Biomedical subjects

P N Devreotes

Publications and source records attributed to P N Devreotes.

At least 55 records · Page 3Linked to original sources

PubMed1995

Seven helix cAMP receptors stimulate Ca2+ entry in the absence of functional G proteins in Dictyostelium.

Surface cAMP receptors (cARs) in Dictyostelium transmit a variety of signals across the plasma membrane. The best characterized cAR, cAR1, couples to the heterotrimeric guanine nucleotide-binding protein (G protein) alpha-subunit G alpha 2 to mediate activation of adenylyl and guanylyl cyclases and cell aggregation. cAR1 also elicits other cAMP-dependent responses including receptor phosphorylation, loss of ligand binding (LLB), and Ca2+ influx through a G alpha 2-independent pathway that may not involve G proteins. Here, we have expressed cAR1 and a related receptor, cAR3, in a g beta- strain (Lilly, P., Wu. L., Welker, D. L., and Devreotes, P. N. (1993) Genes & Dev. 7,986-995), which lacks G protein activity. Both cell lines failed to aggregate, a process requiring the G alpha 2 and G beta- subunits. In contrast, cAR1 phosphorylation in cAR1/g beta- cells showed a time course and cAMP dose dependence indistinguishable from those of cAR1/G beta+ controls. cAMP-induced LLB was also normal in the cAR1/g beta- cells. Finally, cAR1/g beta- cells and cAR3/g beta- cells showed a Ca2+ response with kinetics, agonist dependence, ion specificity, and sensitivity to depolarization agents that were like those of G beta+ controls, although they accumulated fewer Ca2+ ions per cAMP receptor than the control strains. Together, these results suggest that the G beta-subunit is not required for the activation or attenuation of cAR1 phosphorylation, LLB, or Ca2+ influx. It may, however, serve to amplify the Ca2+ response, possibly by modulating other intracellular Ca2+ signal transduction pathways.

Animals↗

PubMed1995

Occupancy of the Dictyostelium cAMP receptor, cAR1, induces a reduction in affinity which depends upon COOH-terminal serine residues.

Many G-protein-coupled receptors display a rapid decrease in ligand binding following pretreatment with agonist. cAR1, a cAMP receptor expressed early in the developmental program of Dictyostelium, mediates chemotaxis, activation of adenylyl cyclase, and gene expression changes that bring about the aggregation of 10(5) amoebae to form a multicellular structure. Occupancy of cAR1 by cAMP initiates multiple desensitization processes, one of which is an apparent reduction in binding sites. In transformed cells expressing cAR1 constitutively, Scatchard analyses revealed that this apparent loss of ligand binding is largely due to a significant reduction in the affinity of cAR1 for cAMP. A parallel increase in the dose dependence of cAR1-mediated cAMP uptake was observed. Consistent with these findings, proteolysis of intact cells and immunofluorescence suggested that cAR1 remains on the cell-surface following cAMP treatment. Finally, agonist-induced loss of ligand binding is impaired in cAR1 mutants lacking a cluster of cytoplasmic serine residues, which are targets of cAMP-induced phosphorylation.

Amino Acid Sequence↗

PubMed1995

Chemoattractant and GTP gamma S-mediated stimulation of adenylyl cyclase in Dictyostelium requires translocation of CRAC to membranes.

