Growth factor-induced c-fos expression defines distinct subsets of midbrain dopaminergic neurons

Growth factors are considered pivotal for the development, maintenance, and function of mesencephalic dopaminergic neurons. Recent studies have identified a plethora of growth factors which support the survival and differentiation of embryonic dopaminergic neurons. However, the exact cellular targets of these growth factors, and, thus, their precise mechanisms of action, remain largely unknown. To identify these cellular targets, we analysed, at the single cell level, growth factor-induced c-fos expression in dissociated mesencephalic cell cultures derived from a fos-lac Z transgenic mouse line. Pharmacological interference with cell-cell communication was utilized to control for direct growth factor effects. beta-Galactosidase-expressing cells were phenotypically characterized by immunocytochemistry to specific neural cell markers. Glia cell line-derived neurotrophic factor, basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin-3 directly induced Fos expression in differently sized, yet overlapping, populations of tyrosine hydroxylase-immunoreactive dopaminergic neurons. In an additional subpopulation of dopaminergic neurons, neurotrophin-3 induced fos-lac Z expression indirectly through a glutamate-mediated activation of N-methyl-D-aspartate receptors. Consistent with their proposed glial-mediated mode of action, transforming growth factor alpha and platelet-derived growth factor induced Fos expression predominantly in glia but only in a very small number of dopaminergic neurons. These findings demonstrate that individual dopaminergic neurons represent the direct targets of different sets of extracellular growth factors. Our findings further establish that growth factors affect dopaminergic neurons by indirect mechanisms which require specific cell-cell communication. These data also suggest a potential role for growth factors in the establishment of the morphological and functional diversity of midbrain dopaminergic neurons.     

Olfactory G proteins: simple and complex signal transduction

In both vertebrates and invertebrates, olfactory perception is mediated by G-protein-coupled receptors. Recent work, in both mouse and Caenorhabditis elegans, sheds light on the role of specific G proteins in olfactory signal transduction, neuronal morphology and axon guidance.     

Role of focal adhesion proteins in signal transduction and oncogenesis

A monoamine pathophysiological hypothesis for paraphilias in males is based on the following data: (i) the monoamines norepinephrine, dopamine, and serotonin are involved in the appetitive dimension of male sexual behavior in laboratory animals; (ii) data gathered from studying the side effect profiles of antidepressant psychostimulant, and neuroleptic drugs in humans suggest that alteration of central monoamine neurotransmission can have substantial effects on human sexual functioning, including sexual appetite; (iii) monoamine neurotransmitters appear to modulate dimensions of human and animal psychopathology including impulsivity, anxiety, depression, compulsivity, and pro/antisocial behavior, dimensions disturbed in many paraphiliacs; (iv) pharmacological agents that ameliorate psychiatric disorders characterized by the aforementioned characteristics, especially central serotonin enhancing drugs, can ameliorate paraphilic sexual arousal and behavior.     

Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction

SOCS-1 (suppressor of cytokine signaling-1) is a representative of a family of negative regulators of cytokine signaling (SOCS-1 to SOCS-7 and CIS) characterized by a highly conserved C-terminal SOCS box preceded by an SH2 domain. This study comprehensively examined the ability of several SOCS family members to negatively regulate the gp130 signaling pathway. SOCS-1 and SOCS-3 inhibited both interleukin-6 (IL-6)- and leukemia inhibitory factor (LIF)-induced macrophage differentiation of murine monocytic leukemic M1 cells and LIF induction of a Stat3-responsive reporter construct in 293T fibroblasts. Deletion of amino acids 51-78 in the N-terminal region of SOCS-1 prevented inhibition of LIF signaling. The SOCS-1 and SOCS-3 N-terminal regions were functionally interchangeable, but this did not extend to other SOCS family members. Mutation of SH2 domains abrogated the ability of both SOCS-1 and SOCS-3 to inhibit LIF signal transduction. Unlike SOCS-1, SOCS-3 was unable to inhibit JAK kinase activity in vitro, suggesting that SOCS-1 and SOCS-3 act on the JAK-STAT pathway in different ways. Thus, although inhibition of signaling by SOCS-1 and SOCS-3 requires both the SH2 and N-terminal domains, their mechanisms of action appear to be biochemically different.     

