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.     

Immunological analyses of the chemotactic receptor of Dictyosteleum discoideum. Identification of cDNA clones

The cell surface cAMP receptor was excised from preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to generate a polyclonal antiserum. The antiserum immunoprecipitates the two molecular weight forms of the cAMP receptor. Both forms are phosphorylated. Western blot analyses show that the antiserum is highly specific and recognizes only the two molecular weight forms of the cAMP receptor. Immunological studies indicate that both forms of the receptor are phosphorylated. Vegetative amoebae possess low levels of the cAMP receptor. Levels of the antigen increase in differentiated cells which express high cell surface cAMP binding activity. The antiserum was also used to isolate 6 lambda gt11 cDNA clones. One of those clones contains a 1.1-kilobase pair cDNA fragment which encodes for a protein of approximately 30,000-35,000 daltons. The antibody which binds to the fusion protein also recognizes the two molecular weight forms of the receptor.     

Down-regulation of cell surface cyclic AMP receptors and desensitization of cyclic AMP-stimulated adenylate cyclase by cyclic AMP in Dictyostelium discoideum. Kinetics and concentration dependence

cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylatecyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of cAMP, and cAMP binding to surface receptors and cAMP-induced activation of adenylate cyclase were measured. cAMP could induce maximally 65% loss of binding activity and complete desensitization of cAMP-stimulated adenylate cyclase activity. Half-maximal effects for down-regulation were observed at 50 nM cAMP and for desensitization at 5 nM cAMP. Down-regulation was rapid with half-times of 4, 2.5, and 1 min at 0.1, 1, and 10 microM cAMP, respectively. Similar kinetic data have been reported for desensitization (Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561). Down-regulation and desensitization were not reversible at 0 degrees C. Down-regulation reversed slowly at 20 degrees C with a half-time of about 1 h. Resensitization of adenylate cyclase was biphasic showing half-times of 4 min and about 1 h, respectively; the contribution of the rapidly resensitizing component was diminished when down-regulation of receptors was enhanced. These results suggest that cAMP-induced down-regulation of receptors and desensitization of adenylate cyclase stimulation proceed by at least two steps. One step is rapidly reversible, occurs at low cAMP concentrations, and induces desensitization without down-regulation, while the second step is slowly reversible, requires higher cAMP concentrations, and also induces down-regulation.     

Kinetics and nucleotide specificity of a surface cAMP binding site in Dictyostelium discoideum, which is not down-regulated by cAMP

Dictyostelium cells exhibit four types of kinetically distinct surface cAMP binding sites, the AH, AL, BS, and BSS sites, which are down-regulated during persistent stimulation with cAMP. Although most cAMP-induced responses are subject to desensitization during constant stimulation, some responses, notably the induction of post-aggregative gene expression, require persistent cAMP stimulation. The kinetics and specificity of residual cAMP-binding activity in cells treated for 4 h with micromolar cAMP were investigated. It was found that around 4000 rapidly dissociating binding sites per cell with an affinity of about 300 nM are retained after down-regulation. The nucleotide specificity of the remaining sites was very similar, but not completely identical to the AH, AL and B sites, suggesting that these sites belong to the same class of cell surface cAMP receptors and may be utilized to mediate responses requiring continuous cAMP stimulation.     

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.     

Adenosine 3', 5'-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum

cAMP induces the activation and subsequent desensitization of adenylate cyclase in Dictyostelium discoideum. cAMP also induces down-regulation of surface cAMP receptors. Desensitization of adenylate cyclase is composed of a rapidly reversible component (adaptation) and a slowly reversible component related to down-regulation of surface cAMP receptors (Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7700-7704). The agonistic and antagonistic activities of the cAMP derivative adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) for these responses were investigated. (Rp)-cAMPS competes with cAMP for binding to different receptor forms with an apparent Ki = 5 microM. (Rp)-cAMPS does not activate adenylate cyclase and antagonizes the cAMP-induced activation with an apparent Ki = 5 microM. (Rp)-cAMPS induces down-regulation of surface cAMP receptors with EC50 = 5 microM. (Rp)-cAMPS induces desensitization of adenylate cyclase, which is not rapidly reversible. These results indicate that desensitization of adenylate cyclase by (Rp)-cAMPS is due to down-regulation of surface cAMP receptors and not to adaptation. We conclude that down-regulation of surface cAMP receptors does not require their activation or modification involved in adaptation.     

