PubMed Health⌕ Search

Biomedical subjects

P N Devreotes

Publications and source records attributed to P N Devreotes.

At least 91 records · Page 5Linked to original sources
PubMed1989

G-protein-linked signal transduction systems control development in Dictyostelium.

G-protein-linked cAMP receptors play an essential role in Dictyostelium development. The cAMP receptors are proposed to have seven transmembrane domains and a cytoplasmic C-terminal region. Overexpression of the receptor in cells, when the endogenous receptor is not present, results in a 10- to 50-fold increase in cAMP-binding sites. Antisense cell lines, which lack cAMP receptors, do not enter the developmental program. Ligand-induced phosphorylation is proposed to occur on serine and threonine residues in the receptor C-terminus. The kinetics of receptor phosphorylation and dephosphorylation correlate closely with the shift of receptor mobility and the adaptation of several cAMP-induced responses. Two alpha-subunits, G-alpha-1 and G-alpha-2, have been cloned and specific antisera developed against each. Both subunits are expressed as multiple RNAs with different developmental time courses. The mutant Frigid A has a functional defect in G-alpha-2 which prevents it from entering development. We propose that G-protein-linked receptor systems will be a major component in the development of many organisms.

Amino Acid Sequence↗
PubMed1988

Ligand-induced phosphorylation of the cAMP receptor from Dictyostelium discoideum.

The cell surface cAMP receptor of Dictyostelium discoideum exists as a doublet of low (D) and high (R) electrophoretic mobility forms, both of which are phosphorylated in vivo. The R form is phosphorylated in a ligand-independent manner, while conversion of the R to D forms, induced by the chemoattractant, is accompanied by at least a 4-fold increase in the level of phosphorylation. When cells are stimulated with saturating levels of cAMP, increased phosphorylation is detectable within 5 s and reaches maximum levels by 5 min with a t1/2 of 45 s. Dephosphorylation of receptor, initiated by removal of the stimulus, is detectable within 30 s, has a half-time of 2 min, and reaches a plateau by 20 min. At half-maximal occupancy, phosphorylation occurred more slowly than at saturation, t1/2 = 1.5 min, and remained at intermediate levels until the cAMP concentration was increased. Accompanying electrophoretic mobility shifts occurred in all cases with similar, though not identical, kinetics. Both phosphorylation and mobility shift were half-maximal at 5 nM cAMP and saturated at 100 nM. Estimation of the specific activity of each receptor form indicates that not all sites are phosphorylated during the R to D transition; at least half of the sites are phosphorylated after the transition is completed. The rate of incorporation of phosphates into the receptor, held in the D form by cAMP, was less than one-third the rate of ligand-induced incorporation starting with the R form and was approximately twice the basal rate of incorporation. These results are compatible with ligand-induced receptor phosphorylation being an early event in the adaptation of other cAMP-induced responses.

Cyclic AMP↗
PubMed1988

A chemoattractant receptor controls development in Dictyostelium discoideum.

During the early stages of its developmental program, Dictyostelium discoideum expresses cell surface cyclic adenosine monophosphate (cyclic AMP) receptors. It has been suggested that these receptors coordinate the aggregation of individual cells into a multicellular organism and regulate the expression of a large number of developmentally regulated genes. The complementary DNA (cDNA) for the cyclic AMP receptor has now been cloned from lambda gt-11 libraries by screening with specific antiserum. The 2-kilobase messenger RNA (mRNA) that encodes the receptor is undetectable in growing cells, rises to a maximum at 3 to 4 hours of development, and then declines. In vitro transcribed complementary RNA, when hybridized to cellular mRNA, specifically arrests in vitro translation of the receptor polypeptide. When the cDNA is expressed in Dictyostelium cells, the undifferentiated cells specifically bind cyclic AMP. Cell lines transformed with a vector that expresses complementary mRNA (antisense) do not express the cyclic AMP receptor protein. These cells fail to enter the aggregation stage of development during starvation, whereas control and wild-type cells aggregate and complete the developmental program within 24 hours. The phenotype of the antisense transformants suggests that the cyclic AMP receptor is essential for development. The deduced amino acid sequence of the receptor reveals a high percentage of hydrophobic residues grouped in seven domains, similar to the rhodopsins and other receptors believed to interact with G proteins. It shares amino acid sequence identity and is immunologically cross-reactive with bovine rhodopsin. A model is proposed in which the cyclic AMP receptor crosses the bilayer seven times with a serine-rich cytoplasmic carboxyl terminus, the proposed site of ligand-induced receptor phosphorylation.

