RESUMEN

BACKGROUND: Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC is essential to sense the environment of food-activated spores. RESULTS: Plc-null spores germinate at alkaline pH, reduced temperature or increased osmolarity, conditions at which the emerging amoebae can not grow. In contrast, food-activated wild-type spores return to dormancy till conditions in the environment allow growth. The analysis of inositol 1,4,5-trisphosphate (IP3) levels and the effect of added IP3 uncover an unexpected mechanism how PLC regulates spore germination: i) deletion of PLC induces the enhanced activity of an IP5 phosphatase leading to high IP3 levels in plc-null cells; ii) in wild-type spores unfavourable conditions inhibit PLC leading to a reduction of IP3 levels; addition of exogenous IP3 to wild-type spores induces germination at unfavourable conditions; iii) in plc-null spores IP3 levels remain high, also at unfavourable environmental conditions. CONCLUSIONS: The results imply that environmental conditions regulate PLC activity and that IP3 induces spore germination; the uncontrolled germination of plc-null spores is not due to a lack of PLC activity but to the constitutive activation of an alternative IP3-forming pathway.

Asunto(s)

Dictyostelium/enzimología , Dictyostelium/fisiología , Isoenzimas/fisiología , Fosfolipasas de Tipo C/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Eliminación de Gen , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/genética , Cinética , Presión Osmótica , Fosfolipasa C delta , Esporas Protozoarias/crecimiento & desarrollo , Temperatura , Fosfolipasas de Tipo C/genética

RESUMEN

Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells, the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied by altered intracellular protein mobility.

Asunto(s)

Actinas/fisiología , Polaridad Celular/fisiología , Citoesqueleto/fisiología , Dictyostelium/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas/metabolismo , Actinas/farmacología , Actinas/ultraestructura , Animales , Citoesqueleto/ultraestructura , Difusión/efectos de los fármacos , Proteínas Fluorescentes Verdes , Indicadores y Reactivos , Cinética , Proteínas Luminiscentes/efectos de los fármacos , Presión Osmótica , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Traducciones

RESUMEN

Small chemotactic cells like Dictyostelium and neutrophils transduce shallow spatial chemoattractant gradients into strongly localized intracellular responses. We show that the capacity of a second messenger to establish and maintain localized signals, is mainly determined by its dispersion range, lambda = the square root of D(m)/k(-1), which must be small compared to the cell's length. Therefore, short-living second messengers (high k(-1)) with diffusion coefficients D(m) in the range of 0-5 microm(2) s(-1) are most suitable. Additional to short dispersion ranges, gradient sensing may include positive feedback mechanisms that lead to local activation and global inhibition of second-messenger production. To introduce the essential nonlinear amplification, we have investigated models in which one or more components of the signal transduction cascade translocate from the cytosol to the second messenger in the plasma membrane. A one-component model is able to amplify a 1.5-fold difference of receptor activity over the cell length into a 15-fold difference of second-messenger concentration. Amplification can be improved considerably by introducing an additional activating component that translocates to the membrane. In both models, communication between the front and the back of the cell is mediated by partial depletion of cytosolic components, which leads to both local activation and global inhibition. The results suggest that a biochemically simple and general mechanism may explain various signal localization phenomena not only in chemotactic cells but also those occurring in morphogenesis and cell differentiation.

Asunto(s)

Quimiotaxis , Modelos Biológicos , Sistemas de Mensajero Secundario/fisiología , Animales , Membrana Celular/metabolismo , Citosol/metabolismo , Dictyostelium/citología , Dictyostelium/metabolismo , Difusión , Neutrófilos/citología , Neutrófilos/metabolismo , Receptores de Superficie Celular/metabolismo , Factores de Tiempo

RESUMEN

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) triphosphate (GTPgammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPgammaS stimulated the adenylyl cyclase activity of DdGCA(kqd) with properties similar to those of the wild-type enzyme (decrease of K(m) and increase of V(max)), demonstrating that GTPgammaS stimulation is independent of substrate specificity. Furthermore, GTPgammaS activation of DdGCA(kqd) is retained in several null mutants of Galpha and Gbeta proteins, indicating that GTPgammaS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.