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.     

Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down-regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhibitory effects of GTP gamma S and GDP beta S on cAMP binding are reduced; the stimulatory effect of cAMP on GTP gamma S binding is lost in fgd A mutants. (d) Basal high-affinity GTPase activity is reduced 40% and the stimulatory effect of cAMP is decreased from 40% in wild type to 30% in fgd A. (e) GTP-mediated stimulation and inhibition of adenylate cyclase is normal in mutant membranes. The results suggest a defective interaction between cell surface cAMP receptors and a specific G-protein in fgd A mutants. This interaction appears to be essential for nearly all signal transduction pathways in Dictyostelium discoideum.     

Agonist regulation of beta-adrenergic receptor mRNA. Analysis in S49 mouse lymphoma mutants

Agonist-promoted down-regulation of beta-adrenergic receptor mRNA was investigated in S49 mouse lymphoma variants with mutations in elements of hormone-sensitive adenylate cyclase. In wild-type cells steady-state levels of beta-adrenergic receptor mRNA were established by DNA-excess solution hybridization to be 1.72 +/- 0.08 (n = 8) amol/microgram total cellular RNA. Receptor mRNA levels declined 35-45% in response to stimulation by the beta-adrenergic agonist (-)isoproterenol or forskolin as described previously in DDT1 MF-2 cells (Hadcock, J. R., and Malbon, C. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5021-5025). Agonist-promoted cAMP accumulation and down-regulation of receptor mRNA were analyzed in three variants with mutations in Gs alpha (H21a, unc, cyc-) and a single variant lacking cAMP-dependent protein kinase activity (kin-). H21a (Gs alpha coupled to receptor, but not to adenylate cyclase), unc (Gs alpha uncoupled from receptor), and cyc- (lacking Gs alpha) variants accumulated cAMP and down-regulated beta AR mRNA in response to forskolin. In unc and cyc- cells isoproterenol failed to stimulate cAMP; accumulation and down-regulation of receptor mRNA was not observed. H21a cells, in contrast, displayed agonist-promoted regulation of beta-adrenergic receptor mRNA but only basal levels of cAMP accumulation in response to isoproterenol. The kin- cells displayed cAMP accumulation in response to forskolin as well as to isoproterenol but no down-regulation of receptor mRNA or receptor expression. Taken together these data demonstrate several features of agonist-promoted down-regulation of mRNA: (i) cAMP-dependent protein kinase activity is required for down-regulation of mRNA (kin-), although elevated cAMP accumulation is not (H21a); (ii) functional receptor-Gs coupling is required (H21a), and clones lacking Gs alpha (cyc-) or receptor Gs coupling (unc) lack the capacity to down-regulate mRNA in response to agonist; and (iii) in the presence of basal levels of cAMP and cAMP-dependent protein kinase activity, functional receptor-Gs coupling (H21a) to some other effector other than adenylate cyclase may be propagating the signal.     

Processing of human choriogonadotropin and its receptors by cultured pig Leydig cells. Role of cyclic AMP and protein synthesis

