60(IRS3) will reveal a new member of the IRS protein family which mediates insulin receptor signals in a narrow range of tissues.

The detailed branching pattern and synaptic organization of the uniglomerular projection neurons of the antennal lobe, the first processing center of the olfactory pathway, of the moth Manduca sexta were studied with laser scanning confocal microscopy and a technique combining laser scanning confocal microscopy and electron microscopy. Uniglomerular projection neurons, identified electrophysiologically or morphologically, were stained intracellularly with neurobiotin or biocytin. Brains containing the injected neurons were treated with streptavidin-immunogold to label the injected material for electron microscopy and with Cy3-streptavidin to label the neurons with fluorescence for laser scanning confocal microscopy, and then embedded in Epon. Labeled neurons were imaged and reconstructed with laser scanning confocal microscopy (based on the retained fluorescence of the labeled neuron in the Epon block), and thin sections were cut at selected optical levels for correlation of light microscopic data and electron microscopic detail. Each neuron had a cell body in one of the three cell-body clusters of the antennal lobe, a primary neurite that extended across the coarse neuropil at the center of the antennal lobe and then formed a dense tuft of processes within a single glomerulus, and an axon that emanated from the primary neurite and projected from the antennal lobe via the antenno-cerebral tract to the lateral horn of the ipsilateral protocerebrum and, collaterally, to the calyces of the mushroom body. In the electron microscope, the fine dendritic branches in the apical zones of the glomeruli, where sensory axons terminate, were found to receive many input synapses. In deeper layers across the glomeruli, the processes participated in both input and output synapses, and the bases of the glomeruli, the most proximal, thickest branches formed output synapses. In both of the protocerebral areas in which axonal branches terminated, those branches formed exclusively output synapses. Our findings indicate that, in addition to conveying olfactory information to the protocerebrum, uniglomerular projection neurons in the antennal lobes of M. sexta participate in local intraglomerular synaptic circuitry.

The antennae of the sphinx moth Manduca sexta are multimodal sense organs, each comprising three segments: scape, pedicel, and flagellum. Each antenna is moved by two systems of muscles, one controlling the movement of the scape and consisting of five muscles situated in the head capsule (extrinsic muscles), and the other system located within the scape (intrinsic muscles) and consisting of four muscles that move the pedicel. At least seven motoneurons innervate the extrinsic muscles, and at least five motoneurons innervate the intrinsic muscles. The dendritic fields of the antennal motoneurons overlap one another extensively and are located in the neuropil of the antennal mechanosensory and motor center. The density of motoneuronal arborizations is greatest in the lateral part of this neuropil region and decreases more medially. None of the motoneurons exhibits a contralateral projection. The cell bodies of motoneurons innervating the extrinsic muscles are distributed throughout an arching band of neuronal somata dorsal and dorsolateral to the neuropil of the antennal mechanosensory and motor center, whereas the cell bodies of motoneurons innervating the intrinsic muscles reside mainly among the neuronal somata situated dorsolateral to that neuropil.

Signal transduction by insulin and IGF-1, several interleukins (IL-2, IL-4, IL-9, IL-13), interferons, GH, and other cytokines involves IRS proteins, which link the receptors for these factors to signaling molecules with Src homology-2 domains (SH2-proteins). We recently reported the amino acid sequence of murine IRS-2; in order to examine a potential genetic role for this molecule in disease, we isolated the murine IRS-2 gene and compared the expression pattern of IRS-2 against IRS-1. Like IRS-1, IRS-2 is encoded by a single exon. Whereas IRS-1 is located on murine chromosome 1, IRS-2 is located on murine chromosome 8 near the insulin receptor. IRS-2 is expressed together with IRS-1 in many cells and tissues; however, IRS-2 predominates in murine hematopoietic cells where it may be essential for cytokine signaling; IRS-1 predominates in adipocytes and differentiated 3T3-L1 cells where it contributes to the normal insulin response. In 32D cells, IRS-1 and IRS-2 undergo differential tyrosine phosphorylation during insulin or IL-4 stimulation, as assessed indirectly by interaction with various recombinant SH2 domains. Thus, signaling specificity through the IRS proteins may be accomplished by specific expression patterns and distinct phosphorylation patterns during interaction with various activated receptors.

