Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide

The effects of oleamide, an amidated lipid isolated from the cerebrospinal fluid of sleep-deprived cats, on serotonin receptor-mediated responses were investigated in cultured mammalian cells. In rat P11 cells, which endogenously express the 5-hydroxytryptamine2A (5HT2A) receptor, oleamide significantly potentiated 5HT-induced phosphoinositide hydrolysis. In HeLa cells expressing the 5HT7 receptor subtype, oleamide caused a concentration-dependent increase in cAMP accumulation but with lower efficacy than that observed by 5HT. This effect was not observed in untransfected HeLa cells. Clozapine did not prevent the increase in cAMP elicited by oleamide, and ketanserin caused an ≈65% decrease. In the presence of 5HT, oleamide had the opposite effect on cAMP, causing insurmountable antagonism of the concentration-effect curve to 5HT, but had no effect on cAMP levels elicited by isoproterenol or forskolin. These results indicate that oleamide can modulate 5HT-mediated signal transduction at different subtypes of mammalian 5HT receptors. Additionally, our data indicate that oleamide acts at an apparent allosteric site on the 5HT7 receptor and elicits functional responses via activation of this site. This represents a unique mechanism of activation for 5HT G protein-coupled receptors and suggests that G protein-coupled neurotransmitter receptors may act like their iontropic counterparts (i.e., γ-aminobutyric acid type A receptors) in that there may be several binding sites on the receptor that regulate functional activity with varying efficacies.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway.

Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-Å resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 Å from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora. This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.

Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle

We present evidence that a bacterial signal transduction cascade that couples morphogenesis with cell cycle progression is regulated by dynamic localization of its components. Previous studies have implicated two histidine kinases, DivJ and PleC, and the response regulator, DivK, in the regulation of morphogenesis in the dimorphic bacterium Caulobacter crescentus. Here, we show that the cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical localization that culminates in asymmetric distribution of DivK within the two cell types that are characteristic of the Caulobacter cell cycle; DivK is dispersed throughout the cytoplasm of the progeny swarmer cell and is localized to the pole of the stalked cell. The membrane-bound DivJ and PleC histidine kinases, which are asymmetrically localized at the opposite poles of the predivisional cell, control the temporal and spatial localization of DivK. DivJ mediates DivK targeting to the poles whereas PleC controls its release from one of the poles at times and places that are consistent with the activities and location of DivJ and PleC in the late predivisional cell. Thus, dynamic changes in subcellular location of multiple components of a signal transduction cascade may constitute a novel mode of prokaryotic regulation to generate and maintain cellular asymmetry.

Changes in Phosphoinositide Metabolism with Days in Culture Affect Signal Transduction Pathways in Galdieria sulphuraria1

The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP2) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP2 in the cells was 910 ± 100 pmol g−1 fresh weight; by 12 d, PIP2 levels increased to 1200 ± 150 pmol g−1 fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min−1 mg−1 protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP3) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP3 was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP3 signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP2 biosynthesis in cells with already-high PIP2 levels suggests that the PIP2 present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP2.

Pro-apoptotic gene expression mediated by the p38 mitogen-activated protein kinase signal transduction pathway

Neurotrophic factor deprivation causes apoptosis by a mechanism that requires macromolecular synthesis. This fact suggests that gene expression is necessary to achieve cell death. To identify mRNA that is expressed in apoptotic cells we used subtractive hybridization with cDNA prepared from neuronal pheochromocytoma cells. Monoamine oxidase (MAO) expression was increased in cells during nerve growth factor withdrawal-induced apoptosis. The increased apoptosis and induction of MAO was prevented by inhibition of the p38 mitogen-activated protein (MAP) kinase pathway. MAO may contribute to the apoptotic process because inhibition of MAO activity suppressed cell death. Together, these data indicate that MAO may be a target of pro-apoptotic signal transduction by the p38 MAP kinase pathway.

A Model for Signal Transduction during Gamete Release in the Fucoid Alga Pelvetia compressa1

Fucoid algae release gametes into seawater following an inductive light period (potentiation), and gamete expulsion from potentiated receptacles of Pelvetia compressa began about 2 min after a light-to-dark transition. Agitation of the medium reversed potentiation, with an exponential time course completed in about 3 h. Light regulated two signaling pathways during potentiation and gamete expulsion: a photosynthetic pathway and a photosynthesis-independent pathway in which red light was active but blue light was not. Uptake of K+ appears to have an important role in potentiation, because a 50% inhibition of potentiation occurred in the presence of the tetraethylammonium ion, a K+-channel blocker. A central role of anion channels in the maintenance of potentiation is suggested by the premature release of gametes in the light when receptacles were incubated with inhibitors of slow-type anion channels. An inhibitor of tyrosine kinases, tyrphostin A63, also inhibited potentiation. A model for gamete release from P. compressa is presented that proposes that illumination results in the accumulation of ions (e.g. K+) throughout the cells of the receptacle during potentiation, which then move into the extracellular matrix during gamete expulsion to generate osmomechanical force, resulting in gamete release.