We have examined the process by which human choriogonadotropin/luteinizing hormone (hCG/LH) receptors are regulated in cultured porcine Leydig cells. Treatment of Leydig cells with human choriogonadotropin, cholera toxin, forskolin and cyclic 8-bromoAMP (8-BrcAMP) produced a loss of surface receptors without modification of the binding affinity. This negative regulation of the number of receptors mediated by maximal concentrations of hCG was higher than that induced by the other agents. The extent of receptor loss in cells treated with increasing concentrations of hCG was highly correlated with their capacity to stimulate cAMP production. However, there was little correlation between down-regulation and cAMP production of these cells treated by hCG plus forskolin or cholera toxin plus forskolin, where a synergistic cAMP production was obtained. Following exposure of Leydig cells to both hCG and 8-BrcAMP, the surface receptor disappearance began after an initial lag period of about 6-8 h. Thereafter a 50% loss of surface receptor was observed in the next 8-h incubation. Monensin with hCG shortens this lag period before initiation of receptor loss. Kinetic studies with 125I-hCG, in the presence or absence of monensin, showed that the half-life of the receptor-bound hormone complexes at the cell surface was 10.5 h and 8 h respectively. Therefore, the steady state of the surface receptor during the lag phase of 8 h is probably related to recycling of internalized receptors and/or translocation of performed receptors. Cycloheximide and actinomycin D inhibit hCG-mediated and 8-BrcAMP-mediated down-regulation. Cycloheximide lengthens ligand-receptor complexes at the surface by slowing down the rate of internalization (half-life of 20 h), but this mechanism is not enough per se to explain the effect of cycloheximide. Pulses of hCG or 8-BrcAMP for 4 h and 8 h sufficed to induce nearly maximal down-regulation. However, it was possible to attenuate this triggering effect by adding cycloheximide after pulse of the cells. Thus, even after removal of the triggering agent (hCG or 8-BrcAMP), the loss of surface receptor could be triggered by a protein-sensitive signal. Taken as a whole these results indicate that a coordinated interaction is involved in the cell-surface hCG/LH receptor regulation. The apparent steady state of the number of receptors during the first hours of stimulation passed through a reuptake of internalized receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.     

The cell density factor CMF regulates the chemoattractant receptor cAR1 in Dictyostelium

Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called conditioned medium factor (CMF) reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form of the cAMP receptor and the loss of ligand binding, which is the first step of ligand-induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess approximately 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a 10-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF binding; disruption of the cAR1 gene abolishes both cAMP and CMF binding. In wild-type cells, downregulation of cAR1 with high levels of cAMP also downregulates CMF binding, and CMF similarly downregulates cAMP and CMF binding. This suggests that the cAMP binding and CMF binding are closely linked. Binding of approximately 200 molecules of CMF to starved cells affects the affinity of the majority of the cAR1 cAMP receptors within 2 min, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cARs. In cells lacking the G-protein beta subunit, cAMP induces a loss of cAMP binding, but not CMF binding, while CMF induces a reduction of CMF binding without affecting cAMP binding, suggesting that the linkage of the cell density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G-protein.     

Identification of separate determinants on the thyrotropin receptor reactive with Graves' thyroid-stimulating antibodies and with thyroid-stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors.

Deletions, substitutions, or mutations of the rat TSH receptor extracellular domain between residues 20 and 107 (all residue numbers are determined by counting from the methionine start site) have been made by site-directed mutagenesis of receptor cDNA. After transfection in Cos-7 cells, constructs were evaluated for their ability to bind [125I]TSH or respond to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients in assays measuring cAMP levels of the transfected cells. Assay results were compared to results from Cos-7 cells transfected with wild-type receptor constructs or vector alone. We identify threonine-40 as a TSAb-specific site whose mutation to asparagine, but not alanine, reduces TSAb activity 10-fold, but only minimally affects TSH-increased cAMP levels. We show that thyroid-stimulating blocking antibodies (TSBAbs), which block TSH or TSAb activity and are found in hypothyroid patients with idiopathic myxedema, continue to inhibit TSH-stimulated cAMP levels when threonine-40 is mutated to asparagine or alanine, suggesting that TSBAbs interact with different TSH receptor epitopes than the TSAb autoantibodies in Graves' patients. This is confirmed by the demonstration that these TSBAbs interact with high affinity TSH-binding sites previously identified at tyrosine-385 or at residues 295-306 of the extracellular domain of the TSH receptor. This is evidenced by a loss in the ability of TSBAbs to inhibit TSAb activity when these residues are mutated or deleted, respectively. Since the TSAb and TSBAb epitopes are in regions of the extracellular domain of the TSH receptor that have no homology in gonadotropin receptors, these data explain at least in part the organ-specific nature of TSH receptor autoantibodies in autoimmune thyroid disease. Data are additionally provided which indicate that residues 30-37 and 42-45, which flank the TSAb epitope at threonine-40, appear to be ligand interaction sites more important for high affinity TSH binding than for the ability of TSH to increase cAMP levels and that cysteine-41 is critical for TSH receptor conformation and expression on the surface of the cell. Thus, despite unchanged maximal values for TSH-increased cAMP levels, substitution of residues 42-45 or deletion of residues 30-37 results in receptors, which, by comparison to wild-type constructs, exhibit significantly worsened Kd values for TSH binding than EC50 values for TSH- or TSAb-increased cAMP activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pertussis toxin inhibits cAMP surface receptor-stimulated binding of [35S]GTP gamma S to Dictyostelium discoideum membranes