We have previously reported that activation of adenylyl cyclase by chemoattractant receptors in Dictyostelium requires, in addition to a heterotrimeric G-protein, a cytosolic protein, designated CRAC (Lilly, P., and P. N. Devreotes. 1994. J. Biol. Chem. 269:14123-14129; Insall, R. H., A. Kuspa, P. J. Lilly, G. Schaulsky, L. R. Levin, W. F. Loomis, and P. N. Devreotes. 1994. J. Cell Biol. 126:1537-1545). In this report, we show that in intact cells, chemoattractants promote translocation of CRAC from the cytosolic to the membrane fraction. However, CRAC is not required at the time of receptor stimulation; it can be added to lysates of activated cells. Treatment of membranes with guanine nucleotides creates binding sites for CRAC. These binding sites can be generated in mutants lacking each of the components of the pathway except the beta-subunit, suggesting that free or "activated" beta gamma-subunits may be a part of the binding site. This hypothesis is consistent with previous observations that CRAC contains a pleckstrin homology domain and that the beta gamma-subunits likely mediate activation of adenylyl cyclase in this system. Thus, CRAC may serve as an adapter, linking the G-protein beta gamma-subunits to activation of the enzyme. GTP gamma S cannot generate CRAC-binding sites when the adenylyl cyclase pathway has been adapted by prior chemoattractant stimulation, suggesting that this is a point of downstream adaptation.

Adenylyl Cyclases↗

PubMed1995

The G protein beta subunit is essential for multiple responses to chemoattractants in Dictyostelium.

Increasing evidence suggests that the beta gamma-subunit dimers of heterotrimeric G proteins play a pivotal role in transducing extracellular signals. The recent construction of G beta null mutants (g beta-) in Dictyostelium provides a unique opportunity to study the role of beta gamma dimers in signaling processes mediated by chemoattractant receptors. We have shown previously that g beta- cells fail to aggregate; in this study, we report the detailed characterization of these cells. The g beta- cells display normal motility but do not move towards chemattractants. The typical GTP-regulated high affinity chemoattractant-binding sites are lost in g beta- cells and membranes. The g beta- cells do not display chemoattractant-stimulated adenylyl cyclase or guanylyl cyclase activity. These results show that in vivo G beta links chemoattractant receptors to effectors and is therefore essential in many chemoattractant-mediated processes. In addition, we find that G beta is required for GTP gamma S stimulation of adenylyl cyclase activity, suggesting that the beta gamma-dimer activates the enzyme directly. Interestingly, the g beta- cells grow at the same rate as wild-type cells in axenic medium but grow more slowly on bacterial lawns and, therefore, may be defective in phagocytosis.

Adenylyl Cyclases↗

PubMed1994

Random chimeragenesis of G-protein-coupled receptors. Mapping the affinity of the cAMP chemoattractant receptors in Dictyostelium.

G-protein-coupled receptors mediate a wide variety of responses to extracellular stimuli in eucaryotic cells. Binding of the ligand to these receptors is thought to involve contacts within a pocket from the sequences of these genes. A family of four surface cAMP receptors that mediate responses to secreted cAMP coordinates the developmental program of Dictyostelium. A large difference in affinity for cAMP exists between cAR1 (25 and 230 nM) and cAR2 (> 5 microns). To understand the basis for this affinity difference, we generated an extensive series of cAR1/cAR2 and cAR2/cAR1 chimeras using a technique designated "random chimeragenesis." When a linearized plasmid was transformed into Escherichia coli, tandemly positioned cAR1 and cAR2 genes crossed over at homologous regions. The cAMP binding properties and EC50 values for agonist-induced phosphorylation of each of the chimeras were characterized in order to map the domains that determine the affinity. These studies implicated a domain in the second extracellular loop in which only 5 residues differ between the two receptors as the major determinant of affinity. A secondary domain including residues 110-147 (11 residue differences) was identified as a minor determinant of affinity.

Animals↗

PubMed1994

Serine 113 is the site of receptor-mediated phosphorylation of the Dictyostelium G protein alpha-subunit G alpha 2.