During metamorphic development, the arrival at the olfactory (antennal) lobe of olfactory receptor axons initiates the process of glomerulus formation. The glomeruli are discrete spheroidal regions of neuropil that are the sites of synaptic interactions among receptor neurons and their target antennal-lobe neurons. The process of glomerulus formation begins as groups of receptor axons form protoglomeruli. These dense clusters of terminal branches mostly are discrete entities from the time they can be recognized, although a few branches from neighboring protoglomeruli overlap laterally. A previous study by Schweitzer et al. [Schweitzer E. S., Sanes J. R. and Hildebrand J. G. (1976) Ontogeny of electroantennogram responses in the moth, Manduca sexta. J. Insect Physiol. 22, 955-960] has shown that odor-induced activity in the receptor neurons can be detected first in recordings from the axons in the antennal nerve only in the last few days of metamorphic development and thus could not influence the process of glomerulus formation. In this study, we have tested directly the possibility that an earlier presence of spontaneous activity in either the receptor axons or the antennal-lobe neurons could affect the process. Tetrodotoxin, a Na(+)-channel blocker, was injected into the hemolymph prior to the onset of glomerulus formation to block any spontaneous Na(+)-dependent activity. Subsequent intracellular recordings from antennal-lobe neurons revealed no spike activity. Comparison with vehicle-injected control animals at stages during and after glomerulus formation revealed no differences in the localization of receptor-axon terminal branches in the glomeruli, in the border of glial cells that forms around each glomerulus, or in the morphology of the tufted glomerular arbors of one of the antennal-lobe neurons. We conclude that: (1) the process of glomerulus formation is largely independent of activity; and (2) glomeruli as modular units of the CNS more closely resemble cortical barrels than cortical columns, both in their ontogeny and in the lack of an obvious effect of activity on the morphology of the neurons arborizing within them.

Angiotensin II (AII), acting via its G-protein linked receptor, is an important regulator of cardiac, vascular, and renal function. Following injection of AII into rats, we find that there is also a rapid tyrosine phosphorylation of the major insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) in the heart. This phenomenon appears to involve JAK2 tyrosine kinase, which associates with the AT1 receptor and IRS-1/IRS-2 after AII stimulation. AII-induced phosphorylation leads to binding of phosphatidylinositol 3-kinase (PI 3-kinase) to IRS-1 and IRS-2; however, in contrast to other ligands, AII injection results in an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity. The latter occurs without any reduction in insulin receptor or IRS phosphorylation or in the interaction of the p85 and p110 subunits of PI 3-kinase with each other or with IRS-1/IRS-2. These effects of AII are inhibited by AT1 receptor antagonists. Thus, there is direct cross-talk between insulin and AII signaling pathways at the level of both tyrosine phosphorylation and PI 3-kinase activation. These interactions may play an important role in the association of insulin resistance, hypertension, and cardiovascular disease.

Experimentally determining the synaptic interconnections between neurons in the nervous system is laborious and difficult in any animal species, but especially so in many invertebrates, including insects, where neurons generally have large, finely branching neuritic trees that form both pre- and postsynaptic specializations in dense neuropils with other neuritic trees. Electron microscopy is needed to identify synapses, but correlation of synapse type and location with the overall branching patterns of neurons, which are visible readily only in the light microscope or through extensive reconstruction of serial electron-microscope sections, is very difficult. In this paper, we present a simple method that we have developed (Sun et al. (1995) J. Histochem. Cytochem., 43: 329-335) that combines laser scanning confocal microscopy and electron microscopy for the study of synaptic relationships of neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Briefly, neurons are labeled by intracellular injection with neurobiotin or biocytin, and then processed with a gold-particle tag for electron microscopic study and a fluorescent tag for confocal microscopy, and embedded in plastic. The fluorescence of the labeled neuron in the plastic blocks is imaged in three dimensions with laser scanning confocal microscopy and then the neuron is thin-sectioned at precisely chosen depths for electron microscopic study. The fluorescence pattern can be monitored repeatedly between episodes of thin-sectioning, and subtraction of a fluorescence image from the previous fluorescence image reveals which fluorescent processes have been sectioned. In this way, electron microscopic detail can be mapped onto a three-dimensional light microscopic image of the neuron.

Tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) by the activated receptors for insulin, IGF-1, and various cytokines creates binding sites for signaling proteins with Src homology 2 domains (SH2 proteins). Determining the role of specific SH2 proteins during insulin signaling has been difficult because IRS-1 possesses as many as 18 potential tyrosine phosphorylation sites, several of which contain redundant motifs. Using 32D cells, which contain no endogenous IRS proteins, we compared the signaling ability of an IRS-1 molecule in which 18 potential tyrosine phosphorylation sites were replaced by phenylalanine (IRS-1(F18)) with two derivative molecules which retained three YMXM motifs (IRS-1(3YMXM)) or the two COOH-terminal SHP2-Fyn binding sites (IRS-1(YCT)). During insulin stimulation, IRS-1(F18) failed to undergo tyrosine phosphorylation or mediate activation of the phosphotidylinositol (PI) 3'-kinase or p70(s6k); IRS-1(YCT) was tyrosine phosphorylated but also failed to mediate these signaling events. Neither IRS-1(3YMXM) nor IRS-1(YCT) mediated activation of mitogen-activated protein kinases. IRS-1(F18) and IRS-1(YCT) partially mediated similar levels of insulin-stimulated mitogenesis at high insulin concentrations, however, suggesting that IRS-1 contains phosphotyrosine-independent elements which effect mitogenic signals, and that the sites in IRS-l(YCT) do not augment this signal. IRS-1(3YMXM) mediated the maximal mitogenic response to insulin, although the response to insulin was more sensitive with wild-type IRS-1. By contrast, the association of IRS-1(3YMXM) with PI 3'-kinase was more sensitive to insulin than the association with IRS-1. Thus, the binding of SH2 proteins (such as PI 3'-kinase) by YMXM motifs in IRS-1 is an important element in the mitogenic response, but other elements are essential for full mitogenic sensitivity.

Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.

Binding of interferon alpha (IFN alpha) to its receptor induces activation of the Tyk-2 and Jak-1 tyrosine kinases and tyrosine phosphorylation of multiple downstream signaling elements, including the Stat components of the interferon-stimulated gene factor 3 (ISGF-3). IFN alpha also induces tyrosine phosphorylation of IRS-1, the principle substrate of the insulin receptor. In this study we demonstrate that various Type I IFNs rapidly stimulate tyrosine phosphorylation of IRS-2. This is significant since IRS-2 is the major IRS protein found in hematopoietic cells. The IFN alpha-induced phosphorylated form of IRS-2 associates with the p85 regulatory subunit of the phosphatidylinositol 3'-kinase, suggesting that this kinase participates in an IFN alpha-signaling cascade downstream of IRS-2. We also provide evidence for an interaction of IRS-2 with Tyk-2, suggesting that Tyk-2 is the kinase that phosphorylates this protein during IFN alpha stimulation. A conserved region in the pleckstrin homology domain of IRS-2 may be required for the interaction of IRS-2 with Tyk-2, as shown by the selective binding of glutathione S-transferase (GST) fusion proteins containing the IRS-2-IH1PH or IRS-1-IH1PH domains to Tyk-2 but not other Janus kinases in vitro.

The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

Interleukin-13 (IL-13) induced a potent mitogenic response in IL-3-dependent TF-1 cells and DNA synthesis to a lesser extent in MO7E and FDC-P1 cells. IL-13 stimulation of these lines, like IL-4 and insulin-like growth factor-1 (IGF-1), resulted in tyrosine phosphorylation of a 170-kD substrate. The tyrosine-phosphorylated 170-kD substrate strongly associated with the 85-kD subunit of phosphoinositol-3 (PI-3) kinase and with Grb-2. Anti-4PS serum readily detected the 170-kD substrate in lysates from both TF-1 and FDC-P1 cells stimulated with IL-13 or IL-4. These data provide evidence that IL-13 induces tyrosine phosphorylation of the 4PS substrate, providing an essential interface between the IL-13 receptor and signaling molecules containing SH2 domains. IL-13 and IL-4 stimulation of murine L cell fibroblasts, which endogenously express the IL-4 receptor (IL-4R alpha) and lack expression of the IL-2 receptor gamma subunit (IL-2R gamma), resulted in tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1)/4PS. Enhanced tyrosine phosphorylation of IRS-1/4PS was observed in response to IL-4, but not IL-13 treatment of L cells transfected with the IL-2R gamma chain. These results indicate that IL-13 does not use the IL-2R gamma subunit in its receptor complex and that expression of IL-2R gamma enhances, but is not absolutely required for mediating IL-4-induced tyrosine phosphorylation of IRS-1/4PS.

Insulin receptor substrate-1 (IRS-1) is the major cytoplasmic substrate of the insulin and insulin-like growth factor (IGF)-1 receptors. Transgenic mice lacking IRS-1 are resistant to insulin and IGF-1, but exhibit significant residual insulin action which corresponds to the presence of an alternative high molecular weight substrate in liver and muscle. Recently, Sun et al. (Sun, X.-J., Wang, L.-M., Zhang, Y., Yenush, L. P., Myers, M. G., Jr., Glasheen, E., Lane, W.S., Pierce, J. H., and White, M. F. (1995) Nature 377, 173-177) purified and cloned 4PS, the major substrate of the IL-4 receptor-associated tyrosine kinase in myeloid cells, which has significant structural similarity to IRS-1. To determine if 4PS is the alternative substrate of the insulin receptor in IRS-1-deficient mice, we performed immunoprecipitation, immunoblotting, and phosphatidylinositol (PI) 3-kinase assays using specific antibodies to 4PS. Following insulin stimulation, 4PS is rapidly phosphorylated in liver and muscle, binds to the p85 subunit of PI 3-kinase, and activates the enzyme. Insulin stimulation also results in the association of 4PS with Grb 2 in both liver and muscle. In IRS-1-deficient mice, both the phosphorylation of 4PS and associated PI 3-kinase activity are enhanced, without an increase in protein expression. Immunodepletion of 4PS from liver and muscle homogenates removes most of the phosphotyrosine-associated PI 3-kinase activity in IRS-1-deficient mice. Thus, 4PS is the primary alternative substrate, i.e. IRS-2, which plays a major role in physiologic insulin signal transduction via both PI 3-kinase activation and Grb 2/Sos association. In IRS-1-deficient mice, 4PS/IRS-2 provides signal transduction to these two major pathways of insulin signaling.