GTP-binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTP gamma S greater than GTP greater than 8-N3-GTP; the binding of GTP gamma S and GTP, but not of 8-N3-GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (Kd = 2.6 microM, Bmax = 85 nM). More detailed studies with [35S]GTP gamma S showed heterogeneous binding composed of two forms of binding sites with respectively high (Kd = 0.2 microM) and low (Kd = 6.3 microM) affinity. cAMP derivatives enhanced GTP gamma S binding by increasing the affinity and the number of the high-affinity sites, while the low-affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTP gamma S binding showed a close correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTP gamma S binding, but eliminated the cAMP stimulation of GTP and GTP gamma S binding. These results indicate that D. discoideum cells have a pertussis toxin-sensitive GTP-binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G-protein in D. discoideum.     

Two distinct pathways for cAMP-mediated down-regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level

We have studied cyclic AMP-mediated regulation of the beta 2-adrenergic receptor (beta 2AR). The effects of cAMP were assessed in Chinese hamster fibroblast (CHW) cells expressing either the wild type human beta 2AR receptor (CH-beta 2) or mutated forms of the receptor lacking the consensus sequences for phosphorylation by the cAMP-dependent protein kinase. Treatment of the CH-beta 2 cells with the cAMP analogue dibutyryl cAMP (Bt2cAMP) induces a time-dependent "down-regulation" of the number of beta 2AR. This down-regulation of the receptors is accompanied by a decline in the steady state level of beta 2AR mRNA. Moreover, the treatment with Bt2cAMP induces an increase in the phosphorylation level of the membrane-associated beta 2AR. Both the reduction in beta 2AR mRNA and the enhanced phosphorylation of the receptor are rapid and precede the loss of receptor. The down-regulation of beta 2AR induced by Bt2cAMP is concentration-dependent and mimicked by the other biologically active cyclic nucleotide analogue, 8-Br-cAMP, by forskolin, and by the phosphodiesterase inhibitor, isobutylmethylxanthine. In the CHW cell lines expressing receptors lacking the putative protein kinase A phosphorylation sites, the Bt2cAMP-induced phosphorylation of beta 2AR is completely abolished. In these cells the down-regulation of beta 2AR receptor number produced by cAMP is significantly slowed, whereas the reduction in beta 2AR mRNA level is equivalent to that observed in CH-beta 2 cells. These data indicate that there are at least two pathways by which cAMP may decrease the number of beta 2ARs in cells: one involves phosphorylation of the receptor by the cAMP-dependent protein kinase and the other leads to a reduction in steady state beta 2AR mRNA levels.     

Photoaffinity labeling of the cell surface adenosine 3':5'-monophosphate receptor of Dictyostelium discoideum and its modification in down-regulated cells