The G protein alpha-subunit G alpha 2 is essential to the developmental program of Dictyostelium. G alpha 2 is transiently phosphorylated on a serine residue(s) following stimulation with extracellular cAMP (Gundersen, R. E., and Devreotes, P.N. (1990) Science 248, 591-593). To aid in defining the function of alpha-subunit phosphorylation, we identified the site of G alpha 2 phosphorylation. Comparison of the isoelectric points (pI) of the phosphorylated and nonphosphorylated forms indicated that a single mole of phosphate is added to G alpha 2. Cleavage at tryptophan residues and immunoprecipitation with a specific peptide antibody localized the phosphorylated serine in the N-terminal 119 residues. Analysis of a series of G alpha 1 and G alpha 2 chimeras further confined the site between amino acids 33 and 215. Site-directed mutagenesis of serines between amino acids 33 and 119 produced two mutants that were not phosphorylated, S45A and S113A. Ser113 was identified as the site by sequential Edman degradation of 32P-radiolabeled G alpha 2 digested with endoproteinase Glu-C. We have expressed the G alpha 2 mutants S113A, S113I, S113T, and S113D in a G alpha 2 null cell line to examine the function of phosphorylation.

Amino Acid Sequence↗

PubMed1994

Identification of CRAC, a cytosolic regulator required for guanine nucleotide stimulation of adenylyl cyclase in Dictyostelium.

As previously reported, guanine nucleotide regulation of adenylyl cyclase activity in the Dictyostelium mutant synag 7 can be restored in vitro by addition of a high-speed supernatant prepared from wild-type cells (Theibert, A., and Devreotes, P. N. (1986) J. Biol. Chem. 261, 15121-15125). We have designated this activity CRAC, for cytosolic regulator of adenylyl cyclase. Trypsinization of partially purified material demonstrated that this activity contains a protein. We report here a 50,000-fold purification of this protein using Q and S Sepharose fast flow and P11 cellulose chromatography, followed by sucrose gradient centrifugation and separation on SDS-polyacrylamide gel electrophoresis. Purification of wild-type and mutant supernatants in parallel allowed identification of an 88-kDa protein required for reconstituting activity. A polyclonal antibody was raised against this protein; it stains a band in unfractionated wild-type, but not mutant, supernatants. Immunoblots of fractions from each purification step show that activity and the immunostaining band cochromatograph. We have determined a short N-terminal sequence of the 88-kDa CRAC polypeptide, which matches a portion of the deduced N terminus of a gene, dagA, isolated from a mutant similar to synag 7. Expression of the dagA cDNA in synag 7 cells restores both the 88 kDa band and CRAC activity.

Adenylyl Cyclases↗

PubMed1994

Mutation of the third intracellular loop of the cAMP receptor, cAR1, of Dictyostelium yields mutants impaired in multiple signaling pathways.

Seven-membrane span receptors transduce a wide range of signals across the plasma membrane. One member of this family, the cAMP receptor, cAR1, of Dictyostelium, mediates some responses (e.g. adenylyl cyclase activation, multicellular aggregation) which require G-proteins and others (e.g. Ca2+ influx, loss of ligand binding, cAR1 phosphorylation) which appear to be G-protein-independent. In this study, we randomly mutagenized the NH2-terminal eight amino acids of the third intracellular loop of cAR1 and examined the ability of these mutants to exhibit the three G-protein-independent responses listed above. Most mutants (classes I, II) exhibited wild-type or midly defective responses. Several mutants (class III), however, were severely impaired in all three processes but not in cAMP binding. Furthermore, these mutants failed to couple productively with G-proteins and could not replace cAR1 in a car1- cell. For these reasons, we propose that class III mutations interfere with the formation of an "active" conformation of the receptor.

Amino Acid Sequence↗

PubMed1994

G protein-linked signaling pathways control the developmental program of Dictyostelium.

The similarity of the signal transduction systems controlling early development in Dictyostelium with those mediating the action of hormones and neurotransmitters in mammals suggests that these strategies were quickly refined as eukaryotic cells began to communicate. These simple, genetically tractable organisms thus offer a great opportunity to elucidate these pathways further. Combinations of the null mutants are being studied to address questions of redundancy, cross-talk, and networking. Since cAR1, cAR2, G alpha 2, G beta, ACA, CRAC, PKA, and PDE are essential to the program, the capacity to rescue these phenotypes also serves as a convenient screen for functional mutations in these proteins. Finally, random mutagenesis by the recently developed method of restriction enzyme-mediated insertion provides a means to isolate new genes (Kuspa et al., 1992). The clear phenotypes of the null mutants observed so far indicate that the Dictyostelium developmental program can be used as a guide to isolate novel components of G protein-linked pathways.