Amino Acid Sequence↗
PubMed1988

Ligand-induced modification of a surface cAMP receptor of Dictyostelium discoideum does not require its occupancy.

In Dictyostelium discoideum amoebae, cAMP-induced phosphorylation of the surface cAMP receptor is associated with a discrete transition in its electrophoretic mobility. The native and modified forms of the receptor are designated R and D (Mr = 40,000 and 43,000). The relationship of the number of receptors which are modified as a function of the receptors which bind cAMP was investigated. Modification was assessed by determining the amounts of R and D forms in Western blots which detect all receptors whether or not they are exposed on the surface. Cyclic AMP or the analog, adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS), induced a loss of cAMP-binding activity (down-regulation), which was not accompanied by a loss of the receptor protein. About 60% of the receptors do not bind cAMP in the absence of Ca2+ and are unmasked by 10 mM Ca2+. However, the fraction of receptors which are modified in response to cAMP is equal in the absence or presence of Ca2+. (Rp)-cAMPs induces down-regulation (50%) but not modification. Addition of cAMP, following down-regulation by (Rp)-cAMPS, causes all receptors to be modified. cAMP induces both down-regulation (80%) and modification. Modification is more readily reversed than down-regulation: 30 min after removal of cAMP, receptors remain down-regulated (57%) but are found in the R form. All receptors shift to the D form when cAMP is readded to the cells. These results indicate that exposed, as well as cryptic and down-regulated receptors, are modified in response to the cAMP stimulus.

Calcium↗
PubMed1988

Structure and expression of the cAMP cell-surface receptor.

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.

Amino Acid Sequence↗
PubMed1988

Localization of chemoattractant receptors on Dictyostelium discoideum cells during aggregation and down-regulation.

cAMP chemoattractant receptors on the surface of Dictyostelium discoideum cells are visualized by means of immunocytochemistry. Receptor antigen is virtually absent from growing cells and begins to accumulate after about 6 hr of starvation, concomitant with the increase in surface cAMP binding activity. In aggregating cells, the antigen is uniformly distributed over the cell surface. Persistent cAMP stimulation, which leads to down-regulation of cAMP binding activity, induces a striking rearrangement of receptor antigen into patches or internal vesicles. A similar patching of receptor antigen is observed during tight aggregate formation, when surface cAMP binding activity decreases. These observations indicate that receptor down-regulation involves receptor agglomeration and suggest that receptor down-regulation takes place in vivo, when tight aggregates are being formed.

Cell Aggregation↗
PubMed1987

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity.

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.

Amino Acids↗
PubMed1986

Surface receptor-mediated activation of adenylate cyclase in Dictyostelium. Regulation by guanine nucleotides in wild-type cells and aggregation deficient mutants.

GTP and GTP analogs produced significant (up to 17-fold) and persistent activation of adenylate cyclase in lysates of Dictyostelium discoideum amoeba. The activation was enhanced 2- to 4-fold by cAMP (the agonist for receptor-mediated adenylate cyclase activation), was specific for guanine nucleoside triphosphates, and was inhibited by guanosine 5'-(O-2-thio)diphosphate. The order of potency of guanine nucleotides was guanosine 5'-(O-3-thio)triphosphate greater than guanyl-5'-yl imidodiphosphate greater than GTP; half-maximal activation was observed with 1-10 microM guanine nucleotide. Maximal activation occurred when the guanine nucleotide was added within seconds after cell lysis and the lysate was preincubated for 5 min prior to assay. Under these optimal in vitro conditions, the capacity of guanine nucleotides to activate decreased, closely correlating with adaptation or desensitization induced by exposure of intact cells to cAMP during a period of 10 min. These data strongly support that regulation of adenylate cyclase in Dictyostelium occurs via a receptor-linked GTP/GDP exchange protein. Two mutants, designated synag 7 and 49 were isolated in which cAMP and/or guanine nucleotides were not sufficient to activate adenylate cyclase. The wild-type pattern of guanine nucleotide regulation was restored to synag 7 lysates by the addition of a high-speed supernatant from wild-type cells. Characterization of these mutants demonstrates that activation of adenylate cyclase is not required for growth or cell-type specific differentiation but is essential for cellular aggregation and influences morphogenesis and pattern formation. This suggests that Dictyostelium may provide a model suitable for detailed genetic analysis of surface receptor-guanine nucleotide-binding regulatory protein linked adenylate cyclase systems and for determining the role of these systems in development.

