Differential regulation and relocalization of the platelet P2Y receptors after activation: a way to avoid loss of hemostatic properties?

In the present study, we investigated the desensitization and trafficking of the P2Y1 and P2Y12 receptors after agonist-induced stimulation of platelets or astrocytoma cells transfected with the P2Y1 or P2Y12 receptors fused to green fluorescent protein. In platelets and in transfected cells, exposure to 10 microM ADP caused desensitization of the P2Y1 receptor-driven calcium signal, whereas the P2Y12 receptor-mediated inhibition of cAMP formation was not affected. Plasma membranes from ADP-stimulated platelets also retained P2Y12 activity. Agonist-induced P2Y1 receptor desensitization was accompanied by its internalization in platelets and transfected cells. In contrast, although a substantial fraction of P2Y12 receptors was rapidly and transiently internalized, most of the P2Y12 receptors remained at the plasma membrane. Activated P2Y1 receptors were internalized through a clathrin-dependent pathway in cells and platelets, whereas the P2Y12 receptors seemed to use a distinct, clathrin-independent pathway. Together, these data indicate that the P2Y1 and P2Y12 receptors are differentially regulated upon activation. The absence of desensitization of the Gi protein-coupled P2Y12 receptor-dependent responses could represent a mechanism to preserve the hemostatic properties of otherwise unresponsive platelets.     

GRK2: multiple roles beyond G protein-coupled receptor desensitization

G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or interacting directly with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo).     

PKCdelta associates with and is involved in the phosphorylation of RasGRP3 in response to phorbol esters

RasGRP is a family of guanine nucleotide exchange factors that activate small GTPases and contain a C1 domain similar to the one present in protein kinase C (PKC). In this study, we examined the interaction of RasGRP3 and PKC in response to the phorbol ester PMA. In Chinese hamster ovary or LN-229 cells heterologously expressing RasGRP3, phorbol 12-myristate 13-acetate (PMA) induced translocation of RasGRP3 to the perinuclear region and a decrease in the electrophoretic mobility of RasGRP3. The mobility shift was associated with phosphorylation of RasGRP3 on serine residues and seemed to be PKCdelta-dependent because it was blocked by the PKCdelta inhibitor rottlerin as well as by a PKCdelta kinase-dead mutant. Using coimmunoprecipitation, we found that PMA induced the physical association of RasGRP3 with PKCdelta and, using in situ methods, we showed colocalization of PKCdelta and RasGRP3 in the perinuclear region. PKCdelta phosphorylated RasGRP3 in vitro. Previous studies suggest that ectopic expression of RasGRP3 increases activation of Erk1/2. We found that overexpression of either PKCdelta or RasGRP3 increased the activation of Erk1/2 by PMA. In contrast, coexpression of PKCdelta and RasGRP3 yielded a level of phosphorylation of Erk1/2 similar to that of control vector cells. Our results suggest that PKCdelta may act as an upstream kinase associating with and phosphorylating RasGRP3 in response to PMA. The interaction between RasGRP3 and PKCdelta points to the existence of complex cross-talk between various members of the phorbol ester receptors which can have important impact on major signal transduction pathways and cellular processes induced by phorbol esters or DAG     

Regulation of recombinant gamma-aminobutyric acid (GABA)(A) and GABA(C) receptors by protein kinase C

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate induced a continuous decrease in the gamma-aminobutyric acid (GABA)-activated current amplitude from recombinant GABA receptors (formed by rho1 or alphabetagamma subunits) expressed in Xenopus oocytes. This decline was due to internalization of receptors from the plasma membrane as confirmed by a decrease in surface fluorescence with green fluorescence protein-tagged receptors as well as a concomitant decrease in surface [(3)H]GABA binding. PMA specifically caused internalization of GABA receptors, but not neuronal acetylcholine receptors (alpha(7) or alpha(4)beta(2)), indicating the internalization was not a general, nonspecific phenomenon. Mutation of rho1 PKC phosphorylation sites, identified by in vitro phosphorylation, did not prevent GABA receptor internalization, nor did coexpression of the rho1 M3-M4 intracellular loop along with rho1 GABA receptors. It is likely that PKC-mediated phosphorylation of other proteins, rather than rho1 itself, was required for the internalization. Both rho1 and alphabetagamma receptors did not degrade after phorbol 12-myristate 13-acetate-induced internalization, but returned to the membrane surface within 24 h. These data suggest internalized receptors can exist in an intracellular compartment that can be delivered back to the plasma membrane. Thus, by regulating GABA receptor surface expression, PKC may play a key role in the regulation of GABA-mediated inhibition.     

