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-Opioid Receptor Signals through Src and Focal Adhesion Kinase to Stimulate c-Jun N-Terminal Kinases in Transfected COS-7 Cells and Human Monocytic THP-1 Cells
Abstract
Opioid peptides exert diverse physiological functions through their cognate receptors. One subtype of the opioid receptors, kappa-opioid receptor, is endogenously expressed in human monocytic THP-1 cells. Stimulation of the THP-1 cells with a kappa-opioid receptor-selective agonist exerted a Gi-dependent activation of c-Jun N-terminal kinase (JNK). To further investigate the signaling mechanism by which the kappa-opioid receptor regulates JNK activity, heterologous expression assays in COS-7 cells were utilized. Overexpression of Galphat in COS-7 cells clearly suppressed kappa-opioid receptor-stimulated JNK activity, indicating that the pathway is primarily regulated by Gbetagamma. In both THP-1 and transfected COS-7 cells, pretreatment of the selective Src family kinase inhibitor pyrazolopyrimidine PP1 abolished the JNK activation, whereas the epidermal growth factor receptor inhibitor AG1478 [N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinanine] failed to do that. Furthermore, the JNK activation in response to kappa-opioid receptor was suppressed by an autophosphorylation-resistant mutant of focal adhesion kinase (FAK). Consistently, activated kappa-opioid receptor induced Src stimulation and FAK autophosphorylation and promoted the formation of Src-FAK complex. The participation of small GTPases as well as a guanine nucleotide exchange factor was also implicated because dominant-negative mutants of Rac, Cdc42, and Son-of-sevenless (Sos) attenuated the agonist-induced activation of JNK. These studies demonstrate that the activation of JNK by kappa-opioid receptors is routed via Gbetagamma, Src, FAK, Sos, Rac, and Cdc42.
... GPCRs, particularly those selectively coupled to the G i and G q families of G proteins, are efficiently linked to the activation of JNK. G i -coupled receptors mainly require a Gβγ/Src-dependent mechanism to stimulate the JNK cascade (Chan and Wong, 2004a;Kam et al., 2004), while G q -coupled receptors utilize both Gβγ/Src and Ca 2+ signals to regulate the kinase activity (Chan and Wong, 2004b). In addition to the participation of Src-like kinases, functional activities of PI3K isoforms and the trans-activation of epidermal growth factor receptors (EGFR) have been proposed as alternative routes for the Gβγ-mediated pathway (Lopez-Ilasaca et al., 1998;Murga et al., 2000;Pierce et al., 2001). ...
... We have previously demonstrated that transfected Cos-7 cells transiently expressing GPCRs are useful cellular models to study the activation of JNK mediated by G i -and G q -coupled receptors Chan et al., 2002;Chan and Wong, 2004a), with the experimental results highly consistent with those obtained from endogenous cellular systems (Kam et al., 2003;Kam et al., 2004;Chan and Wong, 2004b). In this report, we utilized Cos-7 cells transfected with G s -coupled dopamine D 1 receptor (D 1 R) to illustrate the signaling mechanism for JNK activation, and further investigated the effects of co-stimulation of G scoupled receptor with G i -or G q -coupled receptors on the JNK activity. ...
... In Cos-7 cells, both G i -and G qcoupled receptors trigger JNK activation via a Gβγ/Src-dependent mechanism, which can be suppressed by dominant negative mutants of both Sos (i.e. Son of Sevenless) and small GTPases (Chan and Wong, 2004b;Kam et al., 2004). These findings clearly support the involvement of GEFs and small GTPases in the activation of JNK by GPCRs. ...
... GPCRs, particularly those selectively coupled to the G i and G q families of G proteins, are efficiently linked to the activation of JNK. G i -coupled receptors mainly require a G␥/Src-dependent mechanism to stimulate the JNK cascade (Chan and Wong, 2004a;Kam et al., 2004), whereas G q -coupled receptors use both G␥/Src and Ca 2ϩ signals to regulate the kinase activity (Chan and Wong, 2004b). In addition to the participation of Src-like kinases, functional activities of PI3K isoforms and the transactivation of epidermal growth factor receptors (EGFRs) have been proposed as alternative routes for the G␥-mediated pathway (Lopez-Ilasaca et al., 1998;Murga et al., 2000;Pierce et al., 2001). ...
... We have demonstrated previously that transfected COS-7 cells transiently expressing GPCRs are useful cellular models to study the activation of JNK mediated by G i -and G qcoupled receptors Wong, 2000, 2004a;Chan et al., 2002), with the experimental results highly consistent with those obtained from endogenous cellular systems (Kam et al., 2003(Kam et al., , 2004Chan and Wong, 2004b). In this report, we used COS-7 cells transfected with G s -coupled dopamine D 1 receptor (D 1 R) to illustrate the signaling mechanism for JNK activation and further investigated the effects of costimulation of G s -coupled receptor with G i -or G q -coupled receptors on the JNK activity. ...
... The activity of Ras-GRF1 (a GEF for Ras and Rac) can be promoted through tyrosine phosphorylation by Src, which in turn leads to activation of JNK in a Rac-dependent manner (Kiyono et al., 2000). In COS-7 cells, both G i -and G q -coupled receptors trigger JNK activation via a G␥/Src-dependent mechanism, which can be suppressed by dominant-negative mutants of both Sos (i.e., Son of Sevenless) and small GTPases (Chan and Wong, 2004b;Kam et al., 2004). These findings clearly support the involvement of GEFs and small GTPases in the activation of JNK by GPCRs. ...
G protein-coupled receptors (GPCRs) of Gi- or Gq-coupling specificity are effectively linked to activation of the c-Jun N-terminal kinase (JNK) cascade. However, little is known with regard to the regulation of JNK by Gs-coupled receptors. In this report, we used COS-7 cells transfected with the dopamine D1 receptor (D1R) to illustrate the signaling mechanism for Gs-mediated JNK activation. Stimulation of D1R triggered a weak but significant elevation of JNK activity in a time- and dose-dependent manner. This D1R-mediated JNK activation required the participation of Gbetagamma, Src-like kinases, and small GTPases, whereas disruptions of cAMP-, phosphoinositide-3-kinase-, and epidermal growth factor receptor-mediated signaling had no effect. Costimulation of D1R with GPCRs of other coupling specificities resulted in differential activation profiles of JNK. Activation of Gs-coupled D1R weakly potentiated the JNK activation induced by the Gi-coupled opioid receptor-like receptor, but it exhibited a significant inhibitory effect on the kinase activity triggered by the Gq-coupled gastrin-releasing peptide-preferring bombesin receptor (GRPR). Administration of Spadenosine-3',5'-cyclic monophosphorothioate triethylamine (a cAMP analog that mimics the Gs/cAMP signal) also suppressed the JNK activation mediated by Gq-coupled GRPR, as well as the Ca2+-induced kinase activation upon thapsigargin treatment. Moreover, the Ca2+ signal from GRPR synergistically potentiated the D1R-triggered cAMP elevation when the two receptors were stimulated simultaneously. Taken together, our results demonstrated that stimulation of Gs-coupled receptors in COS-7 cells not only enhanced the JNK activity, but also exhibited a "tuning" effect on the kinase activation mediated by GPCRs of other coupling specificities.
... JNK activation requires phosphorylation of a threonine and tyrosine residue in the activation loop and the canonical mitogen-activated protein kinase kinases (MAPKK) MKK4 and MKK7 have been shown to phosphorylate these residues directly [13,14]. Additional kinases have been implicated in opioid receptor mediated JNK activation upstream of the MAPKKs, including PKC and Src [1,[15][16][17]. In some instances, arrestin has been shown to act as a scaffold for JNK activation recruiting MKK4 and MKK7, in addition to ASK1, in close proximity with JNK [18][19][20]. ...
... In addition to an arrestin-dependent mechanism, Src kinase has also been implicated in JNK activation by opioid receptors [15][16][17]. To investigate the role of Src kinases, MOR-GFP expressing HEK293 cells were treated with vehicle, 10μM morphine, or 10μM fentanyl for 30 min. ...
G protein-coupled receptor desensitization is typically mediated by receptor phosphorylation by G protein receptor kinase (GRK) and subsequent arrestin binding; morphine, however, was previously found to activate a c-Jun N-Terminal Kinase (JNK)-dependent, GRK/arrestin-independent pathway to produce mu opioid receptor (MOR) inactivation in spinally-mediated, acute anti-nociceptive responses Melief et. al., 2010 [1]. In the current study, we determined that JNK2 was also required for centrally-mediated analgesic tolerance to morphine using the hotplate assay. We compared JNK activation by morphine and fentanyl in JNK1(-/-), JNK2(-/-), JNK3(-/-), and GRK3(-/-) mice and found that both compounds specifically activate JNK2 in vivo; however, fentanyl activation of JNK2 was GRK3-dependent, whereas morphine activation of JNK2 was GRK3-independent. In MOR-GFP expressing HEK293 cells, treatment with either arrestin siRNA, the Src family kinase inhibitor PP2, or the protein kinase C (PKC) inhibitor Gö6976 indicated that morphine activated JNK2 through an arrestin-independent Src- and PKC-dependent mechanism, whereas fentanyl activated JNK2 through a Src- GRK3/arrestin2-dependent and PKC-independent mechanism. This study resolves distinct ligand-directed mechanisms of JNK activation by mu opioid agonists and understanding ligand-directed signaling at MOR may improve opioid therapeutics.
Copyright © 2015. Published by Elsevier Inc.
... for GPCR-mediated MAPK activation (Lopezllasaca et al. 1998). However, we have demonstrated that some GPCRs do not require PI3K for stimulating MAPKs (Chan and Wong 2000;Kam et al. 2004), and the lack of effect of wortmannin (Fig. 3) supports this notion. Other upstream activators, such as PKC and CaMK II , have also been demonstrated to play critical roles in the ERK pathway (Brandlin et al. 2003;Illario et al. 2003). ...