Growth and invasion of differentiated thyroid gland carcinoma: importance of signal transduction

To define the long-term dental sequelae of therapy for childhood rhabdomyosarcoma of the head and neck, and to identify factors in their development, we retrospectively reviewed the serial panoramic radiographs and clinical records of 22 survivors of head or neck rhabdomyosarcoma who had been followed for at least 5 years. Patients were divided into four groups based upon age at the time of therapy and three groups based upon radiation doses. All patients had received similar multiagent chemotherapy. Dental sequelae of oncotherapy occurred in over half of the long-term survivors of head and neck rhabdomyosarcoma. The abnormalities comprised root stunting in 54%, microdontia in 23%, and hypodontia in 50% of patients; 36% had multiple abnormalities. Microdontia and multiple abnormalities were more prevalent in patients treated at the earliest age, and abnormalities tended to be more prevalent with increasing doses of radiation. Five patients (23%) developed severe cosmetic and/or functional sequelae necessitating surgical and/or orthodontic intervention. The high frequency of dental sequelae we observed suggests that meticulous long-term dental and radiographic follow-up are needed. Early detection and treatment of the complications of therapy will expedite their correction and minimize morbidity.     

Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins

Microglial interaction with amyloid fibrils in the brains of Alzheimer's and prion disease patients results in the inflammatory activation of these cells. We observed that primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar beta-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The tyrosine kinases Lyn and Syk are activated by the fibrillar peptides and initiate a signaling cascade resulting in a transient release of intracellular calcium that results in the activation of classical PKC and the recently described calcium-sensitive tyrosine kinase PYK2. Activation of the MAP kinases ERK1 and ERK2 follows as a subsequent downstream signaling event. We demonstrate that PYK2 is positioned downstream of Lyn, Syk, and PKC. PKC is a necessary intermediate required for ERK activation. Importantly, the signaling response elicited by beta-amyloid and prion fibrils leads to the production of neurotoxic products. We have demonstrated in a tissue culture model that conditioned media from beta-amyloid- and prion-stimulated microglia or from THP-1 monocytes are neurotoxic to mouse cortical neurons. This toxicity can be ameliorated by treating THP-1 cells with specific enzyme inhibitors that target various components of the signal transduction pathway linked to the inflammatory responses.     

The SOCS proteins: a new family of negative regulators of signal transduction

An asymptomatic tumor in the pelvis was incidentally found by ultrasonography in a 67-year-old woman while being examined after presenting with a common cold. Further examinations revealed a presacral cystic tumor, which measured 10 x 12cm in size. The cyst was thus removed in the normal manner for such cases. The pathological diagnosis was an epidermoid cyst. An analysis of 15 other cases previously reported in the literature indicated that large epidermoid cysts should normally be excised through an abdominal approach alone, provided that the tumor is benign.     

Apolipoprotein E inhibits platelet-derived growth factor-induced vascular smooth muscle cell migration and proliferation by suppressing signal transduction and preventing cell entry to G1 phase

The anti-atherogenic effects of apolipoprotein (apo) E have been attributed to its ability to reduce plasma cholesterol level and to limit foam cell formation. The purpose of this study was to ascertain if apoE also may have cytostatic functions that could attenuate vascular occlusive diseases. Purified apoE inhibited smooth muscle cell migration directed to platelet-derived growth factor (PDGF) or oxidized LDL (oxLDL) (p < 0.0001). The purified apoE also suppressed PDGF- and oxLDL-induced smooth muscle cell proliferation (p < 0.001). These apoE inhibitory effects were not because of suppression of PDGF binding to its receptors on the smooth muscle cells, but was correlated with a significant reduction in agonist-stimulated mitogen-activated protein kinase activity (p < 0.01). ApoE also inhibited PDGF-induced cyclin D1 mRNA expression, suggesting that the apoE effect was mediated by growth arrest at the G0 to G1 phase. Taken together, these results suggest that apoE has cytostatic functions in the vessel wall and may protect against vascular diseases through inhibition of cell signaling events associated with growth factor-induced smooth muscle cell migration and proliferation.     

