A feasibility study comparing two treatment approaches for chronic fatigue syndrome in adolescents.
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Adolescent↗
Biomedical subjects
B Ashby
Publications and source records attributed to B Ashby.
Agonist-induced internalization and mitogen-activated protein kinase activation of the human prostaglandin EP4 receptor.
We examined the pathway of prostaglandin E(2) (PGE(2))-induced internalization of the prostaglandin EP4 receptor in HEK 293 cells. Co-expression of dominant negative beta-arrestin (319-418) or dynamin I (K44A) with the EP4 receptor reduced internalization. The activated receptor co-localized with GFP-arrestin 2 and GFP-arrestin 3, confirming the requirement for beta-arrestins in internalization. Inhibition of clathrin-coated vesicle-mediated internalization resulted in inhibition of sequestration, whereas inhibition of caveola-mediated internalization had no effect. PGE(2) stimulation of the EP4 receptor resulted in rapid mitogen-activated protein (MAP) kinase activation. Examination of an internalization-resistant mutant and co-expression of mutant accessory proteins with EP4 revealed that MAP kinase activation proceeds independently of internalization.
Acetic Acid↗
Patterns of cyclic AMP formation by coexpressed D1 and D2L dopamine receptors in HEK 293 cells.
Recent studies using immunofluorescence confocal microscopy (Aizman et al., Nature neuroscience (2000) 3, 226-230) present compelling evidence for colocalization of D1 and D2 dopamine receptors on neurons in the striatum and nucleus accumbens. To examine some of the biochemical consequences of colocalization we coexpressed the D1 and D2 dopamine receptors in HEK293 cells. Dopamine D1 and D2 receptors couple to stimulation and inhibition of adenylyl cyclase, respectively. In cells expressing only the D1 receptor, dopamine stimulated cAMP formation with an EC50 of 2.15 nM. In cells expressing only the D2L receptor, dopamine inhibited cAMP formation by 80% with an EC50 of 0.02 nM. The effect of dopamine on the D2L receptor was antagonized by the selective antagonist spiperone with an IC50 of 0.31 nM. In cells coexpressing both the D1 and D2L receptors, dopamine caused an increase in cAMP that was only 20% of that observed with the D1 receptor alone. In this case, increasing concentrations of spiperone caused a change in the dose-response curve from hyperbolic to bell-shaped as the concentration of spiperone was increased. Using pharmacological constants determined from studies on the individually expressed receptors, the curves obtained in cells co-expressing the two receptors could be modeled by kinetic expressions derived by summing the contributions from each receptor. The model leads to a re-interpretation of the pharmacology of dopaminergic ligands. Hence, one consequence of colocalization is that D2 receptor antagonists become functional agonists of cAMP formation.
Benzazepines↗
Determination of D1 and D2 dopamine receptor expression by Ntera-2 cells.
There is evidence that D1 and D2 dopamine receptors are co-expressed on some neurons. As a potential model of co-expression we examined Ntera-2 cells using RT-PCR, and showed that they express D2 but not D1 receptors. D2 dopamine receptor expression was confirmed by quinpirole inhibition of forskolin-stimulated cAMP formation. Absence of D1 dopamine receptors was confirmed by the inability of dopamine or SKF 81297 to increase cAMP.
Benzazepines↗
Comparison of agonist-induced internalization of the human EP2 and EP4 prostaglandin receptors: role of the carboxyl terminus in EP4 receptor sequestration.