Asunto(s)

Adenilil Ciclasas/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/fisiología , Guanilato Ciclasa/metabolismo , Adenilil Ciclasas/genética , Secuencia de Aminoácidos , Animales , Dictyostelium/enzimología , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Datos de Secuencia Molecular , Mutagénesis , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

A new Dictyostelium discoideum cyclase gene was identified that encodes a protein (sGC) with 35% similarity to mammalian soluble adenylyl cyclase (sAC). Gene disruption of sGC has no effect on adenylyl cyclase activity and results in a >10-fold reduction in guanylyl cyclase activity. The scg- null mutants show reduced chemotactic sensitivity and aggregate poorly under stringent conditions. With Mn(2+)/GTP as substrate, most of the sGC activity is soluble, but with the more physiological Mg(2+)/GTP the activity is detected in membranes and stimulated by GTPgammaS. Unexpectedly, orthologues of sGC and sAC are present in bacteria and vertebrates, but absent from Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae.

Asunto(s)

Adenilil Ciclasas/genética , Dictyostelium/enzimología , Guanilato Ciclasa/genética , Adenilil Ciclasas/clasificación , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Dictyostelium/genética , Guanilato Ciclasa/clasificación , Guanilato Ciclasa/metabolismo , Humanos , Mamíferos , Datos de Secuencia Molecular , Fenotipo , Filogenia , Homología de Secuencia de Aminoácido , Solubilidad

Asunto(s)

Evolución Molecular , Eliminación de Gen , Transferencia de Gen Horizontal , Genes Bacterianos , Genoma Humano , Animales , Arabidopsis , Caenorhabditis elegans , Dictyostelium , Drosophila melanogaster , Proyecto Genoma Humano , Humanos , Monoaminooxidasa/genética , Saccharomyces cerevisiae , Homología de Secuencia de Ácido Nucleico

RESUMEN

The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.

Asunto(s)

Adenilil Ciclasas/química , Dictyostelium/enzimología , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas Protozoarias , Secuencia de Aminoácidos , Animales , Agregación Celular , Células Cultivadas , Clonación Molecular , GMP Cíclico/metabolismo , Dictyostelium/citología , Dictyostelium/genética , Eliminación de Gen , Guanilato Ciclasa/metabolismo , Guanilato Ciclasa/fisiología , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements, CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single-cell water dynamics.

Asunto(s)

Óxido de Deuterio/química , Agua/química , Animales , Células COS , Permeabilidad de la Membrana Celular , Chlorocebus aethiops , Dictyostelium/química , Difusión , Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador/métodos , Líquido Intracelular/química , Naftalenos , Factores de Tiempo

RESUMEN

In Dictyostelium cAMP and cGMP have important functions as first and second messengers in chemotaxis and development. Two cyclic-nucleotide phosphodiesterases (DdPDE 1 and 2) have been identified previously, an extracellular dual-specificity enzyme and an intracellular cAMP-specific enzyme (encoded by the psdA and regA genes respectively). Biochemical data suggest the presence of at least one cGMP-specific phosphodiesterase (PDE) that is activated by cGMP. Using bioinformatics we identified a partial sequence in the Dictyostelium expressed sequence tag database that shows a high degree of amino acid sequence identity with mammalian PDE catalytic domains (DdPDE3). The deduced amino acid sequence of a full-length DdPDE3 cDNA isolated in this study predicts a 60 kDa protein with a 300-residue C-terminal PDE catalytic domain, which is preceded by approx. 200 residues rich in asparagine and glutamine residues. Expression of the DdPDE3 catalytic domain in Escherichia coli shows that the enzyme has Michaelis-Menten kinetics and a higher affinity for cGMP (K(m)=0.22 microM) than for cAMP (K(m)=145 microM); cGMP does not stimulate enzyme activity. The enzyme requires bivalent cations for activity; Mn(2+) is preferred to Mg(2+), whereas Ca(2+) yields no activity. DdPDE3 is inhibited by 3-isobutyl-1-methylxanthine with an IC(50) of approx. 60 microM. Overexpression of the DdPDE3 catalytic domain in Dictyostelium confirms these kinetic properties without indications of its activation by cGMP. The properties of DdPDE3 resemble those of mammalian PDE9, which also shows the highest sequence similarity within the catalytic domains. DdPDE3 is the first cGMP-selective PDE identified in lower eukaryotes.