The protein IRS-1 acts as an interface between signalling proteins with Src-homology-2 domains (SH2 proteins) and the receptors for insulin, IGF-1, growth hormone, several interleukins (IL-4, IL-9, IL-13) and other cytokines. It regulates gene expression and stimulates mitogenesis, and appears to mediate insulin/IGF-1-stimulated glucose transport. Thus, survival of the IRS-1-/- mouse with only mild resistance to insulin was surprising. This dilemma is provisionally resolved with our discovery of a second IRS-signalling protein. We purified and cloned a likely candidate called 4PS from myeloid progenitor cells and, because of its resemblance to IRS-1, we designate it IRS-2. Alignment of the sequences of IRS-2 and IRS-1 revealed a highly conserved amino terminus containing a pleckstrin-homology domain and a phosphotyrosine-binding domain, and a poorly conserved carboxy terminus containing several tyrosine phosphorylation motifs. IRS-2 is expressed in many cells, including tissues from IRS-1-/- mice, and may be essential for signalling by several receptor systems.

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55PIK, a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identify to those in p85. p55PIK mRNA is expressed early during development, remains abundant in adult mouse brain and testis tissue, and is detectable in adult adipocytes and heart and kidney tissues. p55PIK forms a stable complex with p110, and it associates with IRS-1 during insulin stimulation. Moreover, the activated insulin receptor phosphorylates p55PIK in Sf9 cells, and insulin stimulates p55PIK phosphorylation in CHOIR/p55PIK cells. The unique features of p55PIK suggest that it is important in receptor signaling.

Interferon-alpha (IFN alpha) induces rapid tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), a docking protein with multiple tyrosine phosphorylation sites that bind to the Src homology 2 (SH2) domains of various signaling proteins. During IFN alpha stimulation, the p85 regulatory subunit of the phosphatidylinositol 3'-kinase binds via its SH2 domains to tyrosine-phosphorylated IRS-1, and phosphatidylinositol 3'-kinase activity is detected in association with IRS-1. Thus, IFN alpha responses occur by activation of the IRS signaling system, which it shares with insulin, insulin-like growth factor-1, and interleukin-4.

The NH2 terminus of insulin receptor substrate-1 (IRS-1) contains a pleckstrin homology (PH) domain. We deleted the PH domain in IRS-1 (IRS-1 delta PH) and expressed the mutant in Chinese hamster ovary and 32D cells. During insulin stimulation, IRS-1 delta PH is poorly tyrosine-phosphorylated in CHO cells, but undergoes serine/threonine phosphorylation. Similarly, IRS-1 delta PH fails to undergo insulin-stimulated tyrosine phosphorylation in 32D cells, which uncouples the activation of phosphatidylinositol 3'-kinase and p70s6k from the endogenous insulin receptors. Overexpression of the insulin receptor in 32DIR cells, however, restores tyrosine phosphorylation of IRS-1 delta PH and rescues insulin responses including mitogenesis. Thus, while the PH domain is not required for the engagement of downstream signals, it is one of the elements in the NH2 terminus of IRS-1 that is needed for a sensitive coupling to insulin receptors, especially at ordinary receptor levels found in most cells and tissues.

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.

Serum-free culture of mouse neuroblastoma cells was used as the experimental model for the study of neuronal aging, with flow cytometry of cell cycle, DNA and total cellular protein as the indices of neuronal aging. After addition of dynorphin A (1-8) 10(-7) mol.L-1 into the culture medium, the following general tendencies were obtained: (1) The number of S and G2 + M phase cells was increased and the number of G1 phase cells decreased. (2) The total cellular protein in aged experimental neural cells decreased. The results implies that Dyn A (1-8) Produced effects on cell cycle, DNA and total cellular protein in the direction of delaying neuronal aging. The relation between Dyn A (1-8) and neuronal aging merits further investigation.

Insulin Receptor Substrate-1 (IRS-1) is an endogenous cellular protein that is tyrosine phosphorylated during stimulation of cells with insulin, IGF-1, and interleukin 4 (IL-4). Phosphorylated IRS-1 regulates multiple regulatory pathways by recruiting signaling molecules containing Src homology 2 domains (SH2 proteins). The 32D myeloid progenitor cell line contains few insulin receptors and no detectable IRS-1. Expression of the insulin receptor alone partially mediates insulin-stimulated microtubule-associated protein (MAP) kinase activation, and the addition of IRS-1 enhances this effect (Myers, M. G., Jr., Wang, L.-M., Sun, X. J., Zhang, Y., Yenush, L. P., Schlessinger, J., Pierce, J. H., and White, M. F. (1994) Mol. Cell. Biol. 14, 3577-3587). Alone, insulin receptors mediate phosphatidylinositol (PI) 3'-kinase and p70S6k activation poorly if at all during insulin stimulation. Expression of IRS-1 alone in 32D cells mediates the stimulation of p70S6k by insulin, IGF-1, or IL-4; addition of insulin receptor to these cells increases the sensitivity of the insulin response. In contrast, full insulin stimulation of PI 3'-kinase requires both the insulin receptor and IRS-1, suggesting that a high level of IRS-1 phosphorylation is required for insulin-stimulated PI 3'-kinase activation, whereas a low level of IRS-1 tyrosine phosphorylation transmits an essential signal to p70S6k. Both insulin receptors and IRS-1 are required for mitogenic signaling in 32D cells suggesting that MAP kinase or p70S6k alone are not sufficient, and that both or additional unknown IRS-1-mediated signals are necessary.