Prostaglandin E(2) (PGE(2)) couples to stimulation of adenylyl cyclase through two distinct G protein-coupled receptors designated EP2 and EP4. Although they have similar affinities for PGE(2), the EP(2) and EP4 receptors have distinct structural characteristics. EP2 is a 358-amino-acid protein with short third intracellular loop and C-terminal domains, whereas EP4 consists of 488 amino acids with a long third intracellular loop and a long cytoplasmic tail. The ability of the HA epitope-tagged receptors to undergo PGE(2)-induced internalization was examined by enzyme-linked immunosorbent assay and immunofluorescence microscopy after expression in human embryonic kidney 293 cells. The EP2 receptor did not internalize, whereas the EP4 receptor underwent rapid internalization. Truncation of the EP4 receptor after amino acid 350, which removes 138 residues, abolished internalization. Truncation after amino acid 369 markedly attenuated internalization, whereas truncation after amino acid 383 had little effect. Serine and threonine residues in the region 350 to 383 were mutated to determine their role in internalization. The mutants S370-382A, a full-length receptor containing six serine-to-alanine mutations in the region 370 to 382, and S354-369A, containing four serine mutations and one threonine mutation in the region 350 to 370, both internalized to the same extent as the wild-type. A further mutant, designated S354-382A, containing amino acid substitutions S354A, S359A, S364A, S366G, T369A, S370A, S371A, S374A, S377A, S379A, and S382A, also internalized to the same extent as the wild-type. We conclude that the C terminus of the EP4 receptor is involved in internalization; however, serine and threonine residues do not seem to be involved.
Amino Acid Sequence↗
Identification of a region of the C-terminal domain involved in short-term desensitization of the prostaglandin EP4 receptor.
1. The prostaglandin EP4 receptor, which couples to stimulation of adenylyl cyclase, undergoes rapid agonist-induced desensitization when expressed in CHO-K1 cells. 2. Truncation of the 488-amino acid receptor at residue 350 removes the carboxy-terminal domain and abolishes desensitization. 3. To further delineate residues involved in desensitization, the receptor was truncated at position 408, 383 or 369. Receptors truncated at position 408 or 383 underwent PGE2-induced desensitization, whereas the receptor truncated at position 369 displayed sustained activity, indicating that the essential residues for desensitization lie between 370 and 383. 4. The six serines in the 14-amino acid segment between residues 370 and 383 were mutated to alanine, retaining the entire C-terminal domain. Desensitization was absent in cells expressing this mutant. 5. The results indicate involvement of serines located between 370 and 382 in rapid desensitization of the EP4 receptor.
1-Methyl-3-isobutylxanthine↗
Co-expression of prostaglandin receptors with opposite effects: a model for homeostatic control of autocrine and paracrine signaling.
Prostaglandins are ubiquitous autocrine mediators that exert their effects through a number of G protein-coupled receptors. Many organs and tissues express many of the prostaglandin receptors, and prostaglandins have diverse effects on individual organs and tissues. In some cases, several prostaglandin receptors are expressed on a single cell type. Co-expressed prostaglandin receptors frequently appear to have opposite actions, suggesting homeostatic control of prostaglandin effects. Co-expression of opposing receptors provides a molecular mechanism for weak or partial agonism and explains the action of a drug as a mixed agonist/antagonist. The physiological relevance of co-expressed opposing receptors for a single agonist perhaps can be explained in terms of the difference between endocrine and autocrine mediators. Endocrine hormones are generally produced by cells distant from their site of action so that they are diluted to an elevated but stable concentration by the time they reach their target cells. In contrast, autocoids are produced by the same cell type on which they act and may reach transiently high levels at their sites of action. The presence of a second type of receptor that negates the action of the first receptor would tend to buffer cellular responses to transient extremes of agonist concentration. The slow onset of inhibition would also allow for time-dependent buffering, providing additional control over autocoid release and effect. The mechanism is relevant to other autocrine and paracrine mediators including neurotransmitters, which reach transiently high concentrations in the synaptic cleft.
Amino Acid Sequence↗
Molecular basis for ADP-induced platelet activation. I. Evidence for three distinct ADP receptors on human platelets.
Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, alpha,beta-methylene adenosine 5'-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADP-mediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca2+ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. alpha,beta-Methylene adenosine 5'-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.
Adenosine Diphosphate↗
Distribution of prostaglandin IP and EP receptor subtypes and isoforms in platelets and human umbilical artery smooth muscle cells.
