Barrie Ashby

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, α,β-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. α,β-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.

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 (~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.

Constitutive activity of human prostaglandin E receptor EP3 isoforms

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. 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. 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. 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. 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. 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. 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. 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, EP3γ, 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.

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.

Human prostaglandin EP3 receptor isoforms show different agonist‐induced internalization patterns

The human prostaglandin EP3 receptor comprises eight isoforms that differ in carboxyl‐tail. We show here that the isoforms are trafficked differently. When expressed in HEK293 cells, the isoforms located to the cell surface, although a fraction of some remained in the cell. Upon prostaglandin E2 stimulation, EP3.I internalized almost completely, EP3.II, EP3.V, EP3.VI and EP3.f internalized to a lesser extent and EP3.III and EP3.IV did not internalize. Both EP3.I and EP3.f internalized with β‐arrestin and internalization were blocked by a dominant negative form of Eps15, a clathrin‐associated protein. Although EP3.II internalized, β‐arrestin did not translocate with the receptor and internalization was not blocked by mutant Eps15. EP3.V and EP3.VI internalized to discrete areas of the cell with β‐arrestin.

Agonist‐induced internalization and mitogen‐activated protein kinase activation of the human prostaglandin EP4 receptor

We examined the pathway of prostaglandin E2 (PGE2)‐induced internalization of the prostaglandin EP4 receptor in HEK 293 cells. Co‐expression of dominant negative β‐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 β‐arrestins in internalization. Inhibition of clathrin‐coated vesicle‐mediated internalization resulted in inhibition of sequestration, whereas inhibition of caveola‐mediated internalization had no effect. PGE2 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.

Protein kinase C activation is not a key step in ADP-mediated exposure of fibrinogen receptors on human platelets

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 318220. 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.

Identification of a region of the C-terminal domain involved in short-term desensitization of the prostaglandin EP4 receptor

The prostaglandin EP4 receptor, which couples to stimulation of adenylyl cyclase, undergoes rapid agonist‐induced desensitization when expressed in CHO‐K1 cells. Truncation of the 488‐amino acid receptor at residue 350 removes the carboxy‐terminal domain and abolishes desensitization. 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. 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. The results indicate involvement of serines located between 370 and 382 in rapid desensitization of the EP4 receptor.

Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia.

Summary.  Background: In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective: This study was carried out to investigate the contribution of the ADP receptor P2Y12‐mediated pathway to platelet hyperreactivity due to hypercholesterolemia. Methods: Low‐density lipoprotein receptor‐deficient mice and C57Bl/6 wild‐type mice were fed on normal chow and high‐fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor‐induced functional responses via Gi signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation and secretion, αIIbβ3 receptor activation and the phosphorylation of extracellular signal‐regulated protein kinase (ERK) and Akt were analyzed. Results: Plasma cholesterol levels ranged from 69 ± 10 to 1011 ± 185 mg dL−1 depending on diet in mice with different genotypes. Agonist‐dependent aggregation, dense and α‐granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels, even if elevated thromboxane generation was blocked. These functional responses were induced via increased levels of Gi‐mediated ERK and Akt phosphorylation in hypercholesterolemic mice vs. normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR‐C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared with normocholesterolemic controls. Conclusions: These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia.

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.