Interplay of the proto-oncogene proteins CrkL and CrkII in insulin-like growth factor-I receptor-mediated signal transduction

The closely related proto-oncogene proteins CrkII and CrkL consist of one SH2 and two SH3 domains and share 60% overall homology with the highest identity within their functional domains. In this study we show that CrkL and CrkII may play overlapping but different roles in insulin-like growth factor (IGF)-I receptor-mediated signal transduction. While both proteins are substrates involved in IGF-I receptor signaling, they apparently demonstrate important different properties and different biological responses. Evidence supporting this hypothesis includes (a) the oncogenic potential of CrkL versus the absence of this potential in CrkII overexpressing cell lines, (b) the inhibition of IGF-I-dependent cell cycle progression by overexpression of CrkII, and (c) the differential regulation of the phosphorylation status of selective proteins in CrkII and CrkL overexpressing cell lines. In addition we demonstrate the specific association of CrkL and CrkII with the newly characterized IRS-4 protein, again in a differential manner. Whereas CrkL strongly interacts with IRS-4 via its SH2 and N-terminal SH3 domains, CrkII interacts only via its SH2 domain, possibly explaining the unstable nature of IRS-4-CrkII association. The results obtained allow us to propose a unique mechanism of CrkL and CrkII tyrosine phosphorylation in response to IGF-I stimulation. Thus these highly homologous proteins apparently possess structural features that allow for the differential association of each protein with different effector molecules, thereby activating different signaling pathways and resulting in unique biological roles of these proteins.     

Translational regulation of mRNAs with distinct IRE sequences by iron regulatory proteins 1 and 2

Iron regulatory proteins 1 and 2 (IRP-1, IRP-2) interact with iron-responsive elements (IREs) present in the 5'- or 3'-untranslated regions (UTR) of several mRNAs coding for proteins in iron metabolism. Whereas binding of IRP-1 and -2 to an IRE in the 5'-UTR inhibits mRNA translation in vitro, it has remained unknown whether either endogenous protein is sufficient to control translation in mammalian cells. We analyzed this question by taking advantage of published mutant IREs that are exclusively recognized by either IRP-1 or IRP-2 in vitro. These IREs were inserted into the 5'-UTR of a human growth hormone reporter mRNA, and translational regulation was measured in stably transfected mouse L cells. Cells cultured in iron-rich or -depleted medium were labeled with [35S]methionine, and secreted growth hormone was immunoprecipitated. IREs with loop sequence specific for IRP-1 (UAGUAC), IRP-2 (CCGAGC), or both proteins (GAGUCG and the wild-type CAGUGC sequence) all mediated translational regulation, in contrast to a control sequence (GCUCCG) that binds neither IRP-1 nor IRP-2. Control experiments excluded IRP-1 binding to the IRP-2-specific sequence in vivo. The present data demonstrate that IRP-1 and IRP-2 can independently function as translational repressors in living cells.     

Activation of the mas oncogene involves coupling to human alphoid sequences

We have shown previously that mouse NIH3T3 cells transfected with DNA from a human ovarian carcinoma were rendered tumourigenic by an activated mas oncogene in four independent transfection experiments. In all cases the 5'-noncoding region was rearranged in comparison to the original ovarian tumour DNA. We now report that in all four transfectants the newly acquired sequences consist of human centromeric alpha satellite repeat DNA. In at least three transfectants the alphoid DNA originates from the centromere of chromosome three. Analysis of the sequences of the recombination site in one transfectant revealed that a homologous sequence of five base pairs (CAGCA) is present in both parental strands, and might thus have contributed to the recombinational event. To establish a conclusive role for alphoid DNA in the activation of mas, we performed a co-transfection experiment in NIH3T3 cells with cloned alphoid DNA and the mas coding sequence. We show that the transfectants expressing a transformed phenotype contain amplified mas linked to alphoid DNA. NIH3T3 cells transfected with plasmids that contained alphoid sequences cloned directly upstream of the mas coding sequence, and injected into nude mice, gave rise to tumours with amplified mas sequences (7/7). In six of these tumours the alphoid sequences were amplified as well. Our data suggest a novel mechanism of oncogene activation: recombination with normal alphoid repeat DNA resulting in amplification of the oncogene.     