Prostaglandins act through specific receptors to stimulate cyclic AMP formation which inhibits platelet activation and relaxes vascular smooth muscle. We have used RT-PCR combined with Southern blot analysis to determine the subtypes of prostaglandin receptor on platelets. Platelets expressed the EP4 rather than the EP2 prostaglandin EP receptor subtype, whereas vascular smooth muscle cells predominantly expressed the EP2 receptor. The IP receptor, which binds prostacyclin and couples to stimulation of adenylyl cyclase, and three isoforms of the inhibitory EP3 receptor were equally expressed in platelets, HEL cells and umbilical artery smooth muscle cells. The EP3-II isoform showed variation in level of expression among the three cell types. As a positive control for the presence of platelet RNA, PCR was performed using primers specific for the alpha chain of the platelet membrane glycoprotein Ib. As a negative control for the absence of T and B cell contamination in the platelet RNA, PCR was performed using primers specific for the cell specific cluster determinants CD2 (a T-cell marker) and CD20 (a B-cell marker). The finding that platelets express both stimulatory and inhibitory prostaglandin receptors provides confirmation of a homeostatic model of regulation of platelet adenylyl cyclase previously proposed.
Base Sequence↗
Agonist-induced desensitization and down-regulation of the human kappa opioid receptor expressed in Chinese hamster ovary cells.
In this study, we examined whether the human kappa opioid receptor stably expressed in Chinese hamster ovary cells underwent desensitization and down-regulation after prolonged exposure to the agonist (-)U50,488H. Pretreatment with (-)U50,488H led to a reduction in the magnitude of increase in [35S]GTPgammaS binding by the subsequent application of (-)U50,488H. The extent of desensitization was related to duration of exposure and (-)U50,488H concentration. Pretreatment with (-)U50,488H also reduced the potency of (-)U50,488H in inhibiting forskolin-stimulated adenylate cyclase. In membranes of (-)U50,488H-pretreated cells, the affinity of (-)U50,488H was lower than that in the untreated control, and GTPgammaS had no effect on (-)U50,488H affinity, consistent with the notion of uncoupling of the receptor-G protein complex by (-)U50, 488H treatment. Down-regulation of the kappa opioid receptor also occurred on exposure to (-)U50,488H. Higher (-)U50,488H concentrations and/or longer incubation periods were required for down-regulation than for desensitization. The degree of down-regulation depended on the agonist concentration and incubation time. (-)U50,488H-induced desensitization and down-regulation were blocked by naloxone. (+)U50,488H, an inactive stereoisomer, did not cause desensitization or down-regulation. These results indicate that both processes were receptor-mediated. After incubation with (-)U50,488H and removal of (-)U50,488H, both (-)U50,488H-induced [35S]GTPgammaS binding and receptor number returned to the control level, which indicates that both processes were reversible. Thus, desensitization and down-regulation of the kappa opioid receptor occur after agonist exposure and represent two different adaptation mechanisms.
3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
Human prostacyclin receptor: cloning and co-expression with EP3 prostaglandin receptor.
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Animals↗
Constitutive activity of human prostaglandin E receptor EP3 isoforms.
1. The human EP3 prostaglandin receptor is a seven transmembrane, G protein-coupled receptor that couples to inhibition of adenylyl cyclase. The receptor occurs as at least six isoforms which result from alternative splicing. The isoforms are identical over the first 359 amino acids, comprising the seven transmembrane helices, but differ in the carboxyl terminal tail which ranges in length from 6 to 65 amino acids beyond the common region. 2. We have stably expressed in CHO-K1 cells four of the isoforms (EP3I-EP3IV) and a form of the EP3 receptor (T-359) truncated at the carboxyl-terminal region defined by the alternative splicing site at amino acid number 359. 3. Isoforms EP3I and EP3II showed concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase in CHO-K1 cells by the EP3 receptor agonist, sulprostone. The IC50 calculated for sulprostone inhibition was 0.2 nM for EP3I and 0.15 nM for EP3II. The maximum extent of inhibition was 80% for both isoforms. 