... FAK and Pyk2) are activated by Ca 2+ or PKC, which in turn recruit intermediates such as Src-family tyrosine kinases and GEFs for signal transduction (Dikic et al. 1996;Blaukat et al. 1999). In fact, the involvements of FAK members in the GPCR-induced MAPK activation have been demonstrated previously (Kam et al. 2004). ...
Mammalian cells often receive multiple extracellular stimuli under physiological conditions, and the various signaling inputs have to be integrated for the processing of complex biological responses. G protein-coupled receptors (GPCRs) are critical players in converting extracellular stimuli into intracellular signals. In this report, we examined the integration of different GPCR signals by mitogen-activated protein kinases (MAPKs) using the SK-N-MC human brain neuroepithelioma cells as a neuronal model. Stimulation of the Gi-coupled neuropeptide Y1 and Gq-coupled muscarinic M1 acetylcholine receptors, but not the Gs-coupled dopamine D1 receptor, led to the activation of extracellular signal-regulated kinase (ERK). All three receptors were also capable of stimulating c-Jun NH2-terminal kinases (JNK) and p38 MAPK. The Gi-mediated ERK activation was completely suppressed upon inhibition of Src tyrosine kinases by PP1, while the Gq-induced response was suppressed by both PP1 and the Ca2+ chelator, BAPTA-AM. In contrast, activations of JNK and p38 by Gs-, Gi-, and Gq-coupled receptors were sensitive to PP1 and BAPTA-AM pretreatments. Simultaneous stimulation of Gi- and Gq-coupled receptors resulted in the synergistic activation of ERK, but not JNK or p38 MAPK. The Gi/Gq-induced synergistic ERK activation was PTX-sensitive, and appeared to be a co-operative effect between Ca2+ and Src family tyrosine kinases. Enhanced ERK activation was associated with an increase in CREB phosphorylation, while the JNK and p38-responsive transcription factor ATF-2 was weakly enhanced upon Gi/Gq-induction. This report provides evidence that G protein signals can be integrated at the level of MAPK, resulting in differential effects on ERK, JNK and p38 MAPK in SK-N-MC cells.
... In addition, κ-OR activates the extracellular signal-regulated kinase (ERK 1/2) and p38 mitogen-activated protein kinase (MAPK) [32,33] and can activate c-Jun amino-terminal kinase (JNK) [34]. Interestingly, prototypical κ-OR antagonists can also activate JNK via a pharmacological process called biased agonism or ligand-directed signaling, whereby ligands can inhibit one intracellular signaling pathway while simultaneously activating another [35,36]. ...
Indoles constitute a large family of heterocyclic compounds widely occurring in nature which are present in a number of bioactive natural and synthetic compounds, including anticancer agents or atypical opioid agonists. As a result, exponential increases in the development of novel methods for the synthesis of indole-containing compounds have been reported in the literature. A series of indole-aryl amide derivatives 1–7 containing tryptamine or an indolylacetic acid nucleus were designed, synthesized, and evaluated as opioid ligands. These new indole derivatives showed negligible to very low affinity for μ- and δ-opioid receptor (OR). On the other hand, compounds 2, 5, and 7 showed Ki values in the low μM range for κ-OR. Since indoles are well known for their anticancer potential, their effect against a panel of tumor cell lines was tested. The target compounds were evaluated for their in vitro cytotoxicity in HT29, HeLa, IGROV-1, MCF7, PC-3, and Jurkat J6 cells. Some of the synthesized compounds showed good activity against the selected tumor cell lines, with the exception of IGROV1. In particular, compound 5 showed a noteworthy selectivity towards HT29 cells, a malignant colonic cell line, without affecting healthy human intestinal cells. Further studies revealed that 5 caused the cell cycle arrest in the G1 phase and promoted apoptosis in HT29 cells.
... The activation of Src kinase under the KOR, DOR and MOR exerts varied physiological outcomes. Upon stimulation, KOR regulates the inflammatory response by inducing a Gβγ-dependent activation of Src resulting in JNK activation routed via RAC1 and CDC42 in THP1 cells (Kam et al. 2004b). Interestingly, under DOR, the Src cascade activates MAPK1/3 which in turn phosphorylates MAPK14, leading to the maintenance of glutamate homeostasis in mouse astrocyte cultures (Liang et al. 2014). ...
Opioid receptors belong to the class A G-protein-coupled receptors and are activated by alkaloid opiates such as morphine, and endogenous ligands such as endorphins and enkephalins. Opioid receptors are widely distributed in the human body and are involved in numerous physiological processes through three major classical opioid receptor subtypes; the mu, delta and kappa along with a lesser characterized subtype, opioid receptor-like (ORL1). Opioids are the most potent analgesics and have been extensively used as a therapeutic drug for the treatment of pain and related disorders. Chronic administration of clinically used opioids is associated with adverse effects such as drug tolerance, addiction and constipation. Several investigations attempted to identify the molecular signaling networks associated with endogenous as well as synthetic opiates, however, there is a paucity of a cumulative depiction of these signaling events. Here, we report a systemic collection of downstream molecules pertaining to four subtypes of opioid receptors (MOR, KOR, DOR and ORL1) in the form of a signaling pathway map. We manually curated reactions induced by the activation of opioid receptors from the literature into five categories- molecular association, activation/inhibition, catalysis, transport, and gene regulation. This led to a dataset of 180 molecules, which is collectively represented in the opioid receptor signaling network following NetPath criteria. We believe that the public availability of an opioid receptor signaling pathway map can accelerate biomedical research in this area because of its high therapeutic significance. The opioid receptors signaling pathway map is uploaded to a freely available web resource, WikiPathways enabling ease of access (https://www.wikipathways.org/index.php/Pathway:WP5093).
... Src family kinases (SFKs) -non-receptor protein tyrosine kinases that are activated after CXCR4 stimulation 54 -are known to regulate MOR and DOR recycling and phosphorylation, and to contribute to the desensitization of these receptors in vitro [55][56][57][58][59] . We have recently demonstrated the in vivo participation of SFKs in opioid-receptor functions 8 . ...
Chemokines and opioids are important regulators of immune, inflammatory and neuronal responses in peripheral and central pain pathways. Recent studies have provided insights into the functional interactions between chemokine receptors and opioid receptors, and their role in pain modulation. In this Progress article, we discuss how crosstalk between these two systems might provide a molecular and cellular framework for the development of novel analgesic therapies for the management of acute and/or chronic pain.
... In COS7 cells co-transfected with JNK and KOPr, activation of KOPr with U50,488H leads to an increase in JNK phosphorylation that is independent of P13K (Kam et al. 2004b). A further study proposed that KOPr activation of JNK occurred through a G i/o G βγ , src tyrosine kinase, focal adhesion kinase and Rac, Cdc42 and son-of-sevenless (sos) dependent pathway (Kam et al. 2004a). KOPr antagonists such as nor-BNI are thought to act by disrupting the KOPr signaling pathway, possibly by activation of the JNK phosphorylation pathway (Bruchas et al. 2007b). ...
... Beside the microglial activation of SFK, we also reported a neuronal activation of Src family kinases in sensory neurons and in nerve terminal containing MOR and DOR. Src family kinases influence MOR and DOR trafficking and signaling in vitro (Kramer et al., 2000;Kam et al., 2004;Walwyn et al., 2007;Archer-Lahlou et al., 2009;Zhang et al., 2009). Blockade of the SFK signaling pathway in mouse primary sensory neuron culture with PP2 (a specific vehicle (saline or saline + DMSO). ...
Functional interactions between the chemokine receptor CXCR4 and opioid receptors have been reported in the brain, leading to a decreased morphine analgesic activity. However the cellular mechanisms responsible for this loss of opioid analgesia are largely unknown. Here we examined whether Src family-kinases (SFK)-linked mechanisms induced by CXCR4 contributed to the loss of acute morphine analgesia and could represent a new physiological anti-opioid signaling pathway. In this way, we showed by immunohistochemistry and western blot that CXCL12 rapidly activated SFK phosphorylation in vitro in primary cultured lumbar rat dorsal root ganglia (DRG) but also in vivo in the DRG and the spinal cord. We showed that SFK activation occured in a sub population of sensory neurons, in spinal microglia but also in spinal nerve terminals expressing mu- (MOR) and delta-opioid (DOR) receptor. In addition we described that CXCR4 is detected in MOR- and DOR-immunoreactive neurons in the DRG and spinal cord. In vivo, we demonstrated that an intrathecal administration of CXCL12 (1 μg) significantly attenuated the subcutaneous morphine (4 mg/kg) analgesia. Conversely, pretreatment with a potent CXCR4 antagonist (5 μg) significantly enhanced morphine analgesia. Similar effects were obtained after an intrathecal injection of a specific SFK inhibitor, PP2 (10 μg). Furthermore, PP2 abrogated CXCL12-induced decrease in morphine analgesia by suppressing SFK activation in the spinal cord. In conclusion, our data highlight that CXCL12- induced loss of acute morphine analgesia is linked to Src family kinases activation.
... Recent reports suggest the interaction of KOR antagonists with the KOR may be unique, where the KOR antagonists nor-BNI, JDTic, and 59-guanidinonaltrindole (as well as selective KOR agonists) activate the c-Jun-N-terminal kinase (JNK) mitogen-activated protein kinase cascade, an action the authors suggest is responsible for prolonged KOR antagonism (Bruchas et al., 2007; Melief et al., 2011 ). Although KORselective agonists activate JNK through a G bg -proteinmediated mechanism (Kam et al., 2004), nor-BNI does not activate G bg -proteins, leaving the mechanism mediating the observed JNK activation unknown. It has been suggested that a time-sensitive mechanism is required for the long-lasting antagonism, possibly involving ligand-specific compensatory changes in second messenger signaling (Bruchas and Chavkin, 2010). ...