Role of CR4 in Mycobacterium tuberculosis-human macrophages binding and signal transduction in the absence of serum

The beta2 integrin CR4 is involved in Mycobacterium tuberculosis phagocytosis by human mononuclear phagocytes through the opsonin C3bi. In this study, we demonstrate that M. tuberculosis can bind directly to monocyte-derived macrophages via CR4 in the absence of any opsonins. CR4-transfected CHO cells gave similar results, suggesting recognition by CR4 of bacterial structure. Furthermore, binding of M. tuberculosis transduced a potent signal, resulting in tyrosine phosphorylation of macrophage proteins, which was in part mediated by CR4.     

The disaggregation theory of signal transduction revisited: further evidence that G proteins are multimeric and disaggregate to monomers when activated

We have compared the sedimentation rates on sucrose gradients of the heterotrimeric GTP-binding regulatory (G) proteins Gs, G(o), Gi, and Gq extracted from rat brain synaptoneurosomes with Lubrol and digitonin. The individual alpha and beta subunits were monitored with specific antisera. In all cases, both subunits cosedimented, indicating that the subunits are likely complexed as heterotrimers. When extracted with Lubrol all of the G proteins sedimented with rates of about 4.5 S (consistent with heterotrimers) whereas digitonin extracted 60% of the G proteins with peaks at 11 S; 40% pelleted as larger structures. Digitonin-extracted Gi was cross-linked by p-phenylenedimaleimide, yielding structures too large to enter polyacrylamide gels. No cross-linking of Lubrol-extracted Gi occurred. Treatment of the membranes with guanosine 5'-[gamma-thio]triphosphate and Mg2+ yielded digitonin-extracted structures with peak sedimentation values of 8.5 S--i.e., comparable to that of purified G(o) in digitonin and considerably larger than the Lubrol-extracted 2S structures representing the separated alpha and beta gamma subunits formed by the actions of guanosine 5'-[gamma-thio]triphosphate. It is concluded that the multimeric structures of G proteins in brain membranes are at least partially preserved in digitonin and that activation of these structures in membranes yields monomers of G proteins rather than the disaggregated products (alpha and beta gamma complexes) observed in Lubrol. It is proposed that hormones and GTP affect the dynamic interplay between multimeric G proteins and receptors in a fashion analogous to the actions of ATP on the dynamic interactions between myosin and actin filaments. Signal transduction is mediated by activated monomers released from the multimers during the activation process.     

Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.     

Activation loop tyrosines contribute varying roles to TrkB autophosphorylation and signal transduction

The TrkB receptor tyrosine kinase (RTK) is a high affinity receptor for the neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Following exposure to BDNF or NT-4/5, TrkB is autophosphorylated on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Based on crystallographic analyses for others RTKs, TrkB tyrosines Y670, Y674, and Y675 are expected to lie within a putative kinase activation loop. Phosphorylation of these activation loop tyrosines is postulated to be a conserved event required for complete RTK activation. Here, we have assessed the importance these activation loop tyrosines play in regulating TrkB autophosphorylation, cytoplasmic signal transduction, and cell proliferation. We show that while tyrosine 670 is dispensable for BDNF-inducible TrkB autophosphorylation and the activation of certain signal transduction events, it is required for complete TrkB-mediated cellular proliferation. Combinatorial mutagenesis of tyrosines 674 and 675 only moderately affects TrkB autophosphorylation, but significantly impairs the BDNF-inducible stimulation of cytoplasmic signaling events and cellular proliferation. The combined mutation of all three activation loop tyrosines results in an inactive receptor, which is unable to autophosphorylate, stimulate signaling events, or induce mitogenesis. The data highlight the varying degrees of importance of the three activation loop tyrosines in TrkB mediated biological responses.     

Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast

Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs). In animal cells, TMD length is a major factor in Golgi retention. In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals. We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p. We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct. Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known. Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane. The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence. A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole. TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points.     