ADP-Ribosylation Factors↗
PubMed1986

A mechanism for exact sensory adaptation based on receptor modification.

We provide a theoretical explanation for the observation that in many sensory systems a step increase in stimulus triggers a response that goes through a maximum and then returns to the basal level. Considered here is a receptor molecule that in the absence of ligand can be found in either of two states R and D. Two more states, RL and DL, are formed upon the addition of ligand L. It is assumed that the receptor triggers activity in a sensory system, and that the activity is proportional to a weighted combination of the fractions of molecules that are in each of the four states. It is shown that judicious choice of the weights can provide both an adequate response and exact adaptation to step increases in stimuli. The interconversion between states may operate without energy expenditure or through covalent modification. In both cases, adaptation is associated with receptor modification that acts as a counterweight to changed external conditions. Application to cAMP secretion in Dictyostelium discoideum and to chemotaxis in bacteria is discussed.

Adaptation, Physiological↗
PubMed1986

A molecular mechanism for sensory adaptation based on ligand-induced receptor modification.

Physiological responses mediated by cell-surface receptors frequently adapt or "desensitize" (i.e., terminate despite persistent occupancy of receptors by ligand). Binding of ligands to the external domains of a wide variety of surface receptors induces covalent modification of their cytoplasmic domains. A mechanism is presented in which the variety of receptor states generated by ligand binding and covalent modification act together to regulate physiological responsiveness. The development of the model is guided by observations of adaptation for chemotaxis in Escherichia coli and adenylate cyclase activation in Dictyostelium. The general features of the marked response and eventual exact adaptation predicted by the model match those observed in the experimental systems.

Adaptation, Physiological↗
PubMed1985

Kinetics and concentration dependence of reversible cAMP-induced modification of the surface cAMP receptor in Dictyostelium.

We have previously reported that extracellular cAMP induced a reversible shift, from apparent Mr = 40,000 to 43,000, in the electrophoretic mobility of a polypeptide identified by photoaffinity labeling with [32P]8-N3-cAMP as the cAMP receptor of Dictyostelium (Klein, P., Theibert, A., Fontana, D., and Devreotes, P. (1985) J. Biol. Chem. 260, 1757-1764). In this report, we examine the kinetics and concentration dependence of this stimulus-induced receptor modification. Prior to stimulation, 90% of the receptors migrated as the higher mobility form (Mr = 40,000) and 10% as the lower mobility form (Mr = 43,000). Following 15 min of persistent stimulation with 1 microM cAMP, the per cent of receptors migrating as the lower mobility form rose to 80%. This transition occurred with a half-time of 2.5 min. Removal of the stimulus initiated a return to the basal state which occurred with a half-time of about 6 min at 22 degrees C. No reversal occurred at 0 degrees C. Addition and removal of a 50 nM cAMP stimulus induced transitions with similar kinetics, but the final plateau value reached was only 40% lower mobility form. The stimulus concentration which induced 50% of the maximal transition from higher to lower mobility forms at steady state was 27 nM, similar to the KD for [3H]cAMP binding. Scatchard analysis of [3H]cAMP binding indicated that, although a 20% down-regulation occurs during cAMP stimulation, there is no significant difference in the affinities of the higher and lower mobility forms of the receptor. The unoccupied higher and lower mobility forms of the receptor, designated R and D, are considered to be in rapid equilibrium with liganded forms, designated RL and DL. The rate constants for interconversion of the receptor forms R (Formula: see text) D and RL (Formula: see text) DL were calculated from the kinetic data: k1 = 0.012, k-1 = 0.104, k2 = 0.222, and k-2 = 0.055. The interconversion steps are not at equilibrium, suggesting that an energy expenditure occurs during the receptor modification. The pattern of modulation of the cAMP-induced receptor modification suggests that it may be the biochemical mechanism of adaptation.

Adaptation, Physiological↗
PubMed1985

Identification and cyclic AMP-induced modification of the cyclic AMP receptor in Dictyostelium discoideum.