In the mouse 55°C warm-water tail-withdrawal assay, a single administration of nor-BNI (10 mg/kg, i.p.) antagonized kappa opioid receptor (KOR) agonist-induced antinociception up to 14 days, whereas naloxone (10 mg/kg, i.p.) mediated antagonism lasted less than a day. In saturation binding experiments, mouse brain membranes isolated and washed 1 or 7, but not 14 days after nor-BNI administration demonstrated a significant time dependent decrease in maximal KOR agonist [(3)H]U69,593 binding. To determine whether brain concentrations of nor-BNI were sufficient to explain the antagonism of KOR-mediated antinociception, mouse blood and perfused brain were harvested at time points ranging from 30 min to 21 days after a single administration and analyzed for the presence of nor-BNI using LC-MS/MS. Nor-BNI was detected in the perfused brain homogenate up to 21 days after administration (30 nmol, i.c.v. or 10 mg/kg, i.p.). Subsequent experiments in which nor-BNI was administered at doses estimated from the amounts detected in the brain homogenates isolated from pretreated mice over time demonstrated significant antagonism of U50,488 antinociception in a manner consistent with the magnitude of observed KOR antagonism. The dose (1.4 nmol) approximating the lowest amount of nor-BNI detected in brain on day 14 did not antagonize U50,488-induced antinociception, consistent with the absence of U50,488 antagonism observed in vivo at this time point after pre-treatment. Overall, the physical presence of nor-BNI in the mouse brain paralleled its in vivo pharmacological profile, suggesting physico-chemical and pharmacokinetic properties of nor-BNI may contribute to the prolonged KOR antagonism.
... KOR activation by endogenous or synthetic agonists can produce inhibition of adenylate cyclase activity (Attali et al. 1989;Konkoy and Childers 1989;Lawrence and Bidlack 1993) and can decrease cell excitability and neurotransmitter release by altering calcium and potassium currents (Gross et al. 1990;Henry et al. 1995;Hjelmstad and Fields 2003;Rusin et al. 1997;Simmons and Chavkin 1996;Tallent et al. 1994). KOR activation has also been shown to activate mitogen activated protein kinase (MAPK) pathways in neurons and astrocytes (Belcheva et al. 2005;Belcheva et al. 1998;Bohn et al. 2000;Bruchas et al. 2007a;Bruchas et al. 2006;Kam et al. 2004). The MAPK family includes several kinases that respond to a variety of cell stimuli and regulate diverse functions such as proliferation, differentiation, apoptosis, and gene expression. ...
Accumulating evidence indicates that brain kappa-opioid receptors (KORs) and dynorphin, the endogenous ligand that binds at these receptors, are involved in regulating states of motivation and emotion. These findings have stimulated interest in the development of KOR-targeted ligands as therapeutic agents. As one example, it has been suggested that KOR antagonists might have a wide range of indications, including the treatment of depressive, anxiety, and addictive disorders, as well as conditions characterized by co-morbidity of these disorders (e.g., post-traumatic stress disorder) A general effect of reducing the impact of stress may explain how KOR antagonists can have efficacy in such a variety of animal models that would appear to represent different disease states.
Here, we review evidence that disruption of KOR function attenuates prominent effects of stress. We will describe behavioral and molecular endpoints including those from studies that characterize the effects of KOR antagonists and KOR ablation on the effects of stress itself, as well as on the effects of exogenously delivered corticotropin-releasing factor, a brain peptide that mediates key effects of stress.
Collectively, available data suggest that KOR disruption produces anti-stress effects and under some conditions can prevent the development of stress-induced adaptations. As such, KOR antagonists may have unique potential as therapeutic agents for the treatment and even prevention of stress-related psychiatric illness, a therapeutic niche that is currently unfilled.
... In addition to the regulatory effects on adenylate cyclase and ion channel conductance typical of G i/o -coupled receptors, sustained activation of -opioid receptors leads to the activation mitogen-activated protein kinase pathway (MAPK) in heterologous systems and native cells (Belsheva et al., 2005;Bruchas et al., 2006). Using in vitro assays, agonists have been shown to activate all three MAPK family members, including the extracellular signal-regulated kinase (ERK), the stress kinase c-Jun N-terminal kinase (JNK), and the stress kinase p38 in transfected cells and primary cultured astrocytes and neurons (Kam et al., 2004;Belsheva et al., 2005;Bruchas et al., 2006). However, KOR-mediated MAPK activation in vivo has not yet been demonstrated, and a possible relationship between p38 MAPK activation and -induced behaviors has not been established. ...
... However, the ability of nor-BNI to block the effects of KOR activation appears to be due to c-Jun N-terminal Kinase (JNK) MAPK phosphorylation (Bruchas et al., 2007b). Although U50,488 also leads to JNK phosphorylation, this activation occurs in a pertussis-toxin-sensitive manner (Kam et al., 2004) whereas nor-BNI induced JNK phosphorylation is insensitive to pertussis toxin (Bruchas et al., 2007b). Although purely speculative at this point, it is possible that restraint-induced anxiogenic-like behaviors observed in rats with a history of ethanol dependence may be regulated independently of KOR-activated p38 MAPK signaling and/or pertussis-toxin-sensitive JNK phosphorylation. ...
Although recent work suggests that the dynorphin/kappa opioid receptor (DYN/KOR) system may be a key mediator in the stress-related effects of alcohol, the regulation of long-term changes associated with protracted withdrawal from ethanol via the DYN/KOR system has yet to be explored. The objective of the present study was to determine the role of the DYN/KOR system in the regulation of anxiety-related behaviors during an extended period of abstinence from ethanol in animals with a history of ethanol dependence. Male Wistar rats (n = 94) were fed an ethanol or control liquid diet for 25-30 days. Six weeks after its removal, rats were exposed to 20 min of immobilization, and the ability of the KOR antagonist nor-binaltorphimine (nor-BNI) (0-20 mg/kg, intraperitoneal [i.p.]) to attenuate the enhanced responsiveness to stress observed in rats chronically exposed to ethanol was investigated using the elevated plus maze. In addition, the ability of U50,488 (0-10 mg/kg, i.p.) to prime anxiety-like behavior during protracted withdrawal was also examined. Rats with a history of ethanol dependence showed a significant decrease in open-arm exploration after exposure to restraint, indicating an anxiety-like state, compared to similarly treated controls, an effect that was blocked by nor-BNI. nor-BNI also selectively decreased center time and open-arm approaches in ethanol-exposed rats. The highest dose of U50,488 decreased open-arm exploration and the total number of arm entries in ethanol-exposed and control rats. Although lower doses of U50,488 did not affect open-arm exploration in either group, the 0.1 mg/kg dose selectively decreased motor activity in the ethanol-exposed rats when compared to similarly pretreated controls. These findings further support the hypothesis that behaviors associated with withdrawal from ethanol are in part regulated by the DYN/KOR system, and suggest that these effects may be long lasting in nature.
... 16 In addition, KOR activates the extracellular signal-regulated kinase (ERK 1/2) and p38 mitogen-activated protein kinase (MAPK) [17][18][19] and can activate c-Jun aminoterminal kinase (JNK). 20 Interestingly, prototypical KOR antagonists can also activate JNK via a pharmacological process called biased agonism or ligand-directed signaling, whereby ligands can inhibit one intracellular signaling pathway while simultaneously activating another. 21,22 Relatively early studies showed that activation of the MOR elevates mood, whereas KOR activation produces dysphoria and psychotomimetic effects in humans 23 and anhedonia-, dysphoria-, and anxiety-like effects in rodents 24 (see ref. 25 for a review). ...
Kappa opioid receptors (KORs) belong to the G-protein coupled class of receptors (GPCRs). They are activated by the endogenous opioid peptide dynorphin (DYN) and expressed at particularly high levels within brain areas implicated in modulation of motivation, emotion, and cognitive function. Chronic activation of KORs in animal models has maladaptive effects including increases in behaviors that reflect depression, the propensity to engage in drug-seeking behavior, and drug craving. The fact that KOR activation has such a profound influence on behaviors often triggered by stress has led to interest in selective KOR antagonists as potential therapeutic agents. This perspective provides a description of preclinical research conducted in the development of several different classes of selective KOR antagonists, a summary of the clinical studies conducted thus far, and recommendations for the type of work needed in the future to determine if these agents would be useful as pharmacotherapies for neuropsychiatric illness.
... It should be noted that the stimulation of KOR suppresses cAMP production through Gi proteins, which leads to the inactivation of PKA (Tso and Wong, 2003). Furthermore, the stimulation of KOR invokes the JNK cascade (Kam et al, 2004). Although the exact mechanism of ...
It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial.
The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth.
Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3β (p-GSK3β).
The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3β.
... Furthermore, functional -, and -opioid receptors belonging to the G-protein-coupled receptor (GPCR) superfamily (Law et al., 2000) have been demonstrated to be expressed by immune cells (Martin-Kleiner and Gabrilovac, 1997). Interestingly, the same intracellular signaling cascade for different opioid receptors (Lawrence and Bidlack, 1993;Sharp et al., 1996;Wang et al., 2003) and multiple downstream signaling mechanisms for the same opioid receptor (Bohn et al., 2000;Kam et al., 2004) have been reported, depending on which mammalian cell lines were studied. With regard to ectotherms, currently there is only one report exploring the intracellular signaling cascade for -endorphin action on immune cells (Kumar et al., 2011). ...