Insulin-receptor substrate 1 (IRS-1) is a principal substrate of the receptor tyrosine kinase for insulin and insulin-like growth factor 1, and a substrate for a tyrosine kinase activated by interleukin 4. IRS-1 undergoes multisite tyrosine phosphorylation and mediates downstream signals by 'docking' various proteins that contain Src homology 2 domains. IRS-1 appears to be a unique molecule; however, 4PS, a protein found mainly in hemopoietic cells, may represent another member of this family.

GRB-2 is a small SH2- and SH3 domain-containing adapter protein that associates with the mammalian SOS homolog to regulate p21ras during growth factor signaling. During insulin stimulation, GRB-2 binds to the phosphorylated Y895VNI motif of IRS-1. Substitution of Tyr-895 with phenylalanine (IRS-1F-895) prevented the IRS-1-GRB-2 association in vivo and in vitro. The myeloid progenitor cell line, 32-D, is insensitive to insulin because it contains few insulin receptors and no IRS-1. Coexpression of IRS-1 or IRS-1F-895 with the insulin receptor was required for insulin-stimulated mitogenesis in 32-D cells, while expression of the insulin receptor alone was sufficient to mediate insulin-stimulated tyrosine phosphorylation of Shc and activation of p21ras and mitogen-activated protein (MAP) kinase. The Shc-GRB-2 complex formed during insulin stimulation is a possible mediator of p21ras and MAP kinase activation in IRS-1-deficient 32-D cells. Interestingly, IRS-1, but not IRS-1F-895, enhanced the stimulation of MAP kinase by insulin in 32-D cells expressing insulin receptors. Thus, IRS-1 contributes to the stimulation of MAP kinase by insulin, probably through formation of the IRS-1-GRB-2 complex at Tyr-895. Our results suggest that the Shc-GRB-2 complex and the activation of p21ras-dependent signaling pathways, including MAP kinase, are insufficient for insulin-stimulated mitogenesis and that the essential function(s) of IRS-1 in proliferative signaling is largely unrelated to IRS-1-GRB-2 complex formation.

The cellular DNA contents of 139 bladder irrigations from 52 cases of bladder tumors were determined by flow cytometry. The results were compared with those of pathological grading and exfoliative cytology. It is suggested that cytological results were less accurate than DNA contents. High aneuploid DNA contents in tumor cells were marked by low grade and deep penetration of the bladder tumor. Total cystectomy was needed for bladder tumor with high aneuploid DNA, and partial cystectomy and TURBT for that tumor showed high recurrence with instillation of BCG. The results showed that DNA content of tumor is correlated with the nuclear grade, especially in poorly differentiated neoplasms, but flow cytometry can not replace cystoscopy examination.

It is reported that intrathecal (it) injection of low dose of dynorphin (Dyn) induces no analgesia while high dose of Dyn induces analgesia and might lead to hindlimb paralysis and loss of reflex via NMDA receptor. We hypothesized that NMDA receptor antagonists may reveal kappa analgesic-potential of subliminal dose of Dyn. Twenty-four hours after intrathecal cannulation, tail flick latency was measured before and after it of drugs. Combination of Dyn A- (1-13) 5 nmol or U50488H 100 nmol, a kappa receptor agonist with either DL-2-amino-5-phosphonovaleric acid (5 and 10 nmol) or kynurenic acid (25 and 50 nmol) it induced synergistic analgesia, which was reversed by nor-binaltorphimine 15 nmol, a kappa receptor antagonist. It is concluded that kappa opioid receptor agonists and NMDA receptor antagonists synergistically induce analgesia via interaction with their receptors.

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.

The two antennal lobes, the primary olfactory centers of the brain, of the moth Manduca sexta each contain one neuron that displays serotonin immunoreactivity. The neuron projects out of the antennal lobe and sends branches into ipsi- and contralateral protocerebral areas. An axon-like process extends from the contralateral protocerebrum to, and terminates in, the contralateral antennal lobe. In order to begin to investigate the possible role of this unique neuron in olfactory information processing, we have used laser scanning confocal microscopic and electron microscopic immunocytochemical techniques to study the ramification pattern, ultrastructural characteristics, and synaptic connections of the neuron in the antennal lobes of female adult Manduca sexta. The neuron ramifies extensively in the antennal lobe contralateral to the cell body. The ramifications, mainly in the base and center of each glomerulus, do not overlap with those of the sensory axons from the antenna. This finding suggests that the serotonin-immunoreactive neuron may not receive direct input from sensory neurons, and that it may modulate the activity of the neurons of the antennal lobe rather than that of the sensory neurons. In the electron microscope, the neuron exhibits large dense-cored vesicles and small, clear round vesicles. In the antennal lobe ipsilateral to the cell body, the primary neurite of the serotonin-immunoreactive neuron is unbranched and lacks detectable synaptic connections. The ramifications in the contralateral antennal lobe, however, participate in synaptic connections. At very low frequency, contralateral branches form synapses onto unlabeled processes and also receive synapses from unidentified neurons in the glomeruli, indicating that the neuron may participate directly in synaptic processing of olfactory information. The high ratio of output to input synapses made by the serotonin-immunoreactive processes in the contralateral antennal lobe is consistent with the idea that this neuron may receive synaptic input via its bilateral branches in the protocerebrum and then send information to the contralateral antennal lobe where the neuron may exert feedback or modulatory influences on olfactory information processing in the glomeruli.

Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-stimulated autophosphorylation of the cytoplasmic juxtamembrane region of the human insulin receptor was examined by Tricine/SDS-PAGE. Various mutant receptor molecules were used to identify two tryptic phosphopeptides associated with the juxtamembrane region which accounts for 15% of the autophosphorylation of partially purified insulin receptor. These phosphopeptides were immunoprecipitated with an antipeptide antibody against the juxtamembrane sequence and were phosphorylated exclusively on tyrosine. Substitution of both Tyr960 and Tyr953 with alanine eliminated insulin-stimulated phosphorylation of the juxtamembrane region without affecting tyrosine autophosphorylation in the C terminus or regulatory regions. Monosubstitution of Tyr960 with phenylalanine or alanine reduced phosphorylation in the juxtamembrane region by more than 50%, and manual Edman degradation indicated that Tyr960 was phosphorylated in wild-type receptor. In vivo, phosphorylation of the juxtamembrane region accounts for one-third of the insulin receptor phosphorylation and contains both phosphoserine and phosphotyrosine. Deletion of Tyr960 and 11 adjacent amino acids eliminated insulin-stimulated phosphorylation of the juxtamembrane region. Substitution of Tyr960 reduced this phosphorylation by more than 50%. The insulin receptor also undergoes serine phosphorylation outside of the juxtamembrane region which depends on the presence of Tyr1151. Together with our previous studies, this report suggests that phosphorylation of Tyr960 may play an important role in signal transduction by the insulin receptor.

Interleukin 4 (IL-4), insulin, and insulin-like growth factor I (IGF-I) efficiently induced DNA synthesis in the IL-3-dependent murine myeloid cell lines FDC-P1 and FDC-P2. Although these factors could not individually sustain long-term growth of these lines, a combination of IL-4 with either insulin or IGF-I did support continuous growth. The principal tyrosine-phosphorylated substrate observed in FDC cells stimulated with IL-4, previously designated 4PS, was of the same size (170 kDa) as the major substrate phosphorylated in response to insulin or IGF-I. These substrates had phosphopeptides of the same size when analyzed by digestion with Staphylococcus aureus V8 protease, and each tightly associated with the 85-kDa component of phosphatidylinositol 3-kinase after factor stimulation. IRS-1, the principal substrate phosphorylated in response to insulin or IGF-I stimulation in nonhematopoietic cells, is similar in size to 4PS. However, anti-IRS-1 antibodies failed to efficiently precipitate 4PS, and some phosphopeptides generated by V8 protease digestion of IRS-1 were distinct in size from the phosphopeptides of 4PS. Nevertheless, IL-4, insulin, and IGF-I were capable of stimulating tyrosine phosphorylation of IRS-1 in FDC cells that expressed this substrate as a result of transfection. These findings indicate that (i) IL-4, insulin, and IGF-I use signal transduction pathways in FDC lines that have at least one major feature in common, the rapid tyrosine phosphorylation of 4PS, and (ii) insulin and IGF-I stimulation of hematopoietic cell lines leads to the phosphorylation of a substrate that may be related to but is not identical to IRS-1.

Insulin stimulates the formation of binary and ternary signaling complexes between the phosphatidylinositol (PtdIns) 3'-kinase, IRS-1, and the insulin receptor in vivo. Binary complex formation between IRS-1 and the PtdIns 3'-kinase occurs in intact cells and requires the tyrosyl phosphorylation IRS-1, as mutant insulin receptors which weakly phosphorylate IRS-1 in vivo do not mediate formation of IRS-1/PtdIns 3'-kinase complexes in transfected CHO cells. Association with IRS-1 involves as much as 70% of total cellular PtdIns 3'-kinase activity. Insulin also stimulates the formation of ternary signaling complexes, as both IRS-1 and the PtdIns 3'-kinase are present in anti-insulin receptor immunoprecipitates from insulin-stimulated cells. Overexpression of IRS-1 in CHO cells increases the amount of PtdIns 3'-kinase activity in alpha IR immunoprecipitates, and IRS-1 markedly increases the in vitro binding of p85 alpha and PtdIns 3-kinase activity to anti-receptor immunoprecipitates. The mechanism for this association is unknown, but appears to involve the binding of IRS-1/PtdIns 3'-kinase complexes to the insulin receptor. The formation of binary and ternary complexes between the insulin receptor, IRS-1 and the PtdIns 3'-kinase may play a critical role in transmission of the insulin signal.