PKCbetaI mediates the inhibition of P2Y receptor-induced inositol phosphate formation in endothelial cells.

1. Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C (PLC)-linked P2Y1 and P2Y2 receptors, for them 2-methylthio-ATP (2MeSATP) and UTP are respective agonists. Here, we have investigated the particular protein kinase C (PKC) isoform(s) responsible for the inhibition of P2Y1 and P2Y2 receptor-evoked inositol phosphate (IP) formation by phorbol 12-myristate 13-acetate (PMA). 2. Although short-term (20 min) pretreatment of cells with PMA attenuated 2MeSATP- and UTP-induced phosphoinositide (PI) breakdown, this inhibition was lost after 15 h. Preincubation with PMA for 24 h, on the contrary, potentiated 2MeSATP and UTP responses. The IP formation stimulated by NaF was unaltered by PMA pretreatment. 3. Western blot analysis showed that treatment of CPAE with PMA resulted in a rapid translocation of PKC isoform betaI, epsilon and mu, but not lambda, from the cytosol to the membrane fraction. 4. Pretreatment of the selective PKC inhibitor Ro 31-8220 attenuated the inhibitory effect of PMA on IP formation. Go 6976 (an inhibitor of conventional PKCalpha, beta and gamma) and LY 379196 (a selective PKCbeta inhibitor) also dose-dependently inhibited the PMA-mediated desensitization. 5. Transfection of PKCbeta-specific antisense oligonucleotide reduced PKCbetaI protein level and inhibited PMA-mediated PI reduction. 6. RT - PCR analysis showed that PMA treatment for 4 - 24 h up-regulated P2Y1 and P2Y2 receptors at the mRNA levels. 7. These results suggest that PKCbetaI may exert a negative feedback regulation on endothelial P2Y1 and P2Y2 receptor-mediated PI turnover. The down-regulation of PKCbetaI and enhanced P2Y receptor expression together might contribute to the late PI enhancing effect of PMA.     

Role of protein kinase Calpha in signaling from the histamine H(1) receptor to the nucleus

Stimulation of histamine H(1) receptors produced a marked activation of inositol phospholipid hydrolysis, intracellular calcium mobilization, and stimulation of the c-fos promoter in CHO-H1 cells expressing the H(1) receptor at a level of 3 pmol/mg protein. The latter response was determined using a luciferase-based reporter gene (pGL3). This response to histamine was not sensitive to inhibition by pertussis toxin but could be completely attenuated by the protein kinase C (PKC) inhibitor Ro-31-8220, or by 24-h pretreatment with the phorbol esters phorbol 12,13-dibutyrate or phorbol-12-myristate-13-acetate. Several isoforms of PKC can be detected in CHO-H1 cells (alpha, delta, epsilon, mu, iota, zeta) but only PKCalpha and PKCdelta were down-regulated by prolonged treatment with phorbol esters. Of the two isoforms that were down-regulated, only protein kinase Calpha was translocated to CHO-H1 cell membranes after stimulation with either histamine or phorbol esters. The PKC inhibitor G? 6976, which inhibits PKCalpha but not PKCdelta, was also able to significantly attenuate the c-fos-luciferase response to histamine. The mitogen-activated protein kinase kinase inhibitor PD 98059 markedly inhibited the response to histamine, suggesting that the likely major target for PKCalpha was the mitogen-activated protein kinase pathway. These data suggest that the histamine H(1) receptor can signal to the nucleus via PKCalpha after activation of phospholipase Cbeta.     