This in vitro study of the wall lizard Hemidactylus flaviviridis demonstrates the role of the opioid peptide dynorphin A((1-17)) [dyn A((1-17))] in the regulation of the phagocytic activity of splenic phagocytes. Dyn A((1-17)) in a concentration-dependent manner inhibited the phagocytic activity, and the maximum inhibition was recorded at a concentration of 10(-9) mol l(-1). To explore the receptor-mediated effect of dyn A((1-17)), cells were treated simultaneously with the non-selective opioid receptor blocker naltrexone and dyn A((1-17)). Naltrexone completely blocked the inhibitory effect of dyn A((1-17)) on phagocytosis. Moreover, the involvement of selective opioid receptors was investigated using selective opioid receptor antagonists. CTAP and naltrindole, selective μ- and δ-opioid receptor blockers, respectively, failed to block the inhibitory effect of dyn A((1-17)) on phagocytosis. However, the selective κ-opioid receptor blocker NorBNI completely antagonized the inhibitory effect of dyn A((1-17)). Regarding the κ-opioid receptor-coupled downstream signaling cascade, the adenylate cyclase (AC) inhibitor SQ 22536 and protein kinase A (PKA) inhibitor H-89 decreased the inhibitory effect of dyn A((1-17)) on phagocytosis. Furthermore, treatment with dyn A((1-17)) caused an increase in intracellular cAMP content in splenic phagocytes. Thus, it can be concluded that, in H. flaviviridis, dyn A((1-17)) negatively regulates the phagocytic activity of splenic phagocytes by acting through κ-opioid receptors that are coupled with the AC-cAMP-PKA signal transduction mechanism.
... Stimulation of KORs activates all three members of the MAPK family of kinases-extracellular regulated kinase (ERK1/2), p38 stress kinase (p38), and c-Jun N-terminal kinase (JNK)-in various cell preparations, including neurons and astrocytes (Bohn et al., 2000;Kam et al., 2004;Belcheva et al., 2005;Bruchas et al., 2006;McLennan et al., 2008). KORs are coupled to inhibitory G αi -proteins that suppress the activity of cAMPdependent kinases and thereby alter ion channel conductances as well as intracellular signaling cascades. ...
Stress is most often associated with aversive states. It rapidly induces the release of hormones and neuropeptides including dynorphin, which activates kappa opioid receptors (KORs) in the central and peripheral nervous systems. In animal models, many aversive effects of stress are mimicked or exacerbated by stimulation of KORs in limbic brain regions. Although KOR signaling during acute stress may increase physical ability (by producing analgesia) and motivation to escape a threat (by producing aversion), prolonged KOR signaling in response to chronic or uncontrollable stress can lead to persistent expression of behavioral signs that are characteristic of human depressive disorders (i.e., "prodepressive-like" signs). Accumulating evidence suggests that KORs contribute to the progressive amplification (sensitization) of stress-induced behaviors that occurs with repeated exposure to stress. Many of the aversive effects of stress are blocked by KOR antagonists, suggesting that these agents may have potential as therapeutics for stress-related conditions such as depression and anxiety disorders. This review summarizes current data on how KOR systems contribute to the acute (rapid), delayed, and cumulative molecular and behavioral effects of stress. We focus on behavioral paradigms that provide insight on interactions between stress and KOR function within each of these temporal categories. Using a simplified model, we consider the time course and mechanism of KOR-mediated effects in stress and suggest future directions that may be useful in determining whether KOR antagonists exert their therapeutic effects by preventing the development of stress-induced behaviors, the expression of stress-induced behaviors, or both.
... Although the PLCβ activation was not completely attenuated by the alanine mutants, diminished flux of intracellular Ca 2+ might be already enough to suppress the JNK activity. Several studies from our laboratory have shown that Gβγ complex is a mediator of JNK activation by various GPCRs [32][33][34][35][36], but such regulation is abolished upon suppression of the Ca 2+ -activated SOS/ Rac pathway [32,33]. The preservation of ERK activation did not alleviate the suppressive effects of the mutants on ...
Galpha16 can activate phospholipase Cbeta (PLCbeta) directly like Galphaq. It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCbeta and TPR1 interact with the same regions on Galpha16. Previous studies on Galphaq have defined two minimal clusters of amino acids that are essential for the coupling to PLCbeta. Cognate residues in Galpha16 might also be essential for interacting with PLCbeta, and possibly contribute to TPR1 interaction and other signaling events.
Alanine mutations were introduced to the two amino acid clusters (246-248 and 259-260) in the switch III region and alpha3 helix of Galpha16. Regulations of PLCbeta and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Galpha16 was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor kappaB (NF-kappaB) were not significantly affected by these mutations. The interactions between the mutants and PLCbeta2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G16 and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the alpha3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gbetagamma release.
The integrity of the switch III region and alpha3 helix of Galpha16 is critical for the activation of PLCbeta, STAT3, and JNK but not ERK or NF-kappaB. Binding of Galpha16 to PLCbeta2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G16. The studied region was shown to bear multiple functionally important roles of G16.
... Agonist stimulation of KOR leads to inhibition of adenylate cyclase, activation of potassium channels and inhibition of calcium channels, typical of G i/o coupled receptors. Using in vitro approaches, kappa agonists have also been shown to activate all three MAPK family members including the extracellular signal-regulated kinase (ERK1/2), the stress kinase c-Jun N-terminal kinase (JNK), and the stress kinase p38 [5,10111213. KOR-mediated p38 MAPK activation requires receptor phosphorylation by G-protein coupled receptor kinase 3 and β-arrestin recruitment in vivo and in vitro [5,11]. ...
Earlier studies identified the dynorphin-kappa opioid receptor (KOR) system as a critical mediator of dysphoria-induced aversion after repeated stress exposure, but the molecular signaling mechanisms were not fully characterized. In this study we report that repeated forced swim stress caused a significant phosphorylation of extracellular signal-regulated kinase (ERK)1/2 a mitogen-activated protein kinase (MAPK) in both the caudate and nucleus accumbens regions of the mouse striatum. Activation was blocked by the KOR antagonist, norbinaltorphimine, and absent in KOR knockout mice. In contrast to p38-MAPK activation by stress-induced dynorphin release, KOR-mediated ERK1/2 phosphorylation was not dependent on G-protein coupled receptor kinase 3 expression. These results indicate stress-induced activation of the dynorphin-KOR systems activates ERK1/2 MAPK signaling, and this may contribute to the behavioral responses to repeated stress exposure.
... Protein loading was controlled with a protein detection kit (Pierce). Although opioid receptors have no intrinsic tyrosine kinase activity, they activate kinases such as Src and the focal adhesion kinase (FAK) (26). To analyze the mechanism involved in DPDPE-mediated Vav-1 activation, we lysed unstimulated and DPDPE-stimulated MonoMac-1 cells, immunoprecipitated cell extracts with anti-DOR Ab, followed by Western blot with anti-Src Ab (Fig. 5B), and observed DPDPE-mediated Src association to DOR. ...
Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.
... Activation of Src kinases is associated with phosphorylation of the tyrosine residues (tyr416 for Src and tyr396 for Lyn) in the catalytic region [Src homology 3 (SH3) and SH2 domains], and phosphorylation of tyrosine residues (tyr527 for Src and tyr507 for Lyn) in the C-terminal tail down-regulates activity of Src kinases [30]. When Src kinases become activated, they interact with receptor tyrosine kinases and other intracellular signaling pathways such as MAPKs [31][32][33][34], which phosphorylate STAT3 on the ser727 residue, providing it with a necessary component for optimal transcriptional activity [35][36][37]. As previous publications reported that alcohol enhanced IL-10 production, with or without concomitant, pathogen-derived stimuli, we focused our attention on the involvement of Src kinases in alcohol-induced IL-10. ...
Alcohol consumption is associated with an imbalance in pro- and anti-inflammatory cytokines and immunosuppression, partially as a result of enhanced IL-10 production. The mechanisms of IL-10 induction by alcohol remain poorly understood. We identified that increased IL-10 production in human monocytes after acute in vivo alcohol consumption or in vitro alcohol treatment was associated with increased STAT3 activation. Alcohol alone induced and in combination with LPS augmented STAT3 phosphorylation at tyrosine 705 (tyr705) and serine 727 (ser727) residues and increased STAT3 binding to DNA. Upstream, alcohol activated the Src kinases, as indicated by an increase in phosphorylated and a decrease in nonphosphorylated Src proteins. STAT3 activation by Src kinases occurred directly at the tyr705 residue and indirectly at the ser727 residue via JNK MAPKs. Using specific Src (PP2), JNK1/2 (SB600125), or p38 (SB203580) inhibitors, we determined that alcohol treatment alone induced and together with LPS, augmented the DNA-binding capacity of the specificity protein-1 (Sp-1) and AP-1 transcription factors involved in IL-10 production via Src-mediated activation of p38 MAPK and JNK, respectively. Our data suggest that acute alcohol activates Src/STAT3 and Src/MAPK/STAT3, AP-1, and Sp-1 pathways as important mechanisms for IL-10-mediated immunomodulation after acute alcohol use.
... As shown in Fig. 9(a), the effect of epinephrine on the phosphorylation of CREB was not inhibited by 10 lmol/L PP1 or 200 nmol/L staurosporine in all three clones. These inhibitors have been commonly applied in such concentrations in various cell systems including PC12 cells and have not been reported to cause any nonspecific effect (Quinn et al. 2002;Kam et al. 2004). In contrast, pretreatment of the cells with these inhibitors significantly increased the epinephrineinduced transcriptional activation of CREB, without modifying the basal level of CREB transcriptional activity (Fig. 9b). ...
On incubation with epinephrine, PC12 cells stably expressing alpha2-adrenergic receptor (alpha2-AR) undergo morphological and biochemical changes characteristic of neuron-like differentiation. The present study shows that alpha2-AR stimulation increases the phosphorylation of the transcription factor cAMP-response element-binding protein (CREB), the activity of a CRE-reporter plasmid and the expression of cyclin D1 with subtype-dependent efficiency (alpha2A approximately alpha2C > alpha2B). The effects of epinephrine were mimicked by cell exposure to forskolin or to exogenous arachidonic acid (AA) and they were abrogated by prior treatment with the inhibitor of phospholipase C (PLC) (U73122) or the inhibitor of cytochrome P450-dependent epoxygenase, ketoconazole. On the other hand, treatment of the cells with epinephrine caused activation of protein kinase A (PKA), which was fully abolished by ketoconazole. Inhibition of PKA activity with H89 or ketoconazole abolished the effects of epinephrine on CREB, suggesting that activation of the cAMP/PKA pathway by AA epoxy-derivatives is responsible for CREB activation by alpha2-ARs. The effects of epinephrine were unaffected by LY294002. Furthermore, treatment with staurosporine, tyrphostin AG1478, PP1 or PD98059 did not change the extent of CREB phosphorylation but enhanced its transcriptional activity. Altogether, our results demonstrate that, in PC12 cells, the alpha2-AR subtypes cause phosphorylation and activation of CREB through a pathway involving stimulation of PLC, AA release, generation of epoxygenase derivative and increase of PKA activity. They also suggest attenuation of CREB transcriptional activity by mitogen-activated protein kinase, protein kinase C and Src kinases.
