G proteins control diverse pathways of transmembrane signaling

Hormones, neurotransmitters, and autacoids interact with specific receptors and thereby trigger a series of molecular events that ultimately produce their biological effects. These receptors, localized in the plasma membrane, carry binding sites for ligands as diverse as peptides (e.g., glucagon, neuropeptides), lipids (e.g., prostaglandins), nucleosides and nucleotides (e.g., adenosine), and amines (e.g., catecholamines, serotonin). These receptors do not interest directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger); rather, they control one or several target systems via the activation of an intermediary guanine nucleotide-binding regulatory protein or G protein. G proteins serve as signal transducers, linking extracellularly oriented receptors to membrane-bound effectors. Traffic in these pathways is regulated by a GTP (on)-GDP (off) switch, which is regulated by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. These approaches, complemented in particular by electrophysiological experiments, have also identified several effectors that are regulated by G proteins. We can safely assume that current lists of G proteins and the functions that they control are incomplete.     

Drosophila melanogaster myotropins have unique functions and signaling pathways

Drosophila melanogaster TDVDHVFLRFamide (DMS), SDNFMRFamide, and pEVRFRQCYFNPISCF (FLT) represent three structurally distinct peptide families. Each peptide decreases heart rate albeit with different magnitudes and time-dependent responses. DMS and FLT are expressed in the crop and decrease crop motility; however, SDNFMRFamide expression and effect on the crop has not been reported. These data suggest the peptides have different physiological roles. The peptides have non-overlapping expression patterns in neural tissue, which suggests different mechanisms regulate their synthesis and release. The structures, expression patterns, and activities of the myotropins suggest they have important but different roles in biology and different signaling pathways.     

PCI complexes: pretty complex interactions in diverse signaling pathways

Three protein complexes (the proteasome regulatory lid, the COP9 signalosome and eukaryotic translation initiation factor 3) contain protein subunits with a well defined protein domain, the PCI domain. At least two (the COP9 signalosome and the lid) appear to share a common evolutionary origin. Recent advances in our understanding of the structure and function of the three complexes point to intriguing and unanticipated connections between the cellular functions performed by these three protein assemblies, especially between translation initiation and proteolytic protein degradation.     

Nitric oxide activates diverse signaling pathways to regulate gene expression

Nitric oxide signaling is crucial for effecting long lasting changes in cells, including gene expression, cell cycle arrest, apoptosis, and differentiation. We have determined the temporal order of gene activation induced by NO in mammalian cells and have examined the signaling pathways that mediate the action of NO. Using microarrays to study the kinetics of gene activation by NO, we have determined that NO induces three distinct waves of gene activity. The first wave is induced within 30 min of exposure to NO and represents the primary gene targets of NO. It is followed by subsequent waves of gene activity that may reflect further cascades of NO-induced gene expression. We verified our results using quantitative real time PCR and further validated our conclusions about the effects of NO by using cytokines to induce endogenous NO production. We next applied pharmacological and genetic approaches to determine the signaling pathways that are used by NO to regulate gene expression. We used inhibitors of particular signaling pathways, as well as cells from animals with a deleted p53 gene, to define groups of genes that require phosphatidylinositol 3-kinase, protein kinase C, NF-kappaB, p53, or combinations thereof for activation by NO. Our results demonstrate that NO utilizes several independent signaling pathways to induce gene expression.     

The signaling helix: a common functional theme in diverse signaling proteins

Background  

The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood.     

Emergence and maintenance of functional modules in signaling pathways

Background  

While detection and analysis of functional modules in biological systems have received great attention in recent years, we still lack a complete understanding of how such modules emerge. One theory is that systems must encounter a varying selection (i.e. environment) in order for modularity to emerge. Here, we provide an alternative and simpler explanation using a realistic model of biological signaling pathways and simulating their evolution.     

Structural and biochemical analysis of Ras-effector signaling via RalGDS.

The structure of the complex of Ras with the Ras-binding domain of its effector RalGDS (RGS-RBD), the first genuine Ras-effector complex, has been solved by X-ray crystallography. As with the Rap-RafRBD complex (Nasser et al., 1995), the interaction is via an inter-protein beta-sheet between the switch I region of Ras and the second strand of the RGS-RBD sheet, but the details of the interactions in the interface are remarkably different. Mutational studies were performed to investigate the contribution of selected interface residues to the binding affinity. Gel filtration experiments show that the Ras x RGS-RBD complex is a monomer. The results are compared to a recently determined structure of a similar complex using a Ras mutant (Huang et al., 1998) and are discussed in relation to partial loss-of-function mutations and the specificity of Ras versus Rap binding.     