The protein kinase C inhibitor Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] potentiates agonist-induced mitogen-activated protein kinase activation through tyrosine phosphorylation of the epidermal growth factor receptor

Protein kinase C (PKC) isoforms are important transducers of signals from G protein-coupled receptors (GPCRs) to diverse cellular targets, including extracellular signal-regulated kinases 1 and 2 (ERK1/2). Clone 9 rat hepatocytes (C9 cells) express receptors for angiotensin II (Ang II) type 1, lysophosphatidic acid (LPA), and epidermal growth factor (EGF), and their stimulation causes transient ERK1/2 phosphorylation through transactivation of the epidermal growth factor receptor (EGF-R). Inhibition of PKC by Go6983 [2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide], or PKC depletion by prolonged phorbol 12-myristate 13-acetate (PMA) treatment, attenuated ERK1/2 activation by Ang II and PMA, but not by LPA and EGF. In contrast, another PKC inhibitor, Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole], enhanced basal and agonist-stimulated phosphorylation of ERK1/2, which was not caused by alteration in receptor binding and internalization, stimulation of inositol phosphate production, or activation of Pyk2 and Src tyrosine kinases. However, Go6976 enhanced agonist-induced tyrosine phosphorylation of the EGF receptor, possibly through inhibition of protein tyrosine phosphatase (PTP), because the PTP inhibitor sodium orthovanadate mimicked the effects of Go6976. Selective blockade of EGF-R kinase by AG1478 [4-(3-chloroanilino)6,7-dimethoxyquinazoline] abolished the ERK1/2 activation induced by Go6976. Similar experiments were conducted in human embryonic kidney 293 cells, which express receptors for LPA and EGF but exhibit no significant cross-communication between them. Although Go6976 caused a significant increase in EGF-induced tyrosine phosphorylation of the EGF-R and subsequent ERK1/2 activation, it had no such effects on LPA-induced responses. In Chinese hamster ovary cells, which express receptors for LPA but not for EGF, Go6976 also had no significant effect on LPA-induced ERK1/2 activation. These data indicate that Go6976 potentiates agonist-induced ERK1/2 activation through stimulation of tyrosine phosphorylation of the EGF-R.     

Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production

The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)-stimulated degranulation of highly purified human eosinophils using the specific PKC activator - phorbol 12-myristate 13-acetate (PMA). C5a-induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration-dependent manner by PMA (IC(50): 3 and 5 nM, respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31-8220 (1 microM). In the presence of phosphodiesterase inhibitor rolipram (5 microM), PMA stimulated a pronounced concentration-dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC(50): 55 nM vs 22.5 microM). The inactive PMA analogue, 4alpha-PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31-8220 (1 microM) and the selective inhibitor of the novel PKCdelta, rottlerin (1-3 microM), but not the selective inhibitor of the classical PKC isoforms, G? 6976 (0.01-0.1 microM). Western blot analysis revealed the presence of six PKC isoforms (alpha, betaI, betaII, delta, iota and zeta) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA-induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKCdelta isoform, can stimulate cAMP production, and that this may be the basis for its potent anti-degranulatory effect.     

Dexamethasone-induced Ras protein 1 negatively regulates protein kinase C delta: implications for adenylyl cyclase 2 signaling