We have recently identified a cell surface cAMP-binding protein by specific photoaffinity labeling of intact Dictyostelium discoideum cells with 8-N3-[32P] cAMP. The major photolabeled protein appears as a doublet (Mr = 40,000-43,000) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. In this study, the doublet is shown to have the characteristics of the cAMP receptor responsible for chemotaxis and cAMP signaling. Both specific photoaffinity labeling of the doublet and binding of 8-N3-[32P]cAMP are saturable (KD = 0.3 microM), the levels of both peak at 5 h, and both are inhibited by cAMP and several cAMP analogs in the same order of potency and with K1 values similar to those measured for inhibition of [3H]cAMP binding. When cAMP-binding activity was partially purified (40-fold) and then photoaffinity labeled, the same bands (Mr = 40,000-43,000) were observed. The relative intensities of the upper and lower bands of the doublet alternated at the same frequency as the spontaneous oscillations in cAMP synthesis. When oscillations were suppressed, the lower band of the doublet predominated. Following addition of cAMP, the relative intensity gradually shifted to the upper band. When cAMP was removed, there was a gradual restoration of the lower band form. We propose that the lower band form of the receptor activates chemotaxis and cAMP signaling and that the upper band form does not. This reversible receptor modification may then be the mechanism of adaptation, the process by which the physiological responses cease to be stimulated by persistent cAMP. Several developmentally regulated genes in D. discoideum have been reported to be induced or suppressed by pulses of cAMP (adaptive regulation) and others by continuous cAMP (nonadaptive regulation). These observations may be explained by the receptor modification reported here if the two forms of the receptor, which bind cAMP with the same affinity, independently influence gene expression.

Affinity Labels↗
PubMed1985

Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium.

Cyclic AMP stimulation of chemotactically competent Dictyostelium amebas labeled with [32P]orthophosphate transiently increases phosphorylation in the heavy chain and the 18,000 dalton light chain of myosin. Immediately before the increase, heavy chain phosphorylation transiently decreases. These phosphorylation changes also occur when cAMP-induced activation of adenylate cyclase is blocked by pretreatment of amebas with caffeine. The time course of these phosphorylation responses correlates with the shape changes induced in amebas exposed to a temporal increase in cAMP concentration. The dose dependence of the phosphorylation responses is the same as that previously determined for chemotaxis. The phosphorylation responses exhibit adaptation properties in common with those of the shape change response and chemotaxis. Increases in the rate of myosin heavy chain and light chain phosphorylation can be observed in vitro by stimulating unlabeled amebas with cAMP and then lysing the cells into a gamma-[32P]ATP-containing reaction mixture.

Adaptation, Physiological↗
PubMed1984

Specific photoaffinity labeling of the cAMP surface receptor in Dictyostelium discoideum.

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.

Azides↗
PubMed1984

cAMP-stimulated adenylate cyclase activation in Dictyostelium discoideum is inhibited by agents acting at the cell surface.

The ability of Dictyostelium discoideum amoebae to synthesize and secrete cAMP in response to exogenous cAMP is called cAMP signaling. Concanavalin A is a potent, rapid, noncompetitive inhibitor of this response, with the rate of inhibition consistent with its rate of binding. The concanavalin A does not deplete cellular ATP, alter cAMP binding to its surface receptors, or affect basal adenylate cyclase activity, but blocks the cAMP-stimulated activation of adenylate cyclase. Therefore, concanavalin A appears to inhibit a step between the receptor and the adenylate cyclase which is necessary for the transduction of the cAMP signal. Wheat germ agglutinin, a polyclonal antibody against an 80-kDa glycoprotein, four monoclonal antibodies against the amoebal surface, and a chemical cross-linking agent which reacts with cell surface primary amines also inhibit signaling. To determine the importance of cross-linking in the inhibition, succinylated concanavalin A and the unlinked, reactive portion of the chemical cross-linker were tested and found to be relatively ineffective inhibitors. Thus it appears that ligands capable of cross-linking molecules on the external surface of D. discoideum amoebae inhibit cAMP signaling. It is proposed that these cross-linking agents prevent membrane or cytoskeletal rearrangement and that this rearrangement must occur before the adenylate cyclase is activated.

Adenylyl Cyclases↗