Glucose controls multiple processes in Saccharomyces cerevisiae through diverse combinations of signaling pathways

We have studied how the lack of glucose sensors in the plasma membrane, or of the enzymes Hxk1, Hxk2, Glk1, which catalyze the first intracellular step in glucose metabolism, affect the different responses of Saccharomyces cerevisiae to glucose. Lack of the G-protein-coupled receptor Gpr1 or of Snf3/Rgt2 did not affect glucose repression of different genes or activation by glucose of plasma membrane ATPase, whereas lack of Gpr1 decreased, in an additive manner with lack of Mth1, the degradation of fructose 1,6-bisphosphatase that takes place in the presence of glucose. In an hxk1 hxk2 glk1 strain, unable to phosphorylate glucose, all of these responses to the sugar were suppressed or strongly reduced. In the absence of Hxk2 (or Hxk1 and Hxk2), glucose repression of SUC2, GAL1 and GDH2 was relieved, but that of FBP1 and ICL1 was maintained. Hxk1 or Hxk2 were needed for activation of plasma membrane ATPase but not for degradation of FbPase.     

Cellular interactions with the extracellular matrix are coupled to diverse transmembrane signaling pathways

Extracellular matrix (ECM) glycoproteins such as laminin, fibronectin, or collagen IV play a major role in cell behavior regulation. The molecular mechanisms taking place at the interface between the ECM and the cell surface are now rather well defined; however, very little is known about intracellular signals induced by these interactions. In order to get insights into the transduction pathways involved in cell-ECM interactions we have investigated the effects of several intracellular kinase inhibitors. Calmodulin-dependent kinase inhibitors, W-7 and sphingosine, have negative effects on cell-matrix interactions. They inhibit adhesion of several cell lines to laminin (IC50 = 4-10 microM), fibronectin and collagen IV (IC50 = 7-25 microM). The effects are immediate, reversible, and also cell specific, certain combinations of cell line-substrate being irresponsive to these inhibitors. In contrast, two inhibitors, H-7 and staurosporine, for which protein kinase C is a common target, increase two- to fourfold the attachment of HT1080, OVCAR-4, and B16F10 cells to laminin but not to fibronectin. Another inhibitor, HA-1004, known to inhibit protein kinase A at low concentrations, has an activating effect only at high concentration (> 200 microM) when it becomes an inhibitor of protein kinase C. These inhibitors are without effect on RuGli and Saos-2 cell adhesion on the three substrates. Altogether these results suggest that calmodulin-dependent kinases and protein kinase C could be separately involved in ECM-induced cellular responses. However, the effects of kinase inhibitors are substrate-specific and cell type-specific, suggesting that the intracellular signals induced by the extracellular matrix vary with the nature of integrin involved in signal transmission.     

Spermidine regulates Vibrio cholerae biofilm formation via transport and signaling pathways

Vibrio cholerae , the causative agent of the devastating diarrheal disease cholera, can form biofilms on diverse biotic and abiotic surfaces. Biofilm formation is important for the survival of this organism both in its natural environment and in the human host. Development of V. cholerae biofilms are regulated by complex regulatory networks that respond to environmental signals. One of these signals, norspermidine, is a polyamine that enhances biofilm formation via the NspS/MbaA signaling system. In this work, we have investigated the role of the polyamine spermidine in regulating biofilm formation in V. cholerae . We show that spermidine import requires PotD1, an ortholog of the periplasmic substrate-binding protein of the spermidine transport system in Escherichia coli . We also show that deletion of the potD1 gene results in a significant increase in biofilm formation. We hypothesize that spermidine imported into the cell hinders biofilm formation. Exogenous spermidine further reduces biofilm formation in a PotD1-independent, but NspS/MbaA-dependent, manner. Our results suggest that polyamines affect biofilm formation in V. cholerae via multiple pathways involving both transport and signaling networks.     

Potential for control of signaling pathways via cell size and shape

BACKGROUND: In order for signals generated at the plasma membrane to reach intracellular targets, activated messengers, such as G proteins and phosphoproteins, must diffuse through the cytoplasm. If the deactivators of these messengers, GTPase activating proteins (GAPs) and phosphatases, respectively, are sufficiently active in the cytoplasm, then the signal could in principle decay before reaching the target and a stable spatial gradient in phosphostate would be generated. Recent experiments document the existence of such gradients in living cells and suggest a role for them in mitotic spindle morphogenesis and cell migration. However, how such systems behave theoretically when embedded in a cell of varying size or shape has not been considered. RESULTS: Here we use a simple mathematical model to explore the theoretical consequences of a plasma membrane bound activator (i.e., guanine nucleotide exchange factor, GEF, or kinase) and a cytoplasmic deactivator (i.e., GAP or phosphatase), and we find that as a model cell grows, the substrate becomes progressively dephosphorylated as a result of decreased proximity to the activator. Conversely, as a cell spreads and flattens, the substrate becomes globally phosphorylated because of increased proximity of the substrate to the activator. Similarly, in the leading edge of polarized cells and in protrusions such as lamellipodia or filopodia, the substrate is highly phosphorylated. As a specific test of the model, we found that the experimentally observed preferential activation of the G protein Cdc42 in the periphery of fibroblasts that was recently reported is consistent with model predictions. CONCLUSIONS: We conclude that cell-signaling pathways can theoretically be turned on and off, both locally and globally, in response to alterations in cell size and shape.     