We identified dexamethasone-induced Ras protein 1 (Dexras1) as a negative regulator of protein kinase C (PKC) delta, and the consequences of this regulation have been examined for adenylyl cyclase (EC 4.6.1.1) type 2 (AC2) signaling. Dexras1 expression in human embryonic kidney 293 cells completely abolished dopamine D2 receptor-mediated potentiation of AC2 activity, which is consistent with previous reports of its ability to block receptor-mediated Gbetagamma signaling pathways. In addition, Dexras1 significantly reduced phorbol 12-myristate 13-acetate (PMA)-stimulated AC2 activity but did not alter Galpha(s)-mediated cAMP accumulation. Dexras1 seemed to inhibit PMA stimulation of AC2 by interfering with PKCdelta autophosphorylation. This effect was selective for the delta isoform because Dexras1 did not alter autophosphorylation of PKCalpha or PKCepsilon. Dexras1 disruption of PKCdelta autophosphorylation resulted in a significant blockade of PKC kinase activity as measured by [gamma-32P]ATP incorporation using a PKC-specific substrate. Moreover, Dexras1 and PKCdelta coimmunoprecipitated from whole-cell lysates. Dexras1 did not alter the membrane translocation of PKCdelta; however, the ability of Dexras1 to interfere with PKCdelta autophosphorylation was isoprenylation-dependent as determined using the farnesyltransferase inhibitor methyl {N-[2-phenyl-4-N [2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate (FTI-277) and a CAAX box-deficient Dexras1 (C277S) mutant. PMA-stimulated AC2 activity was also not affected by Dexras1 C277S. Taken as a whole, these data suggest that Dexras1 functionally interacts with PKCdelta at the cellular membrane through an isoprenylation-dependent mechanism to negatively regulate PKCdelta activity. Moreover our study suggests that Dexras1 acts to modulate the activation of AC2 in an indirect fashion by inhibiting both Gbetagamma- and PKC-stimulated AC2 activity. The current study provides a novel role for Dexras1 in signal transduction.     

Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor

Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.     

Signal transduction by protease-activated receptors

The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and β-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and β-arrestin-dependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR₁ internalizes through a clathrin- and dynamin-dependent pathway independent of β-arrestins. Interestingly, PAR₁ is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, β-arrestins mediate activated PAR₂ internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR₂ is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.     

Identification of endogenous surrogate ligands for human P2Y receptors through an in silico search

G protein-coupled receptors (GPCRs) are distributed widely throughout the human body, and nearly 50% of current medicines act on a GPCR. GPCRs are considered to consist of seven transmembrane alpha-helices that form an alpha-helical bundle in which agonists and antagonists bind. A 3D structure of the target GPCR is indispensable for designing novel medicines acting on a GPCR. We have previously constructed the 3D structure of human P2Y(1) (hP2Y(1)) receptor, a GPCR, by homology modeling with the 3D structure of bovine rhodopsin as a template. In the present study, we have employed an in silico screening for compounds that could bind to the hP2Y(1)-receptor model using AutoDock 3.0. We selected 21 of the 30 top-ranked compounds, and by measuring intracellular Ca(2+) concentration, we identified 12 compounds that activated or blocked the hP2Y(1) receptor stably expressed in recombinant CHO cells. 5-Phosphoribosyl-1-pyrophosphate (PRPP) was found to activate the hP2Y(1) receptor with a low ED(50) value of 15 nM. The Ca(2+) assays showed it had no significant effect on P2Y(2), P2Y(6), or P2X(2) receptors, but acted as a weak agonist on the P2Y(12) receptor. This is the first study to rationally identify surrogate ligands for the P2Y-receptor family.     

Modulation of GABA(A) receptors by benzodiazepines and barbiturates is autonomous of PKC activation

Previous studies have suggested that activation of calcium-phospholipid-dependent protein kinase (PKC) enhances benzodiazepine (BZD)- and pentobarbital (PB)- mediated potentiation of alpha(1)beta(1)gamma(2) GABA(A) receptors (GABA(A)-Rs). To delineate the underlying mechanism(s), voltage-clamp recordings were performed on recombinant alpha(1)beta(1)gamma(2) GABA(A) receptors functionally expressed in Xenopus laevis oocytes. GABA(A)-Rs were tested for their sensitivity to diazepam and PB before and after incubation in phorbol 12-myristate 13-acetate (PMA). PMA (25 nM) significantly attenuated the GABA(A) current (p<0.05, n=12-19) up to 90%. PMA treatment, however, did not alter the sensitivity to diazepam or pentobarbital. Similar results were obtained with recombinant alpha(1)beta(2)gamma(2) GABA receptors. These data suggest that PKC activation does not alter the allosteric modulation of GABA(A)-Rs by benzodiazepines and barbiturates and is consistent with the observation from other studies in oocytes that PMA decreases the amplitude of the GABA-activated currents via receptor internalization rather than modification of receptor kinetics.     