IRS-1 is a unique cytosolic protein that becomes tyrosine phosphorylated during insulin stimulation of intact cells and immediately associates with the phosphatidylinositol 3'-kinase (PtdIns 3'-kinase). The insulin-like growth factor-I (IGF-I) receptor also mediated the tyrosine phosphorylation of IRS-1 and increased the amount of PtdIns 3'-kinase activity bound to IRS-1 in Chinese hamster ovary cells. Purified insulin receptor and IGF-I receptor phosphorylated recombinant baculovirus-produced IRS-1 on similar sites in vitro, and phosphorylated baculovirus-produced IRS-1 bound PtdIns 3'-kinase activity from lysates of quiescent cells. Treatment of cells with IGF-I activated the PtdIns 3'-kinase, suggesting that IGF-I activates the PtdIns 3'-kinase through IRS-1 binding to p85 in a manner similar to insulin. Chinese hamster ovary cells overexpressing IRS-1 demonstrated increased tyrosine phosphorylation of IRS-1, and more PtdIns 3'-kinase activity associated with IRS-1 in these cells. These data demonstrate that IRS-1 is a common element for signal transmission by the IGF-I and insulin receptors.

IRS-1 is a major insulin receptor substrate which may play an important role in insulin signal transmission. The mRNA for IRS-1 in rat cells and tissues is about 9.5 kilobases (kb). Rat liver IRS-1 was stably expressed in Chinese hamster ovary (CHO) cells (CHO/IRS-1). Although its calculated molecular mass is 131 kDa, IRS-1 from quiescent cells migrated between 165 and 170 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IRS-1 was phosphorylated strongly on serine residues and weakly on threonine residues before insulin stimulation. Insulin immediately stimulated tyrosine phosphorylation of IRS-1, and after 10-30 min with insulin its apparent molecular mass increased to 175-180 kDa. Expression of the human insulin receptor and rat IRS-1 together in CHO/IR/IRS-1 cells increased the basal serine phosphorylation of IRS-1 and strongly increased tyrosine phosphorylation during insulin stimulation. Purified insulin receptors directly phosphorylated baculovirus-produced IRS-1 exclusively on tyrosine residues. By immunofluorescence, IRS-1 was absent from the nucleus, but otherwise distributed uniformly before and after insulin stimulation. Some IRS-1 associated with the insulin receptor during insulin stimulation. In addition, a phosphatidylinositol 3'-kinase associated with IRS-1 during insulin stimulation, and this association was more sensitive to insulin in CHO cells overexpressing the insulin receptor (CHO/IR cells), more responsive to insulin to CHO/IRS-1 cells, and both sensitive and responsive in CHO/IR/IRS-1 cells. Similarly, insulin-stimulated DNA synthesis was more sensitive to insulin in CHO/IR cells, and more responsive in CHO/IRS-1 cells; however, insulin-stimulated DNA synthesis was sensitive but poorly responsive to insulin in CHO/IR/IRS-1 cells. Together, these results suggest that IRS-1 is a direct physiologic substrate of the insulin receptor and may play an important role in insulin signal transmission.

IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.

Insulin rapidly stimulates tyrosine phosphorylation of cellular proteins which migrate between 165 and 190 kDa during SDS-PAGE. These proteins, collectively called pp185, were originally found in anti-phosphotyrosine antibody (alpha PY) immunoprecipitates from insulin-stimulated Fao rat hepatoma cells. Recently, we purified and cloned IRS-1, one of the phosphoproteins that binds to alpha PY and migrates near 180 kDa following insulin stimulation of rat liver [Sun, X. J., et al. (1991) Nature 352, 73-77]. IRS-1 and pp185 undergo tyrosine phosphorylation immediately after insulin stimulation and show an insulin dose response similar to that of insulin receptor autophosphorylation. However, IRS-1 was consistently 10 kDa smaller than the apparent molecular mass of pp185. The pp185 contained some immunoblottable IRS-1; however, cell lysates depleted of IRS-1 with anti-IRS-1 antibody still contained the high molecular weight forms of pp185 (HMW-pp185). Furthermore, the tryptic phosphopeptide map of IRS-1 was distinct from that of HMW-pp185, suggesting that at least two substrates migrate in this region during SDS-PAGE. Moreover, the phosphatidylinositol 3'-kinase and its 85-kDa associated protein (p85) bound to IRS-1 in Fao cells, but weakly or not at all to HMW-pp185. Our results show that Fao cells contain at least two insulin receptor substrates, IRS-1 and HMW-pp185, which may play unique roles in insulin signal transmission.

IRS-1 undergoes rapid tyrosine phosphorylation during insulin stimulation and forms a stable complex containing the 85 kDa subunit (p85) of the phosphatidylinositol (PtdIns) 3'-kinase, but p85 is not tyrosyl phosphorylated. IRS-1 contains nine tyrosine phosphorylation sites in YXXM (Tyr-Xxx-Xxx-Met) motifs. Formation of the IRS-1-PtdIns 3'-kinase complex in vitro is inhibited by synthetic peptides containing phosphorylated YXXM motifs, suggesting that the binding of PtdIns 3'-kinase to IRS-1 is mediated through the SH2 (src homology-2) domains of p85. Furthermore, overexpression of IRS-1 potentiates the activation of PtdIns 3-kinase in insulin-stimulated cells, and tyrosyl phosphorylated IRS-1 or peptides containing phosphorylated YXXM motifs activate PtdIns 3'-kinase in vitro. We conclude that the binding of tyrosyl phosphorylated IRS-1 to the SH2 domains of p85 is the critical step that activates PtdIns 3'-kinase during insulin stimulation.