Mitofusin 2 ameliorates hypoxia-induced apoptosis via mitochondrial function and signaling pathways

Mitochondrial dynamics play a critical role in mitochondrial function and signaling. Although mitochondria play a critical role in hypoxia/ischemia, the further mechanisms between mitochondrial dynamics and ischemia are still unclear. The current study aimed to determine the role of mitofusin 2, a key regulator of mitochondrial fusion, in a hypoxic model and to explore a novel strategy for cerebral ischemia via modulation of mitochondrial dynamics. To the best of our knowledge, this is the first study to investigate both mitochondrial function and molecular pathways to determine the role of mitofusin 2 in hypoxia-induced neuronal apoptosis. In vivo, C57BL/6 mice (male, 19–25 g) underwent a permanent middle cerebral artery occlusion for 12 or 24 h (n = 6 per group). In vitro, cobalt chloride was used to mimic hypoxia in immortalized hippocampal neurons. Down- or up-regulation of Mfn2 was induced to investigate the role of Mfn2 in hypoxia, especially in mitochondrial function and signaling pathways. The findings demonstrated that decreased mitofusin 2 occurred both in vivo and in vitro hypoxic models; second, the anti-apoptotic effect of Mfn2 may work via restoration of mitochondrial function; third, the modulation of the B Cell Leukemia 2/Bcl-2 Associated X protein and extracellular signal-regulated kinase 1/2 signaling pathways highlight the role of Mfn2 in signaling pathways beyond fusion. In summary, depletion of mitofusin 2 would lead to apoptosis both in normal or hypoxic conditions; however, mitofusin 2 overexpression could attenuate hypoxia-induced apoptosis, which represents a potential novel strategy for neuroprotection against ischemic brain damage.     

Nitric oxide modulates salt and sugar responses via different signaling pathways

Locusts lay their eggs by digging into a substrate using rhythmic opening and closing movements of ovipositor valves at the end of the abdomen. The digging rhythm is inhibited by chemosensory stimulation of chemoreceptors on the valves. Nitric oxide (NO) modulated the effects of chemosensory stimulation on the rhythm. Stimulation with either sucrose or sodium chloride (NaCl) stopped the digging rhythm, whereas simultaneous bath application of the NO inhibitor, N-nitro-L-arginine methyl ester (L-NAME), increased the duration for which the digging rhythm stopped. Increasing NO levels caused a significant reduction in the cessation of the rhythm in response to the same 2 chemicals. Bath applying cyclic guanosine monophosphate (cGMP), the soluble guanylate inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and the generic protein kinase inhibitor H-7 had no effect on the duration for which the rhythm stopped in response to NaCl stimulation. Conversely, bath application of cGMP and ODQ resulted in a significant decrease and increase, respectively, in the duration for which the digging rhythm stopped when stimulated with sucrose. Moreover, bath application of the selective protein kinase G (PKG) inhibitor KT-5823 also resulted in a significant increase in the duration of cessation of the rhythm when stimulated with sucrose. Results suggest that NO modulates the behavioral responses to NaCl via a cGMP/PKG-independent pathway while modulating the responses to sucrose via a NO-cGMP/PKG-dependent pathway.     

Lactoferrin activates macrophages via TLR4-dependent and -independent signaling pathways

Lactoferrin (LF) is a component of innate immunity and is known to interact with accessory molecules involved in the TLR4 pathway, including CD14 and LPS binding protein, suggesting that LF may activate components of the TLR4 pathway. In the present study, we have asked whether bovine LF (bLF)-induced macrophage activation is TLR4-dependent. Both bLF and LPS stimulated IL-6 production and CD40 expression in RAW 264.7 macrophages and in BALB/cJ peritoneal exudate macrophages. However, in macrophages from congenic TLR4(-/-) C.C3-Tlr4(lps-d) mice, CD40 was not expressed while IL-6 secretion was increased relative to wild-type cells. The signaling components NF-kappaB, p38, ERK and JNK were activated in RAW 264.7 cells and BALB/cJ macrophages after bLF or LPS stimulation, demonstrating that the TLR4-dependent bLF activation pathway utilizes signaling components common to LPS activation. In TLR4 deficient macrophages, bLF-induced activation of NF-kappaB, p38, ERK and JNK whereas LPS-induced cell signaling was absent. We conclude from these studies that bLF induces limited and defined macrophage activation and cell signaling events via TLR4-dependent and -independent mechanisms. bLF-induced CD40 expression was TLR4-dependent whereas bLF-induced IL-6 secretion was TLR4-independent, indicating potentially separate pathways for bLF mediated macrophage activation events in innate immunity.     

Integration of inositol phosphate signaling pathways via human ITPK1

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.