Asunto(s)

3',5'-GMP Cíclico Fosfodiesterasas , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Dictyostelium/enzimología , 1-Metil-3-Isobutilxantina/farmacología , 3',5'-GMP Cíclico Fosfodiesterasas/química , 3',5'-GMP Cíclico Fosfodiesterasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Dominio Catalítico , Cationes Bivalentes , Clonación Molecular , ADN Recombinante , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Aminoácido

RESUMEN

We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTA(s)(*)). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter contains a tetracycline-responsive element, which is the binding site for tTA(s)(*). Tetracycline prevents tTA(s)(*) from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTA(s)(*) binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene.

Asunto(s)

Dictyostelium/genética , Tetraciclina/farmacología , Transactivadores/efectos de los fármacos , Actinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Recombinante/genética , Dictyostelium/citología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos , Proteínas Fluorescentes Verdes , Cinética , Luciferasas/efectos de los fármacos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Luminiscentes/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Transactivadores/genética

RESUMEN

Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-beta gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5-17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5'-sequence of the hCG gene to the Dictyostelium 'Kozak'-like sequence increased expression levels approximately 1.5-fold. Thus, using both codon optimization and 'Kozak' adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG.

Asunto(s)

Gonadotropina Coriónica Humana de Subunidad beta/genética , Dictyostelium/genética , Hormona Folículo Estimulante/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Gonadotropina Coriónica Humana de Subunidad beta/biosíntesis , Codón/genética , Ensayo de Inmunoadsorción Enzimática , Hormona Folículo Estimulante/biosíntesis , Técnicas Genéticas , Humanos , Datos de Secuencia Molecular , Extensión de la Cadena Peptídica de Translación , Proteínas Recombinantes/biosíntesis , Ribosomas/metabolismo

RESUMEN

The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild-type hormones. We also show that structure-function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues.

Asunto(s)

Gonadotropina Coriónica/genética , Hormona Folículo Estimulante/genética , Glicoproteínas/genética , Mutagénesis , Animales , Gonadotropina Coriónica/biosíntesis , Dictyostelium/genética , Relación Dosis-Respuesta a Droga , Hormona Folículo Estimulante/biosíntesis , Pruebas Genéticas , Glicoproteínas/biosíntesis , Humanos , Proteínas Recombinantes/biosíntesis

RESUMEN

Human choriogonadotropin (hCG) is a highly complex glycoprotein consisting of two non-covalently associated subunits. We aimed for the expression of a single-chain hCG in the soil amoebae Dictyostelium discoideum, a host which, in principle, provides simple genetics in combination with complex protein synthesis. To limit anticipated problems in mRNA translation, the first 30 bases of the coding sequence were altered to conform to the Dictyostelium preferred codon usage. We show that, immunologically, active single-chain hCG is indeed produced by Dictyostelium. Furthermore, this single-chain hCG is able to bind to the human luteinizing hormone/CG receptor and elicit a biological response. Its receptor-binding affinity is comparable to single-chain hCG produced by mammalian cells. We conclude that Dictyostelium is able to express bioactive highly complex heterologous glycoproteins.

Asunto(s)

Gonadotropina Coriónica/genética , Dictyostelium/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Gonadotropina Coriónica/farmacología , Clonación Molecular , Cricetinae , Expresión Génica/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica/fisiología , Señales de Clasificación de Proteína/química , Receptores de HL/metabolismo , Proteínas Recombinantes/genética , Transducción de Señal/fisiología , Transformación Genética

RESUMEN

Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a mutant that does not aggregate upon starvation but is rescued by adding extracellular cAMP. Sequencing of the mutated locus revealed a new gene, DdMYB2, whose product contains three Myb repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2-null cells show undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a constitutive promotor rescues differentiation and morphogenesis of Ddmyb2-null mutants. The results suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP.

Asunto(s)

Adenilil Ciclasas/fisiología , Proteínas de Unión al ADN/genética , Dictyostelium/fisiología , Regulación de la Expresión Génica/fisiología , Genes Protozoarios , Oncogenes , Proteínas Protozoarias , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Transducción de Señal/genética

RESUMEN

In the ameboid eukaryote Dictyostelium discoideum, chemotactic stimulation by cAMP induces an increase of intracellular cGMP and subsequently the phosphorylation of myosin heavy chain II. Resistance to high osmotic stress also requires transient increases of intracellular cGMP and phosphorylation of myosin heavy chain II, although the kinetics is much slower than for chemotaxis. To examine if chemotaxis and osmotic stress share common signaling components we systematically analyzed the osmotic cGMP response and survival in chemotactic mutants with altered cGMP signaling. Null mutants with deletions of cell surface cAMP receptors or the associated GTP-binding proteins Galpha2 and Gbeta show no cAMP-induced cGMP response and chemotaxis; in contrast, osmotic stress induces the normal cGMP accumulation and survival. The same result was obtained with the non-chemotactic mutant KI-10, which lacks the activation of guanylyl cyclase by cAMP. This indicates that these components are required for chemotaxis but not osmotic cGMP signaling and survival. The potential guanylyl cyclase null mutant KI-8 shows no chemotaxis, no osmotic cGMP increase and reduced survival in high osmolarity. Two types of cGMP-binding protein mutants, KI-4 and KI-7, also show reduced tolerance during high osmotic stress. Taken together, these observations clarify that chemotactic and osmotic signals are detected by different mechanisms, but share a cGMP signaling pathway.

Asunto(s)

Quimiotaxis , GMP Cíclico/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Transducción de Señal , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Mutación , Ósmosis , Receptores de AMP Cíclico/genética

RESUMEN

Binding of extracellular cAMP to surface receptors induces at least two responses in Dictyostelium discoideum, the G-protein-dependent activation of guanylyl cyclase, and the opening of a plasma membrane Ca2+ channel. Some experiments suggest that intracellular cGMP opens the Ca2+ channel, while others demonstrate that the channel can open in the absence of functional G-proteins (and thus in the absence of cGMP formation). We have analysed 45Ca2+ uptake in three mutants with altered cGMP formation. Mutant stmF shows a prolonged cGMP response due to deletion of an intracellular phosphodiesterase. Uptake of receptor-stimulated 45Ca2+ is enhanced about two-fold in this mutant if compared to wild-type cells, suggesting that cGMP regulates the opening of the channel. Mutant KI-7 has very low levels of surface cAMP receptors, but nevertheless an enhanced receptor-stimulated cGMP response due to a defect in the turn-off of guanylyl cyclase. This mutant shows poor receptor-stimulated 45Ca2+ uptake, suggesting that cGMP alone is not sufficient to open the Ca2+ channel. Finally, mutant KI-8 has no cGMP due to the absence of nearly all guanylyl cyclase activity. The mutant shows significant but reduced 45Ca2+ uptake (19% of wild-type; 60% if corrected for the reduced level of surface cAMP receptors), suggesting that the channel can open in the absence of cGMP. Taken together, the results demonstrate that receptor-stimulated Ca2+ influx is not directly induced by cGMP formation; it can occur in the absence of cGMP, but is potentiated two- to four-fold by cGMP.

Asunto(s)

Canales de Calcio/fisiología , Calcio/metabolismo , AMP Cíclico/farmacología , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Dictyostelium/fisiología , Receptores de AMP Cíclico/fisiología , Animales , Canales de Calcio/efectos de los fármacos , Radioisótopos de Calcio , Quimiotaxis , AMP Cíclico/metabolismo , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Guanilato Ciclasa/deficiencia , Guanilato Ciclasa/genética , Cinética , Modelos Biológicos , Receptores de AMP Cíclico/genética , Transducción de Señal

RESUMEN

In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.

Asunto(s)

Quimiotaxis , Dictyostelium/fisiología , Proteínas de Unión al GTP/metabolismo , Receptores de AMP Cíclico/fisiología , Actinas/metabolismo , Adenilil Ciclasas/metabolismo , Sustitución de Aminoácidos , Animales , Agregación Celular , Diferenciación Celular , Activación Enzimática , Guanilato Ciclasa/metabolismo , Cinética , Mutagénesis Sitio-Dirigida , Fosforilación , Receptores de AMP Cíclico/biosíntesis , Proteínas Recombinantes/biosíntesis , Eliminación de Secuencia , Serina

RESUMEN

The chemoattractant cAMP induces directed cell locomotion in Dictyostelium cells. Several second messenger pathways are activated upon binding of cAMP to G-protein-coupled receptors, including adenylyl cyclase, guanylyl cyclase, phospholipase C, and the opening of plasma membrane Ca2+ channels. These second messenger responses are unaltered in many chemotactic mutants, except for the cGMP response. Activation of guanylyl cyclase depends on G-proteins and is regulated by a cGMP-binding protein in a complex manner. This cGMP-binding protein also mediates intracellular functions of cGMP to activate a PKC-related kinase that phosphorylates myosin II heavy chain, thereby allowing myosin filaments to rearrange during cell movement.

Asunto(s)

Quimiotaxis/fisiología , GMP Cíclico/fisiología , Dictyostelium/fisiología , Sistemas de Mensajero Secundario , Animales , Quimiotaxis/genética , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Proteínas de Unión al GTP/metabolismo , Guanilato Ciclasa/metabolismo , Receptores de Formil Péptido , Receptores Inmunológicos/metabolismo , Receptores de Péptidos/metabolismo , Equilibrio Hidroelectrolítico

RESUMEN

Biochemical and genetic data on the metabolism of inositol phosphates in the microorganism Dictyostelium are combined in a scheme composed of in five subroutes. The first subroute is the inositol cycle as found in other organisms: inositol is incorporated into phospholipids that are hydrolysed by PLC producing Ins(1,4,5)P3 which is dephosphorylated to inositol. The second subroute is the sequential phosphorylation of inositol to InsP6; the Ins(3,4,6)P3 intermediate does not release Ca2+. The third subroute is the sequential phosphorylation of Ins(1,4,5)P3 to InsP6 in a nucleus associated fraction, whereas the fourth subroute is the dephosphorylation of Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 at the plasma membrane. This last route mediates Ins(1,4,5)P3 formation in cells with a disruption of the single PLC gene. Finally, we recognize the formation of InsP7 and InsP8 as the fifth subroute.

Asunto(s)

Dictyostelium/metabolismo , Fosfatos de Inositol/metabolismo , Animales , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Dictyostelium/genética , Fosfatos de Inositol/biosíntesis , Fosfatos de Inositol/genética , Fosfatidilinositoles/biosíntesis , Fosforilación , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo

RESUMEN

Dictyostelium cells have enzyme activities that generate the inositol polyphosphate Ins(1,4,5)P3 from Ins(1,3,4,5,6)P5 via the intermediates Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4. These enzyme activities could explain why cells with a deletion of the single phospholipase C gene (plc- cells) possess nearly normal Ins(1,4,5)P3 levels. In this study the regulation and the subcellular localization of the enzyme activities was investigated. The enzyme activities performing the different reaction steps from Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 are probably due to a single enzyme. Indications for this are the previously shown similar Ca2+ dependencies of the various reaction steps. Furthermore, the activities mediating the complete conversion of Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 co-purify after subcellular fractionation, solubilization, and chromatography of the proteins. Subcellular fractionation studies demonstrate that the enzyme is localized mainly at the inner face of the plasma membrane. The enzyme activity could not be stimulated in vitro by guanosine 5'-(3-thio)triphosphate, a procedure known to activate G-protein-coupled enzymes in Dictyostelium. Still, in plc- cells the level of Ins(1,4,5)P3 was increased significantly after stimulation with high concentrations of the extracellular ligand cAMP. This stimulation is most likely due to the influx of Ca2+ because no increase of Ins(1,4,5)P3 could be detected in the absence of extracellular Ca2+. The results demonstrate the existence of a new receptor-controlled route for the formation of Ins(1,4,5)P3 that is independent of phospholipase C.

Asunto(s)

Calcio/metabolismo , Dictyostelium/metabolismo , Inositol 1,4,5-Trifosfato/biosíntesis , Monoéster Fosfórico Hidrolasas/metabolismo , Receptores de AMP Cíclico/fisiología , Fosfolipasas de Tipo C/metabolismo , Animales , Membrana Celular/enzimología , Activación Enzimática , Inositol 1,4,5-Trifosfato/metabolismo , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Solubilidad , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo