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P Devreotes

Publications and source records attributed to P Devreotes.

17 recordsLinked to original sources
PubMed2001

Single-molecule analysis of chemotactic signaling in Dictyostelium cells.

Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium discoideum cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells.

Animals↗
PubMed2001

Receptor-mediated activation of heterotrimeric G-proteins in living cells.

Receptor-mediated activation of heterotrimeric GTP-binding proteins (G-proteins) was visualized in living Dictyostelium discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between alpha- and beta- subunits fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociated and reassociated upon addition and removal of chemoattractant. During continuous stimulation, G-protein activation reached a dose-dependent steady-state level. Even though physiological responses subsided, the activation did not decline. Thus, adaptation occurs at another point in the signaling pathway, and occupied receptors, whether or not they are phosphorylated, catalyze the G-protein cycle. Construction of similar energy-transfer pairs of mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of newly found G-protein-coupled receptors.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Desensitization of G-protein-coupled receptors. agonist-induced phosphorylation of the chemoattractant receptor cAR1 lowers its intrinsic affinity for cAMP.

Agonist-induced phosphorylation of G-protein-coupled receptors has been shown to facilitate the desensitization processes, such as receptor internalization, decreased efficiency of coupling to G-proteins, or decreased ligand affinity. The lowered affinity may be an intrinsic property of the phosphorylated receptor or it may be the result of altered interactions between the modified receptor and downstream components such as G-proteins or arrestins. To address this issue, we purified cAR1, the major chemoattractant receptor of Dictyostelium discoideum by a strategy that is independent of the ligand binding capacity of the receptor. To our knowledge, this represents the first successful purification of a chemoattractant receptor. The hexyl-histidine-tagged receptor was solubilized from a highly enriched plasma membrane preparation and purified by Ni2+-chelating chromatography. The protocol offers a simple way to purify 100-500 micrograms of a G-protein coupled receptor that can be targeted to the plasma membrane of D. discoideum. The Kd value for the purified cAR1 was about 200 nM, consistent with that of receptors that are not coupled to G-proteins in intact cells. In contrast, the affinity of phosphorylated cAR1, purified from desensitized cells, was about three times lower. Treatment of the phosphorylated receptor with protein phosphatases caused dephosphorylation and parallel restoration of higher affinity. We propose that ligand-induced phosphorylation of G-protein-coupled receptors causes a decrease in intrinsic affinity and may be useful in maintaining the receptor's sensitivity at high agonist levels. This affinity decrease may precede other processes such as receptor internalization or uncoupling from G-proteins.

Alanine↗
PubMed1998

G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton.

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein beta subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type beta subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In beta subunit-null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein-actin transfected cells showed that beta subunit- null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

Actins↗
PubMed1994

Localization of ligand-induced phosphorylation sites to serine clusters in the C-terminal domain of the Dictyostelium cAMP receptor, cAR1.

When Dictyostelium cells are stimulated with cyclic adenosine 3',5'-monophosphate (cAMP), the major surface cAMP receptor expressed in early development, cAR1, undergoes a rapid phosphorylation and parallel decrease in electrophoretic mobility which may serve to regulate the activity of this G protein-coupled receptor. Biochemical analyses indicate the electrophoretic mobility shift is caused by phosphorylation of serine residues within the C-terminal cytoplasmic domain. The 18 serines of this domain are grouped in four clusters, designated 1 to 4 (in N- to C-terminal order). Two approaches were taken to determine the distribution of phosphorylation sites among the serine clusters. First, a proteolytic analysis of the C-terminal domain was performed. Second, mutants lacking various combinations of the serine clusters were created by site-directed mutagenesis and their abilities to undergo ligand-induced modification were determined. Both approaches yielded corroborative results consistent with the following model: the stimulus induces the addition of approximately two phosphates to cluster 1 and one to cluster 2; basal phosphorylation occurs predominantly in cluster 3 and to a lesser extent in cluster 2; and cluster 4 is not phosphorylated. The phosphorylation-deficient receptor mutants should be useful for establishing the role of ligand-induced phosphorylation of cAR1 in chemotaxis, cell-cell signaling, and gene expression.

Amino Acid Sequence↗
PubMed1994

CRAC, a cytosolic protein containing a pleckstrin homology domain, is required for receptor and G protein-mediated activation of adenylyl cyclase in Dictyostelium.

Adenylyl cyclase in Dictyostelium, as in higher eukaryotes, is activated through G protein-coupled receptors. Insertional mutagenesis into a gene designated dagA resulted in cells that cannot activate adenylyl cyclase, but have otherwise normal responses to exogenous cAMP. Neither cAMP treatment of intact cells nor GTP gamma S treatment of lysates stimulates adenylyl cyclase activity in dagA mutants. A cytosolic protein that activates adenylyl cyclase, CRAC, has been previously identified. We trace the signaling defect in dagA- cells to the absence of CRAC, and we demonstrate that dagA is the structural gene for CRAC. The 3.2-kb dagA mRNA encodes a predicted 78.5-kD product containing a pleckstrin homology domain, in agreement with the postulated interaction of CRAC with activated G proteins. Although dagA expression is tightly developmentally regulated, the cDNA restores normal development when constitutively expressed in transformed mutant cells. In addition, the megabase region surrounding the dagA locus was mapped. We hypothesize that CRAC acts to connect free G protein beta gamma subunits to adenylyl cyclase activation. If so, it may be the first member of an important class of coupling proteins.

Adenylyl Cyclases↗
PubMed1989

Dictyostelium discoideum: a model system for cell-cell interactions in development.

The cellular slime mold Dictyostelium discoideum undergoes a transition from single-celled amoebae to a multicellular organism as a natural part of its life cycle. A method of cell-cell signaling that controls chemotaxis, morphogenesis, and gene expression has developed in this organism, and a detailed understanding of this signaling system provides clues to mechanisms of intercellular communication in the development of metazoans.

Cell Communication↗
PubMed1989

Cell-cell interactions in Dictyostelium development.

The development of an organism requires extensive cell-cell communication; however, little is known about the signals transmitted among differentiating cells. Observations of Dictyostelium amoebae reveal that transmembrane signaling systems have been highly conserved in evolution. The signals that cause these cells to differentiate are processed by mechanisms similar to those that process sensory and hormonal stimuli in higher animals.

Cell Communication↗
PubMed1987

Purification of the surface cAMP receptor in Dictyostelium.

We have previously identified and demonstrated reversible ligand-induced modification of the major cell surface cAMP receptor in Dictyostelium discoideum. The receptor, or a subunit of it, has been purified to homogeneity by hydroxylapatite chromatography followed by two-dimensional preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification was monitored by following 32Pi incorporated by photoaffinity labeling with 8-azido-[32P]cAMP or by in vivo labeling with 32Pi. Two interconvertible forms of the receptor, designated R (Mr 40,000) and D (Mr 43,000), co-purified. Two-dimensional peptide maps of independently purified and 125I-iodinated R and D forms of the receptor were nearly identical but did have several distinct peptides. The estimated 6000-fold purification required is consistent with the number of cell surface binding sites assuming there are not multiple binding sites/polypeptide. In the accompanying article we report the generation of a monospecific polyclonal antiserum which has helped to further elucidate the physical properties and developmental regulation of the cAMP receptor.

Affinity Labels↗
PubMed1987

The surface cyclic AMP receptor in Dictyostelium. Levels of ligand-induced phosphorylation, solubilization, identification of primary transcript, and developmental regulation of expression.

A monospecific polyclonal antiserum to the surface cAMP receptor of Dictyostelium has been developed by immunization with purified receptor immobilized on particles of polyacrylamide and on nitrocellulose paper. In Western blots, the antiserum displays high affinity and specificity for both the R (Mr 40,000) and D (Mr 43,000) forms of the receptor previously identified by photoaffinity labeling with 8-azido-[32P] cAMP. These bands, labeled with the photoaffinity label or with 32 Pi, were quantitatively and specifically immunoprecipitated, supporting co-purification data that all represent the same polypeptide. The R form, found in unstimulated cells, contained at least 0.2 mol of phosphate/mol of receptor. The D form, generated by cAMP stimulation of intact cells, contained at least 4 mol of phosphate/mol of receptor. In the absence of detergents, the receptor was exclusively located on membranes. The receptor was solubilized effectively in Triton X-100 and sedimented as a broad peak of 5-7 S on sucrose velocity gradients. Western blots of membranes isolated at different times after starvation indicate that the appearance of cell surface cAMP binding sites during the aggregation stage of development (5-6 h) is due to de novo synthesis of receptor protein. Pulse labeling with [35S]methionine indicated that the receptor is most rapidly synthesized during the preaggregation stage of development (1-3 h), prior to its maximal accumulation in membranes. The serum specifically immunoprecipitates a polypeptide of Mr 37,000 from an in vitro translation reaction using RNA isolated from preaggregation stage cells. The time course of expression of the mRNA coding for the Mr 37,000 polypeptide parallels the rate of receptor synthesis in vivo.

Affinity Labels↗
PubMed1986

Cell-cell interactions in the development of Dictyostelium.

We have described in D. discoideum a highly organized cell aggregation that is mediated by cAMP. After suitable differentiation induced by starvation, the cells develop the capacity to orient in gradients of cAMP and to secrete cAMP in response to cAMP. This signaling response sets up the cell-cell relay of cAMP waves that transiently orients the cells toward the center. Both the signaling response and the chemotactic response, measured in isolated cells, adapt. The kinetics and properties of adaptation of the two responses are similar and may be due to the same mechanism. The mechanism does not involve protein synthesis, a change in the number or affinity of surface receptors, or the activation of adenylate cyclase. Adaptation of signaling is essential for the oscillatory production of cAMP at the aggregation centers and ensures that the cAMP waves move steadily toward the edge of the aggregation territories. Adaptation of the chemotactic response also ensures that cells do not reorient away from the center in the gradient presented by the trailing edge of the wave. We have demonstrated that both chemotaxis and cAMP signaling are mediated by the same surface receptor. The polypeptide containing the binding site of the receptor has been identified by photoaffinity labeling with [32P]-8-N3-cAMP as a diffuse band of 41,000-45,000 Mr. The receptor and adenylate cyclase copurify on a homogeneous class of vesicles resistant to extraction by nonionic detergents. A GTP-binding protein that is a substrate for cholera toxin-catalyzed ADP ribosylation is found in supernatants and membranes and may be similar to the Gs regulatory protein of adenylate cyclase in higher organisms. The mechanism of activation of the adenylate cyclase and chemotactic machinery is unknown. We have been able to inhibit the activation of the adenylate cyclase selectively and rapidly with agents acting to crosslink cell surface components, which may give a clue to the activation mechanism. The elaborate mechanisms of cell-cell communication occurring in D. discoideum are without precedent in biological literature, although models of oscillatory wave propagation have been proposed to account for pattern formation. Although it is unlikely that extracellular cAMP would be involved, it is not inconceivable that such mechanisms occur during the development of more evolutionarily advanced organisms. The organized communication system in D. discoideum is only apparent when cells are plated uniformly on a flat surface; such organized movements occurring in a three-dimensional structure such as an embryo would be very difficult to discern.

Cyclic AMP↗
PubMed1986

The specificity of the cAMP receptor mediating activation of adenylate cyclase in Dictyostelium discoideum.

In Dictyostelium discoideum amoebae, binding of cyclic AMP (cAMP) to surface receptors elicits numerous responses including chemotaxis, cyclic GMP (cGMP) accumulation, and activation of adenylate cyclase. The specificity of the surface cAMP receptor which mediates activation of adenylate cyclase and cAMP secretion was determined by testing the relative effectiveness of a series of 10 cAMP analogs. Each of the 10 analogs elicited cAMP secretion, chemotaxis, and cGMP accumulation in the same dose range. The order of potency for eliciting these responses (cAMP greater than 2'-H-cAMP greater than N1-O-cAMP greater than cAMPS(Sp) greater than 6-Cl-cAMP greater than cAMPN(CH3)2(Sp) greater than 3'-NH-cAMP greater than 8-Br-cAMP greater than cAMPS(Rp) greater than cAMPN(CH3)2(Rp] matches that for binding to the major cell surface cAMP binding sites and differs from that of the cell surface phosphodiesterase and the major intracellular cAMP binding protein.

Adenylyl Cyclases↗
PubMedDate not supplied

Phenotypic changes induced by a mutated ras gene during the development of Dictyostelium transformants.

The ras proto-oncogene, found in all eukaryotes so far examined, encode s a protein with guanine nucleotide-binding and GTPase activity. Gene disruption experiments in yeast indicate that ras is essential for cell growth. Anit-sense mutagenesis approaches suggest that this is also true for Dictyostelium. Most mutations causing an amino-acid substitution for Gly 12 result in decreased GTPase activity and produce a transforming phenotype. In yeast, a Gly 19---- Val 19, missense mutation (Gly 19 is similar to Gly 12 in mammalian and Dictyostelium ras proteins) causes a series of dominant phenotypes, including elevated adenylate cyclase activity. In mammalian cells there is no evidence that ras activates adenylate cyclase activity. D. discoideum contains a single ras gene (Dd-ras) that encodes a protein very similar to the mammalian ras protein and identical to c-ras at the potentially transforming positions. Dd-ras is expressed in vegetative cells and later in development in prestalk cells whereas ras protein is found in vegetative and developing cells. In the migrating pseudoplasmodium, ras protein is found in prestalk but not prespore cells, suggesting it is involved in the function and/or differentiation of the anteriorly localized prestalk cells. In this report we examine the effects of expression of a Dd-ras gene carrying a Gly-12----Thr 12 missense mutation.

Adenylyl Cyclases↗