Animals↗

PubMed1994

Cloning and targeted mutations of G alpha 7 and G alpha 8, two developmentally regulated G protein alpha-subunit genes in Dictyostelium.

GTP-binding protein (G protein)-mediated signal transduction pathways play essential roles during the aggregation and differentiation process of Dictyostelium. In addition to the five known G protein alpha-subunit genes, we recently identified three novel alpha-subunit genes, G alpha 6, G alpha 7, and G alpha 8, using the polymerase chain reaction technique. We present here a more complete analysis of G alpha 7 and G alpha 8. The cDNAs of these two genes were cloned, and their complete nucleotide sequences were determined. Sequence analyses indicate that G alpha 8 possesses some unusual features. It lacks the "TCATDT" motif, a sequence of amino acids highly conserved among G alpha subunits, and has an additional 50 amino acids at its C-terminus consisting of long stretches of asparagine. Moreover, G alpha 8 is unusually resistant to protease digestion, which may indicate a slow GTP hydrolysis rate. The possible functions of these alpha-subunits were assessed by generating mutants lacking G alpha 7 or G alpha 8 by gene targeting through homologous recombination and by overexpressing G alpha 7 or G alpha 8 protein. Overexpression of G alpha 7 resulted in abnormal morphogenesis starting at the slug stage, whereas analysis of the other strains failed to reveal any obvious growth or developmental defects under either normal or stressful conditions. The implications of these results are discussed.

Amino Acid Sequence↗

PubMed1994

Two cAMP receptors activate common signaling pathways in Dictyostelium.

Multiple signal transduction pathways within a single cell may share common components. In particular, seven different transmembrane helix receptors may activate identical pathways by interacting with the same G-proteins. Dictyostelium cells respond to cAMP using one such receptor, cAR1, coupled by a typical heterotrimeric G-protein to intracellular effectors. However, cells in which the gene for cAR1 has been deleted are unexpectedly still able to respond to cAMP. This implies either that certain responses are mediated by a different receptor than cAR1, or alternatively that a second, partially redundant receptor shares some of the functions of cAR1. We have examined the dose response and ligand specificity of one response, cAMP relay, and the dose response of another, cyclic GMP synthesis. In each case, the EC50 was approximately 100-fold higher and the maximal response was smaller in car1- than wild-type cells. These data indicate that cAR1 normally mediates responses to cAMP. The ligand specificity suggests that the responses seen in car1- mutants are mediated by a second receptor, cAR3. To test this hypothesis, we constructed a cell line containing deletions of both cAR1 and cAR3 genes. As predicted, these lines are totally insensitive to cAMP. We conclude that the functions of the cAR1 and cAR3 receptors are partially redundant and that both interact with the same heterotrimeric G-protein to mediate these and other responses.

Adenylyl Cyclases↗

PubMed1994

Extracellular cAMP can restore development in Dictyostelium cells lacking one, but not two subtypes of early cAMP receptors (cARs). Evidence for involvement of cAR1 in aggregative gene expression.

Extracellular cAMP induces expression of several classes of developmentally regulated genes in Dictyostelium. Four highly homologous surface cAMP receptors (cARs) were identified earlier, but involvement of specific cARs in gene regulation has not been clarified. Cells lacking the chemotactic receptor, cAR1, neither aggregate nor express developmentally regulated genes. Expression of aggregative genes is in wild-type cells induced by nanomolar cAMP pulses and repressed by persistent micromolar cAMP stimuli, which induce expression of prespore and prestalk-enriched genes during the postaggregative stages of development. We show here that in cell lines carrying a cAR1 gene disruption, nanomolar pulses cannot induce aggregative gene expression. Remarkably, micromolar cAMP can induce expression of aggregative genes in car1- cells as well as expression of prespore and prestalk-enriched genes, and furthermore restores their ability to form normal slugs and fruiting bodies. These data indicate that cAR1 mediates aggregative but not postaggregative gene expression and morphogenesis, and suggest that after gene disruption, its function is partially taken over by a lower affinity receptor that is not subjected to desensitization. The absence of another early cAMP receptor, cAR3, does not affect development. However, in a car1-/car3- double mutant, cAMP stimulation cannot restore any developmental gene expression, indicating that cAR3 may have substituted for cAR1 in car1- cell lines.