Critical role for the docking-protein FRS2α in FGF receptor-mediated signal transduction pathways

The docking protein FRS2α has been implicated as a mediator of signaling via fibroblast growth factor receptors (FGFRs). We have demonstrated that targeted disruption of FRS2α gene causes severe impairment in mouse development resulting in embryonal lethality at E7.0–E7.5. Experiments with FRS2α-deficient fibroblasts demonstrate that FRS2α plays a critical role in FGF-induced mitogen-activated protein (MAP) kinase stimulation, phosphatidylinositol-3 (PI-3) kinase activation, chemotactic response, and cell proliferation. Following FGF stimulation, tyrosine phosphorylated FRS2α functions as a site for coordinated assembly of a multiprotein complex that includes Gab1 and the effector proteins that are recruited by this docking protein. Furthermore, we demonstrate that different tyrosine phosphorylation sites on FRS2α are responsible for mediating different FGF-induced biological responses. These experiments establish the central role of FRS2α in signaling via FGFRs and demonstrate that FRS2α mediates multiple FGFR-dependent signaling pathways critical for embryonic development.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

SPINDLY, a tetratricopeptide repeat protein involved in gibberellin signal transduction in Arabidopsis.

Gibberellins (GAs) are a major class of plant hormones that control many developmental processes, including seed development and germination, flower and fruit development, and flowering time. Genetic studies with Arabidopsis thaliana have identified two genes involved in GA perception or signal transduction. A semidominant mutation at the GIBBERELLIN INSENSITIVE (GAI) locus results in plants resembling GA-deficient mutants but exhibiting reduced sensitivity to GA. Recessive mutations at the SPINDLY (SPY) locus cause a phenotype that is consistent with constitutive activation of GA signal transduction. Here we show that a strong allele of spy is completely epistatic to gai, indicating that SPY acts downstream of GAI. We have cloned the SPY gene and shown that it encodes a new type of signal transduction protein, which contains a tetratricopeptide repeat region, likely serving as a protein interaction domain, and a novel C-terminal region. Mutations in both domains increase GA signal transduction. The presence of a similar gene in Caenorhabditis elegans suggests that SPY represents a class of signal transduction proteins that is present throughout the eukaryotes.

I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins associate with and transduce signals from TNF receptor 2, CD40, and presumably other members of the TNF receptor superfamily. TRAF2 is required for CD40- and TNF-mediated activation of the transcription factor NF-kappa B. Here we describe the isolation and characterization of a novel TRAF-interacting protein, I-TRAF, that binds to the conserved TRAF-C domain of the three known TRAFs. Overexpression of I-TRAF inhibits TRAF2-mediated NF-kappa B activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator of TRAF function that may act by maintaining TRAFs in a latent state.

Dual signal transduction through delta opioid receptors in a transfected human T-cell line.

Opiates are known to function as immunomodulators, in part by effects on T cells. However, the signal transduction pathways mediating the effects of opiates on T cells are largely undefined. To determine whether pathways that regulate free intracellular calcium ([Ca2+]i) and/or cAMP are affected by opiates acting through delta-type opioid receptors (DORs), a cDNA encoding the neuronal DOR was expressed in a stably transfected Jurkat T-cell line. The DOR agonists, deltorphin and [D-Ala2, D-Leu5]-enkephalin (DADLE), elevated [Ca2+]i, measured by flow cytofluorometry using the calcium-sensitive dye, Fluo-3. At concentrations from 10(-11)-10(-7) M, both agonists increased [Ca2+]i from 60 nM to peak concentrations of 400 nM in a dose-dependent manner within 30 sec (ED50 of approximately 5 x 10(-9) M). Naltrindole, a selective DOR antagonist, abolished the increase in [Ca2+]i, and pretreatment with pertussis toxin was also effective. To assess the role of extracellular calcium, cells were pretreated with EGTA, which reduced the initial deltorphin-induced elevation of [Ca2+]i by more than 50% and eliminated the second phase of calcium mobilization. Additionally, the effect of DADLE on forskolin-stimulated cAMP production was determined. DADLE reduced cAMP production by 70% (IC50 of approximately equal to 10(-11) M), and pertussis toxin inhibited the action of DADLE. Thus, the DOR expressed by a transfected Jurkat T-cell line is positively coupled to pathways leading to calcium mobilization and negatively coupled to adenylate cyclase. These studies identify two pertussis toxin-sensitive, G protein-mediated signaling pathways through which DOR agonists regulate the levels of intracellular messengers that modulate T-cell activation.

Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I.

We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments.