Receptor guanylyl cyclase C (GC-C): regulation and signal transduction

Receptor guanylyl cyclase C (GC-C) is the target for the gastrointestinal hormones, guanylin, and uroguanylin as well as the bacterial heat-stable enterotoxins. The major site of expression of GC-C is in the gastrointestinal tract, although this receptor and its ligands play a role in ion secretion in other tissues as well. GC-C shares the domain organization seen in other members of the family of receptor guanylyl cyclases, though subtle differences highlight some of the unique features of GC-C. Gene knock outs in mice for GC-C or its ligands do not lead to embryonic lethality, but modulate responses of these mice to stable toxin peptides, dietary intake of salts, and development and differentiation of intestinal cells. It is clear that there is much to learn in future about the role of this evolutionarily conserved receptor, and its properties in intestinal and extra-intestinal tissues.

Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.

Toward blueprints for network architecture, biophysical dynamics, and signal transduction

We review mathematical aspects of biophysical dynamics, signal transduction and network architecture that have been used to uncover functionally significant relations between the dynamics of single neurons and the networks they compose. We focus on examples that combine insights from these three areas to expand our understanding of systems neuroscience. These range from single neuron coding to models of decision making and electrosensory discrimination by networks and populations, as well as coincidence detection in pairs of dendrites and the dynamics of large networks of excitable dendritic spines. We conclude by describing some of the challenges that lie ahead as the applied mathematics community seeks to provide the tools that will ultimately underpin systems neuroscience.

Staphylococcal enterotoxins modulate interleukin 2 receptor expression and ligand-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription (Stat proteins).

Staphylococcal enterotoxins (SE) stimulate T cells expressing the appropriate variable region beta chain of (V beta) T-cell receptors and have been implicated in the pathogenesis of several autoimmune diseases. Depending on costimulatory signals, SE induce either proliferation or anergy in T cells. In addition, SE can induce an interleukin-2 (IL-2) nonresponsive state and apoptosis. Here, we show that SE induce dynamic changes in the expression of and signal transduction through the IL-2 receptor (IL-2R) beta and gamma chains (IL-2R beta and IL-2R gamma) in human antigen-specific CD4+ T-cell lines. Thus, after 4 hr of exposure to SEA and SEB, the expression of IL-2R beta was down-regulated, IL-2R gamma was slightly up-regulated, while IL-2R alpha remained largely unaffected. The changes in the composition of IL-2Rs were accompanied by inhibition of IL-2-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription called Stat3 and Stat5. In parallel experiments, IL-2-driven proliferation was inhibited significantly. After 16 hr of exposure to SE, the expression of IL-2R beta remained low, while that of IL2R alpha and IL2R gamma was further up-regulated, and ligand-induced tyrosine phosphorylation of Jak3 and Stat proteins was partly normalized. Yet, IL-2-driven proliferation remained profoundly inhibited, suggesting that signaling events other than Jak3/Stat activation had also been changed following SE stimulation. In conclusion, our data suggest that SE can modulate IL-2R expression and signal transduction involving the Jak/Stat pathway in CD4+ T-cell lines.

Alterations in energy metabolism, neuroprotection and visual signal transduction in the retina of parkinsonian, MPTP-treated monkeys

Parkinson disease is mainly characterized by the degeneration of dopaminergic neurons in the central nervous system, including the retina. Different interrelated molecular mechanisms underlying Parkinson disease-associated neuronal death have been put forward in the brain, including oxidative stress and mitochondrial dysfunction. Systemic injection of the proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to monkeys elicits the appearance of a parkinsonian syndrome, including morphological and functional impairments in the retina. However, the intracellular events leading to derangement of dopaminergic and other retinal neurons in MPTP-treated animal models have not been so far investigated. Here we have used a comparative proteomics approach to identify proteins differentially expressed in the retina of MPTP-treated monkeys. Proteins were solubilized from the neural retinas of control and MPTP-treated animals, labelled separately with two different cyanine fluorophores and run pairwise on 2D DIGE gels. Out of >700 protein spots resolved and quantified, 36 were found to exhibit statistically significant differences in their expression levels, of at least ±1.4-fold, in the parkinsonian monkey retina compared with controls. Most of these spots were excised from preparative 2D gels, trypsinized and subjected to MALDI-TOF MS and LC-MS/MS analyses. Data obtained were used for protein sequence database interrogation, and 15 different proteins were successfully identified, of which 13 were underexpressed and 2 overexpressed. These proteins were involved in key cellular functional pathways such as glycolysis and mitochondrial electron transport, neuronal protection against stress and survival, and phototransduction processes. These functional categories underscore that alterations in energy metabolism, neuroprotective mechanisms and signal transduction are involved in MPTPinduced neuronal degeneration in the retina, in similarity to mechanisms thought to underlie neuronal death in the Parkinson’s diseased brain and neurodegenerative diseases of the retina proper.

Stress at the synapse : signal transduction mechanisms of adrenal steroids at neuronal membranes

As the key neuron-to-neuron interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. However, the signal transduction mechanisms by which stress mediates its lasting effects on synapse transmission and on memory are not fully understood. A key component of the stress response is the increased secretion of adrenal steroids. Adrenal steroids (e.g., cortisol) bind to genomic mineralocorticoid and glucocorticoid receptors (gMRs and gGRs) in the cytosol. In addition, they may act through membrane receptors (mMRs and mGRs), and signal transduction through these receptors may allow for rapid modulation of synaptic transmission as well as modulation of membrane ion currents. mMRs increase synaptic and neuronal excitability; mechanisms include the facilitation of glutamate release through extracellular signal-regulated kinase signal transduction. In contrast, mGRs decrease synaptic and neuronal excitability by reducing calcium currents through N-methyl-D-aspartate receptors and voltage-gated calcium channels by way of protein kinase A- and G protein-dependent mechanisms. This body of functional data complements anatomical evidence localizing GRs to the postsynaptic membrane. Finally, accumulating data also suggest the possibility that mMRs and mGRs may show an inverted U-shaped dose response, whereby glutamatergic synaptic transmission is increased by low doses of corticosterone acting at mMRs and decreased by higher doses acting at mGRs. Thus, synaptic transmission is regulated by mMRs and mGRs, and part of the stress signaling response is a direct and bidirectional modulation of the synapse itself by adrenal steroids.

Multivariate Statistical Analysis Applied to an IL6 Signal Transduction Model in Hepatocytes

This paper introduces the application of linear multivariate statistical techniques, including partial least squares (PLS), canonical correlation analysis (CCA) and reduced rank regression (RRR), into the area of Systems Biology. This new approach aims to extract the important proteins embedded in complex signal transduction pathway models.The analysis is performed on a model of intracellular signalling along the janus-associated kinases/signal transducers and transcription factors (JAK/STAT) and mitogen activated protein kinases (MAPK) signal transduction pathways in interleukin-6 (IL6) stimulated hepatocytes, which produce signal transducer and activator of transcription factor 3 (STAT3).A region of redundancy within the MAPK pathway that does not affect the STAT3 transcription was identified using CCA. This is the core finding of this analysis and cannot be obtained by inspecting the model by eye. In addition, RRR was found to isolate terms that do not significantly contribute to changes in protein concentrations, while the application of PLS does not provide such a detailed picture by virtue of its construction.This analysis has a similar objective to conventional model reduction techniques with the advantage of maintaining the meaning of the states prior to and after the reduction process. A significant model reduction is performed, with a marginal loss in accuracy, offering a more concise model while maintaining the main influencing factors on the STAT3 transcription.The findings offer a deeper understanding of the reaction terms involved, confirm the relevance of several proteins to the production of Acute Phase Proteins and complement existing findings regarding cross-talk between the two signalling pathways.

Analysis of IL6 Signal Transduction Model using Reduced Rank Regression

This work presents the application of reduced rank regression to the field of systems biology. A computational approach is used to investigate the mechanisms of the janus-associated kinases/signal transducers and transcription factors (JAK/STAT) and mitogen activated protein kinases (MAPK) signal transduction pathways in hepatic cells stimulated by interleukin-6. The results obtained identify the contribution of individual reactions to the dynamics of the model. These findings are compared to previously available results from sensitivity analysis of the model which focused on the parameters involved and their effect. This application of reduced rank regression allows for an understanding of the individual reaction terms involved in the modelled signal transduction pathways and has the benefit of being computationally inexpensive. The obtained results complement existing findings and also confirm the importance of several protein complexes in the MAPK pathway which hints at benefits that can be achieved by further refining the model.