... of intracellular calcium (6). Recently, KOR activation has also been shown to stimulate the mitogen-activated protein kinase pathways (MAPK), including extracellular signal-regulated kinase (ERK1/2), p38, and c-Jun N-terminal Kinase (JNK) (7)(8)(9)(10)(11). ...
Norbinaltorphimine (NorBNI), guanidinonaltrindole, and atrans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine (JDTic) are selective kappa opioid receptor (KOR) antagonists having very long durations of action in vivo despite binding non-covalently in vitro and having only moderately high affinities. Consistent with this, we found that antagonist treatment significantly reduced the subsequent analgesic response of mice to the KOR agonist U50,488 in the tail-withdrawal assay for 14-21 days. Receptor protection assays were designed to distinguish between possible explanations for this anomalous effect, and we found that mice pretreated with the readily reversible opioid antagonists naloxone or buprenorphine before norBNI responded strongly in the tail-flick analgesia assay to a subsequent challenge with U50,488 1 week later. Protection by a rapidly cleared reagent indicates that norBNI did not persist at the site of action. In vitro binding of [(3)H]U69,593 to KOR showed that K(d) and Bmax values were not significantly affected by prior in vivo norBNI exposure, indicating that the agonist binding site was intact. Consistent with the concept that the long-lasting effects might be caused by a functional disruption of KOR signaling, both norBNI and JDTic were found to stimulate c-Jun N-terminal kinase (JNK) phosphorylation in HEK293 cells expressing KOR-GFP but not in untransfected cells. Similarly, norBNI increased phospho-JNK in both the striatum and spinal cord in wild type mice but not in KOR knock-out mice. Pretreatment of mice with the JNK inhibitor SP600125 before norBNI attenuated the long acting antagonism. Together, these results suggest that the long duration KOR antagonists disrupt KOR signaling by activating JNK.
... In addition to the regulatory effects on adenylate cyclase and ion channel conductance typical of G i/o -coupled receptors, sustained activation of κ-opioid receptors leads to the activation mitogen-activated protein kinase pathway (MAPK) in heterologous systems and native cells (Belsheva et al., 2005;Bruchas et al., 2006). Using in vitro assays, κ agonists have been shown to activate all three MAPK family members, including the extracellular signal-regulated kinase (ERK), the stress kinase c-Jun N-terminal kinase (JNK), and the stress kinase p38 in transfected cells and primary cultured astrocytes and neurons (Kam et al., 2004;Belsheva et al., 2005;Bruchas et al., 2006). However, KORmediated MAPK activation in vivo has not yet been demonstrated, and a possible relationship between p38 MAPK activation and κ-induced behaviors has not been established. ...
The molecular mechanisms mediating stress-induced dysphoria in humans and conditioned place aversion in rodents are unknown. Here, we show that repeated swim stress caused activation of both kappa-opioid receptor (KOR) and p38 mitogen-activated protein kinase (MAPK) coexpressed in GABAergic neurons in the nucleus accumbens, cortex, and hippocampus. Sites of activation were visualized using phosphoselective antibodies against activated kappa receptors (KOR-P) and against phospho-p38 MAPK. Surprisingly, the increase in P-p38-IR caused by swim-stress exposure was completely KOR dependent; P-p38-IR did not increase in KOR(-/-) knock-out mice subjected to the same swim-paradigm or in wild-type mice pretreated with the KOR antagonist norbinaltorphimine. To understand the relationship between p38 activation and the behavioral effects after KOR activation, we administered the p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (i.c.v.)] and found that it selectively blocked the conditioned place aversion caused by the kappa agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and the KOR-dependent swim stress-induced immobility while not affecting kappa-opioid analgesia or nonselectively affecting associative learning. We found that the mechanism linking KOR and p38 activation in vivo was consistent with our previous in vitro data suggesting that beta-arrestin recruitment is required; mice lacking G-protein-coupled receptor kinase 3 also failed to increase p-p38-IR after KOR activation in vivo, failed to show swim stress-induced immobility, or develop conditioned place aversion to U50488. Our results indicate that activation of p38 MAPK signaling by the endogenous dynorphin-kappa-opioid system likely constitutes a key component of the molecular mechanisms mediating the aversive properties of stress.
The κ‑opioid receptor (KOR) is one of the primary receptors of opioids and serves a vital role in the regulation of pain, anesthesia, addiction and other pathological and physiological processes. KOR is associated with several types of cancer and may influence cancer progression. It has been proposed that KOR may represent a new tumor molecular marker and provide a novel basis for molecular targeted therapies for cancer. However, the association between KOR and cancer remains to be explored comprehensively. The present review introduces KOR and its association with different types of cancer. Improved understanding of KOR may facilitate development of novel antitumor therapies.
The clinical application of opioids may be accompanied by a series of adverse consequences, such as opioid tolerance, opioid-induced hyperalgesia, opioid dependence or addiction. In view of this issue, clinicians are faced with the dilemma of treating various types of pain with or without opioids. In this review, we discuss that Src protein tyrosine kinase plays an important role in these adverse consequences, and Src inhibitors can solve these problems well. Therefore, Src inhibitors have the potential to be used in combination with opioids to achieve synergy. How to combine them together to maximize the analgesic effect while avoiding unnecessary trouble provides a topic for follow-up research.
The dynorphin/κ-opioid receptor system has been implicated in the pathogenesis and pathophysiology of several psychiatric disorders. In the present review, we present evidence indicating a key role for this system in modulating neurotransmission in brain circuits that subserve mood, motivation, and cognitive function. We overview the pharmacology, signaling, post-translational, post-transcriptional, transcriptional, epigenetic and cis regulation of the dynorphin/κ-opioid receptor system, and critically review functional neuroanatomical, neurochemical, and pharmacological evidence, suggesting that alterations in this system may contribute to affective disorders, drug addiction, and schizophrenia. We also overview the dynorphin/κ-opioid receptor system in the genetics of psychiatric disorders and discuss implications of the reviewed material for therapeutics development.
The hematopoietic-specific G(q) subfamily members, Galpha(16) and Galpha(14) proteins have recently been shown to be capable of stimulating the signal transducer and activator of transcription 3 (STAT3) as well as STAT1. In the present study we examined whether this activation was STAT-member specific as well as determining the possible involvement of Gbetagamma dimers. Despite clear stimulation of STAT3, the constitutively active mutants of Galpha(16) (Galpha(16)QL) and Galpha(14) (Galpha(14)QL) failed to induce the phosphorylation of several STAT family members, including STAT2, STAT4 and STAT5 in human embryonic kidney 293 cells. On the other hand, transient expression of specific combinations of Gbetagamma complexes induced STAT3 phosphorylation. Among the 48 combinations tested, 13 permutations of Gbetagamma stimulated STAT3 phosphorylation and all of them contain the neuronal-specific Ggamma(2), Ggamma(4), Ggamma(7) and Ggamma(9). These results suggested that the activation of STAT family members by Galpha(16) or Galpha(14) was selective and that distinct combinations of Gbetagamma complexes can also regulate the STAT signaling pathway.
Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K(ir)3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K(ir)3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wild-type mice, but not in mice lacking the kappa-opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K(ir)3.1-ir. The U50,488-induced increase in pY12-K(ir)3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K(ir)3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K(ir)3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K(ir)3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K(ir)3.1-ir; and the heterologous desensitization of K(ir)3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K(ir)3.1 and channel inhibition through a GRK3-, p38 MAPK- and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.
Recent studies have revealed that in G protein-coupled receptor signalings switching between G protein- and beta-arrestin (betaArr)-dependent pathways occurs. In the case of opioid receptors, the signal is switched from the initial inhibition of adenylyl cyclase (AC) to an increase in AC activity (AC activation) during prolonged agonist treatment. The mechanism of such AC activation has been suggested to involve the switching of G proteins activated by the receptor, phosphorylation of signaling molecules, or receptor-dependent recruitment of cellular proteins. Using protein kinase inhibitors, dominant negative mutant studies and mouse embryonic fibroblast cells isolated from Src kinase knock-out mice, we demonstrated that mu-opioid receptor (OPRM1)-mediated AC activation requires direct association and activation of Src kinase by lipid raft-located OPRM1. Such Src activation was independent of betaArr as indicated by the ability of OPRM1 to activate Src and AC after prolonged agonist treatment in mouse embryonic fibroblast cells lacking both betaArr-1 and -2. Instead the switching of OPRM1 signals was dependent on the heterotrimeric G protein, specifically Gi2 alpha-subunit. Among the Src kinase substrates, OPRM1 was phosphorylated at Tyr336 within NPXXY motif by Src during AC activation. Mutation of this Tyr residue, together with mutation of Tyr166 within the DRY motif to Phe, resulted in the complete blunting of AC activation. Thus, the recruitment and activation of Src kinase by OPRM1 during chronic agonist treatment, which eventually results in the receptor tyrosine phosphorylation, is the key for switching the opioid receptor signals from its initial AC inhibition to subsequent AC activation.
This paper is the 27th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over 30 years of research. It summarizes papers published during 2004 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.