4. Isoforms EP3III and EP3IV showed marked constitutive activity, inhibiting forskolin-stimulated adenylyl cyclase in the absence of agonist. EP3IV also displayed some agonist-dependent inhibition whereas EP3III was fully constitutively active. 5. The truncated receptor T-359 was fully constitutively active, inhibiting forskolin-stimulated adenylyl cyclase by about 70% in the absence of agonist, and showed no agonist-dependent inhibition, in agreement with a similar truncation of the mouse EP3 receptor. 6. To confirm that differences in cyclic AMP level between isoforms represent constitutive activity, we treated cells with pertussis toxin for 6 h to abolish Gi function. Pertussis toxin reversed sulprostone-mediated inhibition of cyclic AMP formation in EP3I and EP3II and abolished constitutive activity of EP3III, EP3IV and T-359 so that the level of forskolin-stimulated cyclic AMP produced was the same in all cells and similar to that obtained in mock-transfected cells. In mock-transfected cells, sulprostone had no effect on forskolin-stimulated cyclic AMP formation. 7. For these experiments we chose clones that showed similar expression levels of each isoform, as determined by binding of [3H]-prostaglandin E2 (PGE2) (EP3I, 0.71; EP3II, 1.47; EP3IV, 1.59 pmol mg-1 protein). Mock-transfected cells showed no detectable binding of [3H]-PGE2. In addition, we performed a detailed study of the effects of expression level on constitutive activity. Over a six fold range of expression there was no change in the properties of each isoform with regard to whether it was constitutively active or not. 8. The degree of constitutive activity correlated with the inverse of the length of the C-terminal tail of the isoforms. However, no correlation was found between isoforms from human and mouse: whereas EP3II shows no constitutive activity, its mouse homologue, EP3 gamma, shows almost complete constitutive activity, even though the C-terminal domains of the receptors following the splice site differ in only 7 of 29 amino acids.
Adenylate Cyclase Toxin↗
The long cytoplasmic carboxyl terminus of the prostaglandin E2 receptor EP4 subtype is essential for agonist-induced desensitization.
The 488-amino acid human prostaglandin E2 receptor EP4 subtype, which couples to stimulation of adenylyl cyclase, shares the major structural features of G protein-coupled receptors, having seven putative transmembrane domains, an extracellular amino terminus, and a cytoplasmic carboxyl terminus. The latter is composed of 156 amino acids and contains 38 serine and threonine residues, which are potential phosphorylation sites. The carboxyl terminus may be important in receptor function; in some receptors, truncation of the cytoplasmic tail abolishes desensitization. In others, truncation leads to constitutive activity, and in other receptors, truncation has no effect on receptor function. To investigate the role of the long cytoplasmic tail of the EP4 receptor, we constructed a mutant EP4 that lacks the last 138 amino acids at the carboxyl terminus, including 36 serine and threonine residues. The truncated EP4 receptor was stably expressed in Chinese hamster ovary cells at levels comparable to that of the wild-type receptor and exhibited a Kd value for [3H]PGE2 binding similar to that of the wild-type receptor. PGE2-mediated adenylyl cyclase activity as a function of PGE2 concentration was similar in cells expressing the wild-type and truncated EP4 receptors. Neither the wild-type receptor nor the truncated form showed any constitutive activity. However, the wild-type EP4 receptor underwent PGE2-induced desensitization fully within 15-20 min, whereas the truncated EP4 receptor, lacking 36 of the 38 carboxyl-terminal serines and threonines, displayed a sustained activation. Despite the continuous presence of PGE2, the rate of cAMP synthesis via stimulation of the truncated receptor remained constant over > or = 20 min. These findings suggest that the cytoplasmic tail of EP4 plays an important role in agonist-induced desensitization.
Amino Acid Sequence↗
Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells.
Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 +/- 6 nM), much less for UTP and ADP (approximately 1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.
Amino Acid Sequence↗
Prostaglandin E2 both stimulates and inhibits adenyl cyclase on platelets: comparison of effects on cloned EP4 and EP3 prostaglandin receptor subtypes.