Protein deficiency and nutritional recovery modulate insulin secretion and the early steps of insulin action in rats

Maternal malnutrition was shown to affect early growth and leads to permanent alterations in insulin secretion and sensitivity of offspring. In addition, epidemiological studies showed an association between low birth weight and glucose intolerance in adult life. To understand these interactions better, we investigated the insulin secretion by isolated islets and the early events related to insulin action in the hind-limb muscle of adult rats fed a diet of 17% protein (control) or 6% protein [low (LP) protein] during fetal life, suckling and after weaning, and in rats receiving 6% protein during fetal life and suckling followed by a 17% protein diet after weaning (recovered). The basal and maximal insulin secretion by islets from rats fed LP diet and the basal release by islets from recovered rats were significantly lower than that of control rats. The dose-response curves to glucose of islets from LP and recovered groups were shifted to the right compared to control islets, with the half-maximal response (EC 50) occurring at 16.9 ± 1.3, 12.4 ± 0.5 and 8.4 ± 0.1 mmol/L, respectively. The levels of insulin receptor, as well as insulin receptor substrate-1 and phosphorylation and the association between insulin receptor substrate-1 and phosphatidylinositol 3-kinase were greater in rats fed a LP diet than in control rats. In recovered rats, these variables were not significantly different from those of the other two groups. These results suggest that glucose homeostasis is maintained in LP and recovered rats by an increased sensitivity to insulin as a result of alterations in the early steps of the insulin signal transduction pathway.

Specificity of somatostatin receptor and G -protein interactions as characterized by somatostatin receptor subtype specific antibodies

The neuropeptide somatostatin is a widely distributed general inhibitor of endocrine, exocrine, gastrointestinal and neural functions. The biological actions of somatostatin are initiated by interaction with high affinity, plasma membrane somatostatin receptors (sst receptors). Five sst receptor subtypes have been cloned and sequence analysis shows they are all members of the G protein coupled receptor superfamily. The G proteins play a pivotal role in sst receptor signal transduction and the specificity of somatostatin receptor-G protein coupling defines the possible range of cellular responses. However, the data for endogenous sst receptor and G protein coupling is very limited, and even when it is available, the sst receptor subtypes involved in G protein coupling and signal transduction are unknown due to the expression of multiple sst receptor subtypes in target cell lines or tissues of somatostatin.^ In an effort to characterize each individual sst receptor subtypes, antisera against unique C-terminal regions of different sst receptor subtypes have been developed in our lab. In this report, antisera made against the sst1, sst2A and sst4 receptors are characterized. They are highly specific to their corresponding receptors and efficiently immunoprecipitate the sst receptors. Using these antibodies, the cell lines expressing these sst receptor subtypes were identified with both immunoprecipitation and Western blot methods. The development of sst receptor subtype specific antibodies make it possible to determine the specificity of the sst receptor subtype and G protein coupling in target cells or tissues expressing multiple sst receptors, two questions were addressed by this thesis: (1) whether different cellular environments affect receptor subtype and G protein coupling; (2) whether different sst receptors couple to different G proteins in similar cellular environments.^ Taken together our findings, both sst1 and sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells, G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in GH$\sb4$C$\sb1$ cells. Further, sst2A receptors couple with G$\alpha\sb{\rm i1},$ G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in AR4-2J cells while sst4 receptors couple with G$\alpha\sb{\rm i2}$ and G$\alpha\sb{\rm i3}$ in CHO cells. Therefore, the G protein coupling of the same sst receptors in different cell lines is basically similar in that they all couple with multiple $\alpha$-subunits of the G$\rm \sb{i}$ proteins, suggesting cellular environment has little effect on receptor and G protein coupling. Moreover, different sst receptors have similar G protein coupling specificities in the same cell line, suggesting components other than receptor and G$\alpha$ subunits in the signal transduction pathways may contribute to specific functions of each sst receptor subtype. This series of experiments represent a novel approach in dissecting signal transduction pathways and may have general application in the field. Furthermore, this is the first systematic study of sst receptor subtype and G protein $\alpha$-subunit interaction in both transfected cells and in normal cell lines. The information generated will be very useful in our understanding of sst receptor signal transduction pathways and in directing future sst receptor research. ^

Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans.

Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology.

Identification of novel cholesteatoma-related gene expression signatures using full-genome microarrays

This study demonstrates that the expression profile of cholesteatoma is similar to a metastatic tumour and chronically inflamed tissue. Based on the investigated profiles we present novel protein-protein interaction and signal transduction networks, which include cholesteatoma-regulated transcripts and may be of great value for drug targeting and therapy development.

Robustness and dissipation of mitogen-activated protein kinases signal transduction network: Underlying funneled landscape against stochastic fluctuations

We uncovered the underlying energy landscape of the mitogen-activated protein kinases signal transduction cellular network by exploring the statistical natures of the Brownian dynamical trajectories. We introduce a dimensionless quantity: The robustness ratio of energy gap versus local roughness to measure the global topography of the underlying landscape. A high robustness ratio implies funneled landscape. The landscape is quite robust against environmental fluctuations and variants of the intrinsic chemical reaction rates.