Animals↗

PubMed1993

The surface cyclic AMP receptors, cAR1, cAR2, and cAR3, promote Ca2+ influx in Dictyostelium discoideum by a G alpha 2-independent mechanism.

Activation of surface folate receptors or cyclic AMP (cAMP) receptor (cAR) 1 in Dictyostelium triggers within 5-10 s an influx of extracellular Ca2+ that continues for 20 s. To further characterize the receptor-mediated Ca2+ entry, we analyzed 45Ca2+ uptake in amoebas overexpressing cAR2 or cAR3, cARs present during multicellular development. Both receptors induced a cAMP-dependent Ca2+ uptake that had comparable kinetics, ion selectivity, and inhibitor profiles as folate- and cAR1-mediated Ca2+ uptake. Analysis of mutants indicated that receptor-induced Ca2+ entry does not require G protein alpha subunits G alpha 1, G alpha 2, G alpha 3, G alpha 4, G alpha 7, or G alpha 8. Overexpression of cAR1 or cAR3 in g alpha 2- cells did not restore certain G alpha 2-dependent events, such as aggregation, or cAMP-mediated activation of adenylate and guanylate cyclases, but these strains displayed a cAMP-mediated Ca2+ influx with kinetics comparable to wild-type aggregation-competent cells. These results suggest that a plasma membrane-associated Ca(2+)-influx system may be activated by at least four distinct chemoreceptors during Dictyostelium development and that the response may be independent of G proteins.

Animals↗

PubMed1993

Extracellular cAMP is sufficient to restore developmental gene expression and morphogenesis in Dictyostelium cells lacking the aggregation adenylyl cyclase (ACA).

Cell movement and cell-type-specific gene expression during Dictyostelium development are regulated by cAMP, which functions both as an extracellular hormone-like signal and an intracellular second messenger. Previous data indicated that aca- mutants, which lack adenylyl cyclase activity, fail to aggregate and do not express cell-type-specific genes. We show here that overexpression of ACG, a constitutively active adenylyl cyclase, which in wild-type cells is only expressed during spore germination, partially restores the coordination of cell movement and completely restores developmental gene expression. The aca- cells can also be induced to develop into viable spores by synergy with wild-type cells and, furthermore, form small but normal fruiting bodies, after a developmentally relevant regimen of stimulation with nanomolar cAMP pulses followed by micromolar cAMP concentrations. 2'-Deoxy cAMP, a cAMP analog that activates the cell-surface cAMP receptors but not cAMP-dependent protein kinase (PKA), also induces fruiting body formation as well as expression of prespore-specific and prestalk-enriched genes in aca- cells. Intracellular cAMP levels were not altered in aca- cells after stimulation with 2'-deoxy cAMP. Our data indicate that ACA is not required to provide intracellular cAMP for PKA activation but is essential to produce extracellular cAMP for coordination of cell movement during all stages of development and for induction of developmental gene expression.

Adenylyl Cyclases↗

PubMed1993

CAR2, a prestalk cAMP receptor required for normal tip formation and late development of Dictyostelium discoideum.