The effects of prostaglandin E2 (PGE2) on platelet cyclic AMP formation were examined and compared with effects on cloned prostaglandin receptors. PGE2 gave a weak stimulation of adenyl cyclase in platelets compared with the PGI2 analog Iloprost. In the presence of the adenyl cyclase stimulator forskolin, the response to PGE2 was amplified in a synergistic manner. By contrast, in the presence of Iloprost, PGE2 inhibited cyclic AMP formation. We postulate that the weak platelet response to PGE2 is due to co-localization of a PGE2 receptor that couples to stimulation of adenyl cyclase with the EP3 prostaglandin receptor that binds PGE2 tightly and inhibits adenyl cyclase. In support of this postulate, we compared the responses obtained with platelets with those of cloned EP4 (stimulatory) and EP3 (inhibitory) prostaglandin receptor subtypes and show similar dose-response curves for stimulation and inhibition of cyclic AMP formation between platelets and cloned receptors.
Adenylyl Cyclase Inhibitors↗
Protein kinase C activation is not a key step in ADP-mediated exposure of fibrinogen receptors on human platelets.
UNLABELLED: A selective inhibitor of protein kinase C (PKC), Ro 31-8220, blocks pleckstrin (P47) phosphorylation in platelets activated with either ADP, ADP plus synthetic thromboxane agonist U46619 and ADP plus U46619 plus epinephrine, while inducing a weak inhibition of platelet aggregation, and no significant effect on the fibrinogen binding. In platelets activated by U46619 alone, P47 phosphorylation, platelet aggregation, fibrinogen binding and serotonin release are all inhibited by Ro 31-8220. In the presence of an ADP scavenger system, U46619 induces pleckstrin phosphorylation, serotonin release and calcium mobilization but not platelet aggregation and fibrinogen binding, unless epinephrine is added. IN CONCLUSION: (1) PKC activation is required for ADP secretion; (2) ADP or epinephrine are essential for fibrinogen receptor exposure induced by U46619; (3) fibrinogen receptor exposure induced by ADP is independent of activation of PKC.
Adenosine Diphosphate↗
Cloning and expression of a prostaglandin E receptor EP3 subtype from human erythroleukaemia cells.
Prostaglandins inhibit platelet activation by stimulating intracellular cyclic AMP formation. We have postulated that intracellular cyclic AMP levels in platelets are buffered by a distinct prostaglandin receptor that mediates inhibition of cyclic AMP formation. In order to provide evidence for the model, we have cloned the cDNA coding for a prostaglandin receptor EP3 subtype, which is coupled to inhibition of adenylate cyclase, from the megakaryocytic cell line human erythroleukaemia (HEL) cells. A PCR-generated hybridization probe, produced using primers based on the sequence of the mouse prostaglandin EP3 receptor published by Sugimoto, Namba, Honda, Hayashi, Negishi, Ichikawa and Narumiya [(1992) J. Biol. Chem. 267, 6463-6466], was used to screen a lambda gt11 HEL cell cDNA library. The composite full-length cDNA clone HEP3, generated from the two partial clones pHEP3-7 and pHEP3-5, is 1.6 kb long with an open reading frame coding for 390 amino acids. This clone is 83% identical to the alpha subtype of the mouse EP3 receptor. The full-length construct was transfected into COS-1 cells. The cloned receptor exhibited the properties of a prostaglandin EP3 subtype, inhibiting forskolin-stimulated cyclic AMP formation in response to prostaglandin E2 (PGE2) and binding PGE2 with high specificity and a Kd of 3.2 nM. Radiolabelled PGE2 could be displaced by prostaglandins in the order PGE2 = PGE1 > iloprost = PGD2. Northern blot analysis revealed that the receptor is also present in human kidney.
Amino Acid Sequence↗
Interactions among prostaglandin receptors.
A single prostaglandin may have multiple effects on the same cell type with each effect showing a different prostaglandin concentration dependence, indicating the presence of separate receptors coupled to different second-messenger systems. The effects may be antagonistic toward each other, suggesting homeostatic control of prostaglandin effects, which may be important in buffering cellular response to elevated prostaglandin levels in inflammation. We have used prostaglandin regulation of cyclic AMP metabolism in platelets and human erythroleukemia (HEL) cells as a model to analyze interactions between stimulatory and inhibitory prostaglandin receptors coupled to adenylate cyclase. Cloning of an EP3 prostaglandin receptor subtype from HEL cells confirmed the presence of an inhibitory receptor distinct from that involved in prostaglandin stimulation of adenylate cyclase.
Adenylyl Cyclases↗