Angiotensin II (AII), injected intracerebroventricularly, has been shown to antagonize opioid analgesia. The mechanism for this was obscure. In the neuroblastoma X glioma NG 108-15 hybrid cell line, the K(+)-induced increase in [Ca2+]i can be suppressed by the delta opioid agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) at 0.01-1 microM, an effect completely reversed by the opioid antagonist naloxone. Angiotensin II (AII) at concentrations of 0.1 and 1 microM mobilized free Ca2+ from an intracellular pool, and this effect was antagonized by the AII receptor antagonist saralasin. All (1 microM) had no significant effect on the increase in [Ca2+]i induced by K+, but it blocked the suppressive effect of DPDPE on the K(+)-induced [Ca2+]i increase. The results indicate that mobilization of intracellular calcium may underlie the anti-opioid effect of AII.

Since the discovery of insulin nearly 70 years ago, there has been no problem more fundamental to diabetes research than understanding how insulin works at the cellular level. Insulin binds to the alpha subunit of the insulin receptor which activates the tyrosine kinase in the beta subunit, but the molecular events linking the receptor kinase to insulin-sensitive enzymes and transport processes are unknown. Our discovery that insulin stimulates tyrosine phosphorylation of a protein of relative molecular mass between 165,000 and 185,000, collectively called pp185, showed that the insulin receptor kinase has specific cellular substrates. The pp185 is a minor cytoplasmic phosphoprotein found in most cells and tissues; its phosphorylation is decreased in cells expressing mutant receptors defective in signalling. We have now cloned IRS-1, which encodes a component of the pp185 band. IRS-1 contains over ten potential tyrosine phosphorylation sites, six of which are in Tyr-Met-X-Met motifs. During insulin stimulation, the IRS-1 protein undergoes tyrosine phosphorylation and binds phosphatidylinositol 3-kinase, suggesting that IRS-1 acts as a multisite 'docking' protein to bind signal-transducing molecules containing Src-homology 2 and Src-homology-3 domains. Thus IRS-1 may link the insulin receptor kinase and enzymes regulating cellular growth and metabolism.

Arginyl residues of human antithrombin III have been implicated to involve in the heparin binding site [Jorgensen, A. M., Borders, C. L., & Fish, W. W. (1985) Biochem, J. 231, 59-63]. We have performed chemical modification of antithrombin with (p-hydroxyphenyl)glyoxal (HPG) in order to determine the locations of these arginine residues. Antithrombin was modified with 12 mM HPG in the absence and presence of heparin (2-fold by weight to antithrombin). In the absence of heparin, about 3-4 mol of arginines/mol of antithrombin were modified within 60 min, and the modification led to the loss of 95% of the inhibitor's heparin cofactor activity as well as heparin-induced fluorescence enhancement and 50% of its progressive inhibitory activity. In the presence of heparin, the extent of modification was diminished by 30% and modified antithrombin retained approximately 70% of its heparin cofactor activity. Peptide mapping and subsequent sequence analysis revealed that selective HPG modification occurred at Arg129 and Arg145 and that their modifications were protected upon binding of heparin to antithrombin. We conclude that Arg129 and Arg145 are situated within the heparin binding site of human antithrombin III.

Abnormal venous leaks are a causative factor of impotence and can be identified by perfusion cavernosography. The perfusion cavernosography was carried out in 47 impotent patients and the venous leakage was found in 21. Among 21 cases found the venous leakage, 20 cases underwent surgical treatment and the results showed that the patient's sexual life (erection) was not satisfactory after simple ligation of the deep dorsal vein and that ligation of the abnormal venous outlets at the level of the crura on penis was an effective way to restore potency. The authors consider that intra-cavernous papaverine injection induced penile erection is an effective means of screening for venous leakage.

Human antithrombin III (AT-III) contains three disulphide linkages (Cys-8-Cys-128, Cys-21-Cys-95 and Cys-247-Cys-430), and two of them (Cys-8-Cys-128 and Cys-21-Cys-95) are situated near the heparin-binding domain of the inhibitor. We demonstrate in this paper that: (i) partially reduced AT-III (with Cys-8-Cys-128 and Cys-21-Cys-95 quantitatively reduced) could be re-oxidized in air to regain 70-80% of its heparin cofactor activity and thrombin-inhibitory activity; (ii) completely reduced AT-III was re-oxidized under similar conditions and recovered 30-35% of it biological activities. Structural analysis of refolded AT-IIIs indicates that restorations of their disulphide contents and conformations (evaluated by chemical modification) are congruent with recoveries of their biological activities.