Divergent signal transduction responses to infection with attaching and effacing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an attaching and effacing pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. Although this organism causes adhesion pedestals, the cellular signals responsible for the formation of these lesions have not been clearly defined. We have shown previously that STEC O157:H7 does not induce detectable tyrosine phosphorylation of host cell proteins upon binding to eukaryotic cells and is not internalized into nonphagocytic epithelial cells. In the present study, tyrosine-phosphorylated proteins were detected under adherent STEC O157:H7 when coincubated with the non-intimately adhering, intimin-deficient, enteropathogenic E. coli (EPEC) strain CVD206. The ability to be internalized into epithelial cells was also conferred on STEC O157:H7 when coincubated with CVD206 ([158 +/- 21] % of control). Neither the ability to rearrange phosphotyrosine proteins nor that to be internalized into epithelial cells was evident following coincubation with another STEC O157:H7 strain or with the nonsignaling espB mutant of EPEC. E. coli JM101(pMH34/pSSS1C), which overproduces surface-localized O157 intimin, also rearranged tyrosine-phosphorylated and cytoskeletal proteins when coincubated with CVD206. In contrast, JM101 (pMH34/pSSS1C) demonstrated rearrangement of cytoskeletal proteins, but not tyrosine-phosphorylated proteins, when coincubated with intimin-deficient STEC (strains CL8KO1 and CL15). These findings indicate that STEC O157:H7 forms adhesion pedestals by mechanisms that are distinct from those in attaching and effacing EPEC. Taken together, these findings point to diverging signal transduction responses to infection with attaching and effacing bacterial enteropathogens.     

Conserved domain structure of beta-neurexins. Unusual cleaved signal sequences in receptor-like neuronal cell-surface proteins

Neurexins, a family of neuronal cell-surface proteins, consist of the longer alpha-neurexins (I alpha, II alpha, and III alpha) and the shorter beta-neurexins (I beta and II beta) with identical C termini but distinct N termini. alpha-Neurexins have the structure of cell surface receptors, but the membrane topology and conservation of beta-neurexins is unknown. We have now characterized cDNA clones encoding bovine neurexins I beta and III beta, thereby demonstrating the presence of a beta-form for neurexin III and the evolutionary conservation of beta-neurexins in mammals. Similar to alpha-neurexins, beta-neurexins were found to be highly O-glycosylated after expression by transfection in COS cells, suggesting that alpha- and beta-neurexins utilize the same O-glycosylation cassette and have similar transmembrane orientations. To determine if beta-neurexins contain a cleaved or uncleaved signal sequence for membrane translocation, beta-neurexin-IgG fusion proteins were expressed in COS cells, and their N termini were directly sequenced. This revealed that the N terminus of all three beta-neurexins contains an unusual cleaved signal sequence. Together our data show that all known neurexin genes generate alpha and beta forms with similar transmembrane organizations and receptor-like structures. Due to the presence of a long atypical cleaved signal peptide, beta-neurexins contain only a short unique sequence before splicing into the alpha-neurexin sequence. Thus, beta-neurexins are essentially N terminally truncated alpha-neurexins.     

Insulin receptors and signal transduction proteins in the hypothalamo-hypophyseal system: a review on morphological findings and functional implications

Receptors for insulin are widely distributed in the brain and pituitary. The current hypothesis on receptor function in these regions points to a role of insulin as a mediator in the communication of the peripheral endocrine system with the brain via various steps of the neuroendocrine axis. Recent data demonstrate that receptor-positive neurons in the brain, i.e. in the hypothalamus, and secretory cells in the anterior pituitary gland possess specific proteins that are thought to be involved in key steps of post receptor signal transduction, in particular insulin receptor substrate-1 and phosphatidylinositol 3'-kinase (PI3k). PI3k is a critical enzyme of the intracellular signaling pathway that is activated by a number of receptor tyrosine kinases, including receptors for insulin and IGF-1. This information further completes the framework indicating in vivo activity of insulin receptors in central neuroendocrine cells and their involvement in one branch of several physiological mechanisms that control body metabolism and nutritional behaviour.