Previous study carried out on PC12 cells expressing each alpha(2)-adrenergic receptor subtype individually (PC12/alpha(2A), /alpha(2B) or /alpha(2C)) have shown that epinephrine causes activation of PI3K and phosphorylation of Erk 1/2. The signal transduction mechanisms whereby each alpha(2)-AR subtype triggers these actions were investigated in the present study. In all three clones, epinephrine-induced phosphorylation of MAPK or Akt was abolished by prior treatment with ketoconazole, but not with indomethacin or nordihydroguaiaretic acid. On the other hand, treatment of the clones with epinephrine caused a rapid increase of AA release, which was fully abolished by the PLC inhibitor U73122, but was unaffected by the PLA(2) inhibitor quinacrine. The effects of epinephrine on MAPK and Akt were mimicked by cell exposure to exogenous AA. Furthermore, whereas U73122 abolished the effects of epinephrine, quinacrine only prevented the effects of epinephrine, suggesting that AA release through PLC and its metabolites are responsible for MAPK and Akt activation by alpha(2)-ARs. Treatment with 1,10-phenanthroline, CRM197, or tyrphostin AG1478 suppressed MAPK and Akt phosphorylation by epinephrine or AA, in a subtype-specific manner. Furthermore, conditioned culture medium from epinephrine-treated PC12/alpha(2) induced MAPK and Akt phosphorylation in wild-type PC12. Inhibition of NGFR tyrosine phosphorylation had no effect but the src inhibitor PP1 abolished MAPK and Akt phosphorylation in all three clones. Our results provide evidence for a putative pathway by which alpha(2)-ARs activate MAPK and Akt in PC12 cells, involving stimulation of PLC, AA release, AA metabolism by cytochrome P450-dependent epoxygenase, stimulation of matrix metalloproteinases and subtype-specific transactivation of EGFR through src activation and heparin-binding EGF-like growth factor release.
New data regarding signal transduction triggered by opioid ligands in immune cells are reviewed, and the signal transduction in neuronal cells is documented. Similar signaling pathways are induced by opioids in immune as well as neuronal cells. Opioids altered second messenger cAMP, intracellular calcium, and second messenger-induced kinases in immune cells. Met-enkephalin, preferentially delta-opioid, was bimodally regulated, while kappa-opioids inhibited these second messengers. delta-, kappa- and micro-opioids altered nitric oxide secretion, inducing cGMP as the second messenger in immune cells. Coupling of opioid agonists to opioid receptors activated mitogen-activated protein/extracellular signal-regulated protein kinases and various transcription factors in immune cells. Activator protein 1 (AP-1), c-fos, and nuclear factor-kappaB (NF-kappaB) are transcription factors shared by neuronal and immune cells. Delta-opioids activated AP-1, c-fos, activating transcription factor 2, Ikaros-1 and Ikaros-2 transcription factors in immune cells. Induction of kappa-opioid receptor gene by retinoic acid resulted in increased binding of Sp1 transcription factor to the promoter of the kappa-opioid receptor. Micro-opioids inhibited synthesis of common transcription factors AP-1, c-fos, NF-kappaB, and nuclear factor of activated T cells in activated or stimulated immune cells, whereas micro-opioids activated NF-kappaB, GATA-3, and Kruppel-like factor 7 transcription factors in non-stimulated immune cells.
AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (gamma-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappa-mediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism.
Hematopoietic restrictive Gα16 has long been known to stimulate phospholipase Cβ (PLCβ) and induce mitogen-activated protein kinase (MAPK) phosphorylation. Recently, we have demonstrated that Gα16 is capable of inducing the phosphorylation and transcriptional activation of transcription factors, such as signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB (NFκB). However, the downstream signaling regulation by Gα16 has not yet been documented. In the present study, we have determined the signaling mechanism by which constitutively active Gα16 mediates c-Fos transcriptional activation in human embryonic kidney (HEK) 293 cells. Overexpression of constitutively active Gα16, Gα16QL, resulted in the stimulation of c-Fos transcriptional activation in HEK 293 cells. The participation of PLCβ, c-Src/Janus kinase 2 (JAK2) and extracellular signal-regulated kinase (ERK) signaling pathways in Gα16QL-induced c-Fos transcriptional activation was demonstrated by the use of their specific inhibitors. However, c-Jun N terminal kinase (JNK), p38 MAPK and phosphatidylinositol-3 kinase (PI3K) were not required. Interestingly, the dominant negative mutant of STAT1, but not STAT3, suppressed c-Fos transcriptional activation induced by Gα16QL, implying that STAT1 was involved in this signaling mechanism. To further examine the role of STAT1 in the signaling pathway of Gα16, we demonstrated that Gα16QL was able to induce STAT1 activation. Also, stimulation of adenosine A1 receptor-coupled Gα16 was shown to induce ERK and STAT1 phosphorylations in a concentration-dependent manner. Using selective inhibitors, PLCβ, c-Src/JAK and ERK, but not JNK, p38 MAPK and PI3K, were shown to be involved in Gα16QL-induced STAT1 activation. Collectively, our results demonstrate for the first time that stimulation of Gα16 can lead to STAT1-dependent c-Fos transcriptional activation via PLCβ, c-Src/JAK and ERK pathways.
Alpha2-adrenergic receptors have been reported to induce subtype-specific neuronal differentiation in vitro, but the signaling mechanisms that mediate this effect have not been characterized. In the present study we found that stimulated alpha2-ARs induce delayed transactivation of TrkA in PC12 cells. The transactivation of TrkA was sensitive to the PP1 inhibitor of the Src family kinases and required prior transactivation of the EGF receptor. Moreover, alpha2-adrenergic receptors induced sustained activation of MAPK and Akt. The sustained activation of Akt, but not of MAPK, was subtype-specific and correlated with the neuronal differentiation of PC12 cells, with the order alpha2A<alpha2B<alpha2C. Furthermore, stimulated alpha2-ARs induced an increased over time expression of the cell cycle associated proteins, p21WAF1 and Cyclin D1 and led to cell cycle arrest in a similar subtype-specific manner. Contrary to sustained activation of MAPK, the persistent activation of Akt and of p21WAF1 and Cyclin D1 as well as neurite outgrowth and expression of the neuronal marker peripherin, were all blocked by K252a an inhibitor of TrkA activity. Together these results demonstrate a novel outcome following alpha2-AR-mediated EGFR transactivation, being the consecutive transactivation of TrkA, and that this event may mediate the subtype-specific differentiation of alpha2-AR-expressing PC12 cells.
Stimulation of a number of cell surface receptors, including integrins and G protein-coupled receptors, results in the activation of a non-receptor tyrosine kinase known as focal adhesion kinase (FAK). In turn, this kinase is believed to play a critical role in signaling to intracellular kinase cascades controlling gene expression such as extracellular signal-regulated kinases (ERKs), by a yet poorly defined mechanism. Furthermore, whether this tyrosine kinase also mediates the activation of other mitogen-activated protein kinase family members, such as c-Jun NH(2)-terminal kinases (JNKs), is still unclear. We show here that the activation of FAK by anchoring to the cell membrane is itself sufficient to stimulate potently both ERK and JNK. These effects were found to be phosphatidylinositol 3-kinase-independent, as FAK effectively stimulated Akt, and wortmannin suppressed Akt but not ERK or JNK activation. As previously reported by others, activation of ERK correlated with the ability of FAK to induce tyrosine phosphorylation of Shc. Surprisingly, however, stimulation of JNK was not dependent on the kinase activity of FAK or on the ability to induce tyrosine phosphorylation of FAK substrates. Instead, we provide evidence that FAK may stimulate JNK through a novel pathway involving the recruitment of paxillin to the plasma membrane and the subsequent activation of a biochemical route dependent on small GTP-binding proteins of the Rho family.
Jun kinases (JNK) are involved in the stress response of mammalian cells. Stimulation of JNK can be induced by stress factors and by agonists of tyrosine kinase and G protein-coupled receptors. G protein-dependent receptors stimulate JNK via Gβγ subunits of heterotrimeric G proteins, but the subsequent signaling reaction has been undefined. Here we demonstrate JNK activation in COS-7 cells by Gβγ-stimulated phosphoinositide 3-kinase γ (PI3Kγ). Signal transduction from PI3Kγ to JNK can be suppressed by dominant negative mutants of Ras, Rac, and the protein kinase PAK. These results identify PI3Kγ as a mediator of Gβγ-dependent regulation of JNK activity.
Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent
activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gβγ subunit-mediated increases in tyrosine phosphorylation
of the Shc adapter protein, Shc·Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity.
We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway
via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gβγ subunits in COS-7
cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase
activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by
cellular expression of Gβγ subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient
association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity
and Shc tyrosine phosphorylation. LPA stimulation or expression of Gβγ subunits resulted in c-Src activation, as assessed
by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase
inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation,
and Shc·Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gβγ subunits,
or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src
family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation,
Shc·Grb2 complex formation, and MAP kinase activation. These data suggest that Gβγ subunit-mediated formation of Shc·c-Src
complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive
G protein-coupled receptors.
Chemotaxis is mediated by activation of seven-transmembrane domain, G protein-coupled receptors, but the signal transduction pathways leading to chemotaxis are poorly understood. To identify G proteins that signal the directed migration of cells, we stably transfected a lymphocyte cell line (300-19) with G protein-coupled receptors that couple exclusively to Galphaq (the m3 muscarinic receptor), Galphai (the kappa-opioid receptor), and Galphas (the beta-adrenergic receptor), as well as the human thrombin receptor (PAR-1) and the C-C chemokine receptor 2B. Cells expressing receptors that coupled to Galphai, but not to Galphaq or Galphas, migrated in response to a concentration gradient of the appropriate agonist. Overexpression of Galpha transducin, which binds to and inactivates free Gbetagamma dimers, completely blocked chemotaxis although having little or no effect on intracellular calcium mobilization or other measures of cell signaling. The identification of Gbetagamma dimers as a crucial intermediate in the chemotaxis signaling pathway provides further evidence that chemotaxis of mammalian cells has important similarities to polarized responses in yeast. We conclude that chemotaxis is dependent on activation of Galphai and the release of Gbetagamma dimers, and that Galphai-coupled receptors not traditionally associated with chemotaxis can mediate directed migration when they are expressed in hematopoietic cells.