Extracellular cAMP serves as a primary signaling molecule to regulate the development of Dictyostelium discoideum. It is required for chemotaxis, aggregation, cytodifferentiation, and morphogenetic movement. The receptors for cAMP are members of the family of cell-surface receptors that are linked to G proteins and characterized by seven putative transmembrane domains. Previously, we have isolated the gene for the cAMP receptor subtype 1 (CAR1) from Dictyostelium and suggested that several genes related to CAR1 were present in the genome. Here, we describe a family of cAMP receptor genes of Dictyostelium and the isolation and function of the gene for the cAMP receptor subtype 2, CAR2. CAR2 is structurally similar to CAR1. Overall, their transmembrane and loop domains are approximately 75% identical in amino acid sequence; however, their carboxyl termini are quite dissimilar; CAR2 possesses homopolymeric runs of histidines and asparagines that are absent from the corresponding region in CAR1. Although CAR1 is maximally expressed during the early stages of development, CAR2 is expressed only after cells have aggregated and, then, preferentially in prestalk cells. Transgenic Dictyostelium that have had their wild-type CAR2 gene replaced by a defective copy using homologous recombination proceed through early development but are detained at the tight mound stage. CAR2 may be required for cAMP-directed sorting of prestalk cells during pattern formation within the aggregation mound. Furthermore, although prestalk genes are expressed normally in aggregates that lack CAR2, they exhibit an enhanced expression of prespore-specific mRNA. Previously, we had shown that there was a requirement for CAR1 during early development. The present results demonstrate that the multiple responses of Dictyostelium to cAMP are regulated by distinct cAMP receptors that are encoded by unique genes.

Amino Acid Sequence↗

PubMed1993

Identification and targeted gene disruption of cAR3, a cAMP receptor subtype expressed during multicellular stages of Dictyostelium development.

Extracellular cAMP acts through cell-surface receptors to coordinate the developmental program of Dictyostelium. A cAMP receptor (cAR1), which is expressed during early aggregation, has been cloned and sequenced previously. We have identified a new receptor subtype, cAR3, that has approximately 56% and 69% amino acid identity with cAR1 and cAR2, respectively. cAR1, cAR2, or cAR3 expressed from plasmid in growing Dictyostelium cells can be photoaffinity labeled with 8-N3[32P]cAMP and phosphorylated when stimulated with cAMP. cAR3 RNA was not present during growth but appeared during late aggregation. Its expression peaked at 9 hr and then fell to a reduced level that was maintained until culmination. The expression of cAR3 protein followed a similar pattern, but with a 3-hr lag, and reached a maximum at the mound stage. In contrast, cAR1 protein was expressed predominantly during early aggregation and at low levels during later stages. At their respective peaks of expression, there were approximately 5 x 10(3) cAR3 sites per cell compared with approximately 7 x 10(4) cAR2 sites per cell. The cAR3 gene was disrupted by homologous recombination in several different parental cell lines. Surprisingly, the car3- cell lines display no obvious phenotype.

Amino Acid Sequence↗

PubMed1993

A G-protein beta-subunit is essential for Dictyostelium development.

Recent studies have demonstrated that G-protein-linked signal transduction pathways play a significant role in the developmental program of the simple eukaryotic organism Dictyostelium. We have reported previously the isolation of a G-protein beta-subunit and present here a more complete analysis of this gene. Low-stringency Southern blots and RFLP mapping studies suggest that the beta-subunit is a unique gene found on linkage group II. Its deduced amino acid sequence of 347 residues is approximately 60% identical to those of the human, Drosophila, and Caenorhabditis elegans beta-subunits. The carboxy-terminal 300 residues are about 70% identical; the amino-terminal 50 residues are quite divergent, containing only 10 identities. At all stages of growth and development, a single 1.9-kb beta-subunit mRNA is present at a high level, and a specific antibody detects a single 37-kD protein. We propose that G-protein heterotrimers are formed when this beta-subunit couples with each of the eight distinct G-protein alpha-subunits that are transiently expressed during development. Targeted disruption of the beta-subunit gene had no effect on the viability of haploid cells, but resulted in the inability of cells to aggregate.

Amino Acid Sequence↗

PubMed1992

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in Dictyostelium development.

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.

Adenylyl Cyclases↗