Acyclic analogues of adenosine bisphosphates as P2Y receptor antagonists: phosphate substitution leads to multiple pathways of inhibition of platelet aggregation

Activation by ADP of both P2Y(1) and P2Y(12) receptors in platelets contributes to platelet aggregation, and antagonists at these receptor subtypes have antithrombotic properties. In an earlier publication, we have characterized the SAR as P2Y(1) receptor antagonists of acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine. In this study, we have focused on antiaggregatory effects of P2Y antagonists related to a 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) scaffold, containing uncharged substitutions of the phosphate groups. For the known nucleotide (cyclic and acyclic) bisphosphate antagonists of P2Y(1) receptors, there was a significant correlation between inhibition of aggregation induced by 3.3 microM ADP in rat platelets and inhibition of P2Y(1) receptor-induced phospholipase C (PLC) activity previously determined in turkey erythrocytes. Substitution of the phosphate groups with nonhydrolyzable phosphonate groups preserved platelet antiaggregatory activity. Substitution of one of the phosphate groups with O-acyl greatly reduced the inhibitory potency, which tended to increase upon replacement of both phosphate moieties of the acyclic derivatives with uncharged (e.g., ester) groups. In the series of nonsymmetrically substituted monophosphates, the optimal antagonist potency occurred with the phenylcarbamate group. Among symmetrical diester derivatives, the optimal antagonist potency occurred with the di(phenylacetyl) group. A dipivaloyl derivative, a representative uncharged diester, inhibited ADP-induced aggregation in both rat (K(I) 3.6 microM) and human platelets. It antagonized the ADP-induced inhibition of the cyclic AMP pathway in rat platelets (IC(50) 7 microM) but did not affect hP2Y(1) receptor-induced PLC activity measured in transfected astrocytoma cells. We propose that the uncharged derivatives are acting as antagonists of a parallel pro-aggregatory receptor present on platelets, that is, the P2Y(12) receptor. Thus, different substitution of the same nucleoside scaffold can target either of two P2Y receptors in platelets.     

PKC modulation of GABAA receptor endocytosis and function is inhibited by mutation of a dileucine motif within the receptor beta 2 subunit

The modulation of GABAA receptors by protein kinase C is complex and involves effects on both ion channel function and receptor trafficking. Although PKC regulates receptor cell surface expression the mechanism is not well understood. Using immunofluorescence studies in HEK 293 cells, we demonstrate that activation of PKC by the phorbol ester PMA promotes receptor endocytosis and is dependent on the presence of a gamma subunit. This endocytosis is blocked by the dominant negative dynamin mutant K44A indicating that PKC-induced receptor endocytosis involves the dynamin endocytic pathway. Mutation of a dileucine motif within the receptor beta2 subunit inhibits the effect of PKC activation on receptor endocytosis. Using patch clamp analysis, we show that PKC activation produces a robust inhibition of GABA-gated chloride currents in cells expressing wildtype GABAA receptors, but it is ineffective in modulating receptors lacking the dileucine motif. Furthermore, the introduction into the patch pipette of a 10-amino acid peptide corresponding to the dileucine motif present in the receptor beta2 subunit prevents PKC modulation of wildtype recombinant receptors. Furthermore, in cerebral cortical neuronal slices inclusion of this peptide in the patch pipette prevents PKC modulation of native GABAA receptors. Using limited chymotrypsin digestion assays, we also show that PKC increases receptor internalization in primary cultures of cerebral cortical neurons. Lastly, PKC inhibitors do not block constitutive receptor endocytosis or affect GABA-gated chloride currents suggesting that PKC-dependent phosphorylation is not required for GABAA receptor endocytosis but plays a modulatory role in the process.     