The cAMP cell surface receptor of Dictyostelium discoideum amoebae was identified by the use of the photoaffinity analogue 8-N3-[32P]cAMP. Labeling by intact cells of one component, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, could be specifically inhibited by the presence of nonradioactive cAMP. The component, P45 (apparent molecular weight of 45,000), was not identified on vegetative cells but was labeled with increasing intensity as cells differentiated and increased their levels of surface cAMP binding sites. Developmental mutants, starved under conditions where they do not express significant levels of cAMP binding sites, did not incorporate radioactivity into this protein. These mutants did label P45 when starved under differentiation-inducing conditions such that their levels of surface cAMP binding sites increased. P45 co-purified with the plasma membrane fraction isolated from cells to which 8-N3-[32p]cAMP had been covalently bound. Down-regulated amoebae, which displayed approximately 25% of the binding activity of untreated cells, did not label P45. These cells did, however, label a new component with an apparent molecular weight of 47,000 (P47).l The appearance of this component represented the only discernible difference in labeling profile under these conditions. As in the case of P45, radioactive incorporation into P47 did not occur if the photoactivation of 8-N3-[32P]cAMP was performed in the presence of nonradioactive cAMP.     

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.     

Direct biochemical measurements of signal relay during Dictyostelium development

Upon starvation, individual Dictyostelium discoideum cells enter a developmental program that leads to collective migration and the formation of a multicellular organism. The process is mediated by extracellular cAMP binding to the G protein-coupled cAMP receptor 1, which initiates a signaling cascade leading to the activation of adenylyl cyclase A (ACA), the synthesis and secretion of additional cAMP, and an autocrine and paracrine activation loop. The release of cAMP allows neighboring cells to polarize and migrate directionally and form characteristic chains of cells called streams. We now report that cAMP relay can be measured biochemically by assessing ACA, ERK2, and TORC2 activities at successive time points in development after stimulating cells with subsaturating concentrations of cAMP. We also find that the activation profiles of ACA, ERK2, and TORC2 change in the course of development, with later developed cells showing a loss of sensitivity to the relayed signal. We examined mutants in PKA activity that have been associated with precocious development and find that this loss in responsiveness occurs earlier in these mutants. Remarkably, we show that this loss in sensitivity correlates with a switch in migration patterns as cells transition from streams to aggregates. We propose that as cells proceed through development, the cAMP-induced desensitization and down-regulation of cAMP receptor 1 impacts the sensitivities of chemotactic signaling cascades leading to changes in migration patterns.     

Induction of post-aggregative differentiation in Dictyostelium discoideum by cAMP: Evidence of involvement of the cell surface cAMP receptor

Exogenous cAMP is known to induce post-aggregative differentiation in Dictyostelium discoideum under conditions that normal development is blocked. We have analysed the cyclic nucleotide specificity, the effect of modulation of the cAMP signal and the dose-response relationship of the induction of two independent markers of post-aggregative differentiation, i.e., a prespore cell-specific antigen detected by a monoclonal antibody, and the activity of glycogen phosphorylase. Our results confirm that high concentrations of cAMP (10(-6)-10(-3)M) are required for the induction of these markers. The cells are shown not to adapt to the cAMP signal. The cyclic nucleotide specificity of induction agrees with the specificity of the cell surface cAMP receptor, but is very dissimilar to the specificity of the intracellular cAMP-dependent protein kinase. It is thus unlikely that cAMP leaks into the cell and activates the cAMP-dependent protein kinase directly. Instead, the induction of post-aggregative differentiation by cAMP seems to be mediated by cell surface cAMP receptors.     

Distribution of cAMP-dependent protein kinase during development in Dictyostelium discoideum

During the developmental cycle of Dictyostelium discoideum cyclic AMP functions as both a chemotactic signal for aggregation and a regulatory molecule during later events of differentiation. Morphological and biochemical data suggest that cAMP may direct cells during morphogenesis and differentiation. We utilized microtechniques to determine the stage- and cell-specific levels of the cAMP-dependent protein kinase, the probable intracellular cAMP receptor. Kinase activity was low and non-cAMP-dependent in amoebae and early aggregates but increased and became cAMP-dependent in aggregates after the formation of tight cell contacts. Maximum kinase activity and cAMP dependency occurred during the slug and culmination stages. The only differential distribution of the kinase within a single stage occurred during culmination when the activity in the stalks was approximately one-fourth of that in the prespore mass. Preliminary evidence indicates that this difference is not due to an inhibitor. In all other stages tested cAMP-dependent protein kinase activity was equal in prespore and prestalk cells.