The small GTP-binding proteins Ras, Rac, and Cdc42 link protein-tyrosine kinases with mitogen-activated protein kinase (MAPK) signaling cascades. Ras controls the activation of extracellular signal-regulated kinases (ERKs), while Rac and Cdc42 regulate the c-Jun N-terminal kinases (JNKs). In this study, we investigated whether small G protein/MAPK cascades contribute to signal transduction by transforming variants of c-Fes, a nonreceptor tyrosine kinase implicated in cytokine signaling and myeloid differentiation. First, we investigated the effects of dominant-negative small G proteins on Rat-2 fibroblast transformation by a retroviral homolog of c-Fes (v-Fps) and by c-Fes activated via N-terminal addition of the v-Src myristylation signal (Myr-Fes). We observed that dominant-negative Ras, Rac, and Cdc42 inhibited v-Fps- and Myr-Fes-induced growth of Rat-2 cells in soft agar, indicating that activation of these small GTP-binding proteins is required for fibroblast transformation by Fps/Fes tyrosine kinases. To determine whether MAPK pathways are activated downstream of these small G proteins, we measured ERK and JNK activity in the v-Fps- and Myr-Fes-transformed Rat-2 cells. Both ERK and JNK activities were elevated in the transformed cells, suggesting that these pathways are involved in cellular transformation. Dominant-negative mutants of Ras (but not Rac or Cdc42) specifically inhibited ERK activation by v-Fps and Myr-Fes, demonstrating that ERK activation occurs exclusively downstream of Ras. All three dominant-negative small G proteins inhibited JNK activation by v-Fps and Myr-Fes, indicating that JNK activation by these tyrosine kinases requires both Ras and Rho family GTPases. These data demonstrate that multiple small G protein/MAPK cascades are involved in downstream signal transduction by Fps/Fes tyrosine kinases.
Heterotrimeric G protein βγ subunit (Gβγ) mediates signals to two types of stress-activated protein kinases, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase, in mammalian cells. To investigate the signaling mechanism
whereby Gβγ regulates the activity of JNK, we transfected kinase-deficient mutants of two JNK kinases, mitogen-activated protein
kinase kinase 4 (MKK4) and 7 (MKK7), into human embryonal kidney 293 cells. Gβγ-induced JNK activation was blocked by kinase-deficient
MKK4 and to a lesser extent by kinase-deficient MKK7. Moreover, Gβγ increased MKK4 activity by 6-fold and MKK7 activity by
2-fold. MKK4 activation by Gβγ was blocked by dominant-negative Rho and Cdc42, whereas MKK7 activation was blocked by dominant-negative
Rac. In addition, Gβγ-mediated MKK4 activation, but not MKK7 activation, was inhibited completely by specific tyrosine kinase
inhibitors PP2 and PP1. These results indicate that Gβγ induces JNK activation mainly through MKK4 activation dependent on
Rho, Cdc42, and tyrosine kinase, and to a lesser extent through MKK7 activation dependent on Rac.
Heterotrimeric G proteins stimulate the activities of two stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase in mammalian cells. In this study, we examined whether alpha subunits of G(i) family activate JNK using transient expression system in human embryonal kidney 293 cells. Constitutively activated mutants of Galpha(i1), Galpha(i2), and Galpha(i3) increased JNK activity. In contrast, constitutively activated Galpha(o) and Galpha(z) mutants did not stimulate JNK activity. To examine the mechanism of JNK activation by Galpha(i), kinase-deficient mutants of mitogen-activated protein kinase kinase 4 (MKK4) and 7 (MKK7), which are known to be JNK activators, were transfected into the cells. However, Galpha(i)-induced JNK activation was not blocked effectively by kinase-deficient MKK4 and MKK7. In addition, activated Galpha(i) mutant failed to stimulate MKK4 and MKK7 activities. Furthermore, JNK activation by Galpha(i) was inhibited by dominant-negative Rho and Cdc42 and tyrosine kinase inhibitors, but not dominant-negative Rac and phosphatidylinositol 3-kinase inhibitors. These results indicate that Galpha(i) regulates JNK activity dependent on small GTPases Rho and Cdc42 and on tyrosine kinase but not on MKK4 and MKK7.
Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein-coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Here we demonstrate that Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetay, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins.
Apoptotic and mitogenic stimuli activate c-Jun NH2-terminal kinases (JNKs) in T cells. Although T cells express both JNK1 and JNK2 isozymes, the absence of JNK2 alone can result in resistance to anti-CD3-induced thymocyte apoptosis and defective mature T cell proliferation. Similar defects in thymocyte apoptosis and mature T cell proliferation, the latter due to reduced interleukin 2 production, are also caused by JNK1 deficiency. Importantly, T cell function was compromised in Jnk1(+/-)Jnk2(+/-) double heterozygous mice, indicating that JNK1 and JNK2 play similar roles in regulating T cell function. The reduced JNK dose results in defective c-Jun NH2-terminal phosphorylation in thymocytes but not in peripheral T cells, in which nuclear factors of activated T cells (NK-ATs)-DNA binding activity is affected. Thus, JNK1 and JNK2 control similar functions during T cell maturation through differential targeting of distinct substrates.
The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-alpha-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2-/- T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2-/- T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.
Plating suspended Swiss 3T3 cells onto fibronectin-coated dishes promoted phosphorylation of endogenous focal adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site, and at Tyr-577, located in the activation loop, as revealed by site-specific antibodies that recognize the phosphorylated form of these residues. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin. Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). Stimulation of Swiss 3T3 cells with bombesin also induced a rapid increase in the phosphorylation of endogenous FAK at Tyr-397. In contrast to the results obtained with fibronectin, PP-2 did not prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a concentration (10 micrometer) that suppressed bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with other G protein-coupled receptor agonists including vasopressin, bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results identify, for first time, the existence of Src-dependent and Src-independent pathways leading to FAK autophosphorylation at Tyr-397 stimulated by adhesion-dependent signals and G protein-coupled receptor agonists in the same cell.
Phosphorylation of the MAPK isoform ERK by G protein-coupled receptors involves multiple signaling pathways. One of these
pathways entails growth factor receptor transactivation followed by ERK activation. This study demonstrates that a similar
signaling pathway is used by the μ-opioid receptor (MOR) expressed in HEK293 cells and involves calmodulin (CaM). Stimulation
of MOR resulted in both epidermal growth factor receptor (EGFR) and ERK phosphorylation. Data obtained with inhibitors of
EGFR Tyr kinase and membrane metalloproteases support an intermediate role of EGFR activation, involving release of endogenous
membrane-bound epidermal growth factor. Previous studies had demonstrated a role for CaM in opioid signaling based on direct
CaM binding to MOR. To test whether CaM contributes to EGFR transactivation and ERK phosphorylation by MOR, we compared wild-type
MOR with mutant K273A MOR, which binds CaM poorly, but couples normally to G proteins. Stimulation of K273A MOR with [d-Ala2,MePhe4,Gly-ol5]enkephalin (10–100 nm) resulted in significantly reduced ERK phosphorylation. Furthermore, wild-type MOR stimulated EGFR Tyr phosphorylation 3-fold
more than K273A MOR, indicating that direct CaM-MOR interaction plays a key role in the transactivation process. Inhibitors
of CaM and protein kinase C also attenuated [d-Ala2,MePhe4,Gly-ol5]enkephalin-induced EGFR transactivation in wild-type (but not mutant) MOR-expressing cells. This novel pathway of EGFR transactivation
may be shared by other G protein-coupled receptors shown to interact with CaM.
Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.
Te mu-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G(16) protein. Given the promiscuous nature of G(16) and the high degree of resemblance of signaling properties of the three opioid receptors, both delta- and kappa-opioid receptors are likely to activate G(16). Interactions of delta- and kappa-opioid receptors with G(16) were examined by coexpressing the opioid receptors and G alpha(16) in COS-7 cells. The delta-selective agonist [D-Pen(2),D-Pen(5)]enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the delta-opioid receptor and G alpha(16). The delta-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of G alpha(16) and exhibited appropriate ligand selectivity and dose dependency. Similar transfection studies revealed only weak stimulation by the mu-opioid receptor, whereas the kappa-opioid receptor produced moderate phospholipase C activity. G alpha(16) thus appeared to interact differentially with the three opioid receptors. Radioligand binding assays indicate that the mu-opioid receptor was expressed at a lower level than those of the delta- and kappa-opioid receptors. To examine if differential coupling to G alpha(16) is prevalent, a panel of G(s)- or G(i)-coupled receptors was coexpressed with G alpha(16) in COS-7 cells and assayed for agonist-induced stimulation of phospholipase C. Activation of alpha(2)- and beta(2)-adrenergic, dopamine D-1 and D-2, adenosine A(1), somatostatin-l and -2, C5a, formyl peptide, and luteinizing hormone receptors all resulted in stimulation of phospholipase C, with maximal stimulations ranging from 1.5- to almost 17-fold. These findings suggest that the promiscuous G alpha(16) can in fact discriminate among different receptors and that such preferential interaction might in part be due to the abundance of receptors.
As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous κ-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the κ-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gβγ subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous κ-opioid receptor in C6 glioma cells.
The Son of Sevenless (Sos) proteins control receptor-mediated activation of Ras by catalyzing the exchange of guanosine diphosphate
for guanosine triphosphate on Ras. The NH2-terminal region of Sos contains a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. In COS-1 cells,
the DH domain of Sos stimulated guanine nucleotide exchange on Rac but not Cdc42 in vitro and in vivo. The tandem DH-PH domain
of Sos (DH-PH-Sos) was defective in Rac activation but regained Rac stimulating activity when it was coexpressed with activated
Ras. Ras-mediated activation of DH-PH-Sos did not require activation of mitogen-activated protein kinase but it was dependent
on activation of phosphoinositide 3-kinase. These results reveal a potential mechanism for coupling of Ras and Rac signaling
pathways.