Cannabinoid receptor trafficking in peripheral cells is dynamically regulated by a binary biochemical switch

The cannabinoid G protein-coupled receptors (GPCRs) CB₁ and CB₂ are expressed in different peripheral cells. Localization of GPCRs in the cell membrane determines signaling via G protein pathways. Here we show that unlike in transfected cells, CB receptors in cell lines and primary human cells are not internalized upon agonist interaction, but move between cytoplasm and cell membranes by ligand-independent trafficking mechanisms. Even though CB receptors are expressed in many cells of peripheral origin they are not always localized in the cell membrane and in most cancer cell lines the ratios between CB₁ and CB₂ receptor gene and surface expression vary significantly. In contrast, CB receptor cell surface expression in HL60 cells is subject to significant oscillations and CB₂ receptors form oligomers and heterodimers with CB₁ receptors, showing synchronized surface expression, localization and trafficking. We show that hydrogen peroxide and other nonspecific protein tyrosine phosphatase inhibitors (TPIs) such as phenylarsine oxide trigger both CB₂ receptor internalization and externalization, depending on receptor localization. Phorbol ester-mediated internalization of CB receptors can be inhibited via this switch. In primary human immune cells hydrogen peroxide and other TPIs lead to a robust internalization of CB receptors in monocytes and an externalization in T cells. This study describes, for the first time, the dynamic nature of CB receptor trafficking in the context of a biochemical switch, which may have implications for studies on the cell-type specific effects of cannabinoids and our understanding of the regulation of CB receptor cell surface expression.     

The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.     

Apparent efficacy of kappa-opioid receptor ligands on serum prolactin levels in rhesus monkeys

ADP produces a series of responses in rabbit platelets such as shape changes, aggregation and intracellular Ca2+ mobilization. In human platelets, the P2X1 receptor mediates a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) upon stimulation with ADP. We investigated whether this phenomenon is also present in rabbit platelets. We found that the P2X1 receptor-mediated response was absent because there was (1) no elevation of [Ca2+]i in response to alpha,beta-methylene-ATP, a selective P2X1 receptor agonist, in fura-2-loaded platelets; (2) no change in the ADP-induced [Ca2+]i increase and platelet aggregation after P2X1 receptor desensitization with alpha,beta-methylene-ATP; (3) complete inhibition of the ADP-induced [Ca2+]i elevation by the P2Y1 receptor specific antagonist, A3'P5'PS, with a similar ID50 value both in the presence and absence of external Ca2+. These results indicate that ADP-induced [Ca2+]i elevation is mainly mediated by P2Y1 receptors in rabbit platelets. We conclude that the P2X1 receptor does not play a significant role in the ADP-induced platelet shape changes and aggregation.     

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.     

Differential regulation of the signaling and trafficking of the two prostaglandin D2 receptors, prostanoid DP receptor and CRTH2

Prostaglandin D2 (PGD2) exerts its actions on two G protein-coupled receptors, the prostanoid DP receptor and CRTH2 (chemoattractant homologous receptor expressed on TH2 cells). Here, we characterize the regulation of the signaling and trafficking of the prostanoid DP receptor and CRTH2. Time-course and dose-response curves showed that both receptors expressed in HEK293 cells internalized maximally after 2 h of stimulation with 1 microM PGD2. Co-expression of the G protein-coupled receptor kinases GRK2, GRK5 or GRK6 increased agonist-induced internalization of CRTH2, while only GRK2 had an effect on the internalization of the prostanoid DP receptor. Protein kinase C (PKC) activation stimulated the internalization of both receptors. Interestingly, only PGD2-induced internalization of CRTH2, and not of prostanoid DP receptor, was decreased by inhibition of PKC or protein kinase A (PKA). Our data also indicate that CRTH2 is subjected to basal phosphorylation by PKA, which appears to be involved in CRTH2 internalization. Prostanoid DP receptor internalization was promoted by co-expression of arrestin-2 and -3, while the internalization of CRTH2 was increased by co-expression of arrestin-3 only. The detection of prostanoid DP receptor and CRTH2 internalization was reduced by the co-expression of Rab4 and Rab11, respectively, suggesting differential regulation of receptor recycling. Moreover, immunofluorescence microscopy experiments showed that the prostanoid DP receptor specifically co-localized with Rab4, and CRTH2 with Rab11. The signaling of the prostanoid DP receptor was regulated by GRK2 overexpression, while that of CRTH2 was modulated by overexpression of GRK2, -5 and -6. Our results show a differential regulation of the prostanoid DP receptor and CRTH2, two receptors for PGD2.