Stimulation of a variety of cell surface receptors enhances the enzymatic activity of mitogen-activated protein kinases (MAPKs). MAPKs have been classified in three subfamilies: extracellular signal-regulated kinases (ERKs), stress-activated protein kinases or c-Jun NH-terminal kinases (SAPKs/JNKs), and p38 kinase. Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated, the mechanism of activation of JNKs is still poorly understood. Recently, we have shown that stimulation of G protein-coupled receptors can effectively induce JNK in NIH 3T3 cells (Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J., and Gutkind, J. S.(1995) J. Biol. Chem. 270, 5620-5624). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the signaling pathway linking G protein-coupled receptors to JNK. We show that stimulation of either muscarinic receptor subtype leads to JNK activation; however, this effect was not mimicked by expression of activated forms of α, α, α, or α G protein α subunits. In contrast, overexpression of Gβ subunits potently induced JNK activity. Furthermore, we show that signaling from m1 and m2 mAChRs to JNK involves β subunits of heterotrimeric G proteins, acting on a Ras and Rac1-dependent pathway.
Abstract Heptahelical opioid receptors utilize Gi proteins to regulate a multitude of effectors including the classical adenylyl cyclases and the more recently discovered mitogen-activated protein kinases (MAPKs). The c-Jun NH2-terminal kinases (JNKs) belong to one of three subgroups of MAPKs. In NG108-15 neuroblastoma × glioma hybrid cells that endogenously express δ-opioid receptors, δ-agonist dose-dependently stimulated JNK activity in a pertussis toxin-sensitive manner. By using COS-7 cells transiently transfected with the cDNAs of δ-opioid receptor and hemagglutinin (HA)-tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gβγ subunits by transducin suppressed the opioid-induced JNK activity. The possible involvement of the small GTPases was also examined. Expression of dominant negative mutants of Rac and Cdc42 blocked the opioid-induced JNK activation, and a partial inhibition was observed in the presence of the dominant negative mutant of Ras. In contrast, the dominant negative mutant of Rho did not affect the opioid-induced JNK activation. In addition, the receptor-mediated JNK activation was dependent on Src family tyrosine kinases, but independent of phosphatidylinositol-3 kinase and EGF receptor tyrosine kinases. Collectively, these results demonstrate functional regulation of JNK by the δ-opioid receptor, and this pathway requires Gβγ, Src kinases and the small GTPases Rac and Cdc42.
Jun kinases (JNK) are involved in the stress response of mammalian cells. Stimulation of JNK can be induced by stress factors
and by agonists of tyrosine kinase and G protein-coupled receptors. G protein-dependent receptors stimulate JNK via Gβγ subunits
of heterotrimeric G proteins, but the subsequent signaling reaction has been undefined. Here we demonstrate JNK activation
in COS-7 cells by Gβγ-stimulated phosphoinositide 3-kinase γ (PI3Kγ). Signal transduction from PI3Kγ to JNK can be suppressed
by dominant negative mutants of Ras, Rac, and the protein kinase PAK. These results identify PI3Kγ as a mediator of Gβγ-dependent
regulation of JNK activity.
The μ-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G16 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both δ- and κ-opioid receptors are likely to activate G16. Interactions of δ- and κ-opioid receptors with G16 were examined by coexpressing the opioid receptors and Gα16 in COS-7 cells. The δ-selective agonist [d-Pen2,d-Pen5]enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the δ-opioid receptor and Gα16. The δ-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of simeter for quality control of blood units and irradiators. 13. Transfusion1993;33: 898–901.14. ButsonMJ, YuPK, CheungT, et al. Dosimetry of blood irradiation with radiochromic film.Transfus Med1999;9: 205–8.15. NathR, BiggsPJ, LingCC, et al. AAPM code of practice for radiotherapy accelerators: Report of AAPM Radiation Therapy Task Group No. 45.Med Phys
Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with μ-, δ-, or κ-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding μ- ([d-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, δ- ([d-Pen2,d-Pen5]enkephalin)-, or κ- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a βγ scavenger, CD8-β-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8-β-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and βγ subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.
The mu-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G16 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both delta- and kappa-opioid receptors are likely to activate G16. Interactions of delta- and kappa-opioid receptors with G16 were examined by coexpressing the opioid receptors and G alpha16 in COS-7 cells. The delta-selective agonist [D-Pen2,D-Pen5] enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the delta-opioid receptor and G alpha16. The delta-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of G alpha16 and exhibited appropriate ligand selectivity and dose dependency. Similar transfection studies revealed only weak stimulation by the mu-opioid receptor, whereas the kappa-opioid receptor produced moderate phospholipase C activity. G alpha16 thus appeared to interact differentially with the three opioid receptors. Radioligand binding assays indicate that the mu-opioid receptor was expressed at a lower level than those of the delta- and kappa-opioid receptors. To examine if differential coupling to G alpha16 is prevalent, a panel of Gs- or Gi-coupled receptors was coexpressed with G alpha16 in COS-7 cells and assayed for agonist-induced stimulation of phospholipase C. Activation of alpha2- and beta2-adrenergic, dopamine D1 and D2, adenosine A1, somatostatin-1 and -2, C5a, formyl peptide, and luteinizing hormone receptors all resulted in stimulation of phospholipase C, with maximal stimulations ranging from 1.5- to almost 17-fold. These findings suggest that the promiscuous G alpha16 can in fact discriminate among different receptors and that such preferential interaction might in part be due to the abundance of receptors.
The present study describes the characterization of an opioid binding site on membranes prepared from the R1.1 cell line, a murine thymoma. Specific (-)[3H]bremazocine binding was saturable, stereoselective, and limited to a single high affinity binding site with a Kd value of 15.2 +/- 1.6 pM and a Bmax value of 54.8 +/- 6.0 fmol/mg of protein. The kappa-selective alkaloids and dynorphin peptides inhibited (-)[3H]bremazocine binding with Ki values of less than 1 nM, in contrast to mu- and delta-selective ligands. The high affinity of this site for alpha-neo-endorphin and U50,488 suggests that this kappa opioid binding site resembles the kappa 1b subtype. NaCl, as well as other mono- and divalent cations, inhibited (-)[3H]bremazocine binding. In the presence of NaCl, the nucleotides GTP, GDP, and the nonhydrolyzable analog guanylyl-5'-imidodiphosphate (Gpp(NH)p) also decreased (-)[3H]bremazocine binding, suggesting that this kappa opioid binding site is coupled to a G-protein. In summary, R1.1 cells possess a single high affinity kappa opioid receptor that shares many properties with brain kappa 1b opioid receptors.
To investigate the role of opioids as direct modulators of the immune response, we have searched for expression of the recently cloned delta, mu and kappa opioid receptors in immune cells. We have devised a reverse transcriptase-polymerase chain reaction strategy which specifically detects a region spanning putative transmembrane regions 2 to 7 for each transcript in both human and mouse immune cells. In human peripheral blood lymphocyte and monocyte preparations, delta was undetectable while the kappa transcript was present. The analysis of human cell lines revealed low but significant levels of delta opioid receptor transcripts in T, B or monocyte cell lines while the kappa transcript was found in B cell lines only. Investigation of murine cells showed the presence of transcript for the delta receptor in splenocytes and in some T and B cell lines. Unexpectedly, no expression of the mu receptor was detected. Sequence analysis of PCR products demonstrated nucleotide identity between immune and neuronal transcripts, indicating that they derive from the same genes. In conclusion, our results lead to the identification of kappa and delta opioid receptor transcripts in immune cells.
Previous reports from this laboratory, and others, have shown that exogenous mu and kappa opioids modulate both cellular and humoral immune responses. Our earlier work has suggested that accessory cells may serve as a target for the direct effects of kappa opioid compounds. In the present study, the function of the macrophage cell line P388D1 was modulated by the kappa-selective opioid agoinst U50,488H (trans-3,4-dichloro-N-methyl-N-[7-(1- pyrrolidinyl)cyclohexyl]benzene-acetamide methanesulfonate). Lipopolysaccharide-induced interleukin (IL)-1 and tumor necrosis factor-alpha production were inhibited after the administration of nanomolar concentrations of U50,488H. Furthermore, inhibition of IL-1 produced by the P388D1 cell line was reversed by both the classical opioid antagonist naloxone and by the kappa opioid receptor antagonist norbinaltorphimine. Examination of IL-1 mRNA levels in P388D1 by northern blot analysis showed that the inhibition mediated by U50, 488H apparently occurred at the level of transcription. On the other hand, U50,488H failed to modulate the production of IL-6 by this macrophage-like cell line. In addition, U50,488H failed to modulate the production of either IL-1 or tumor necrosis factor-alpha from the macrophage-like cell line RAW 264.7, an indication that subpopulations of macrophages exist with different sensitivities to opioids. These results are consistent with a growing body of data which suggests that a component of the inhibition mediated by opioid compounds involves a reduction in the production of cytokines.
We have expressed the cloned rat kappa-opioid receptor in human embryonic kidney 293 cells and studied the ability of kappa-selective ligands to inhibit adenylyl cyclase. In transfected 293 cells, activation of the kappa-opioid receptor by U50,488 and the dynorphins resulted in the inhibition of cAMP accumulation. The inhibitory response was sensitive to pertussis toxin and highly selective for kappa-agonists; neither mu- nor delta-opioids were able to activate the kappa-opioid receptor. Upon co-transfection with the alpha subunit of Gz, inhibition of cAMP accumulation by kappa-agonist became refractory to pertussis toxin, indicating that the kappa-opioid receptor can couple to both G(i) and Gz proteins.
Transduction of a mitogenic signal from the cell membrane to the nucleus involves the adapter proteins SHC and Grb2, which mediate activation of the Ras/mitogen-activated protein (MAP) kinase pathway. In contrast to receptor tyrosine kinases (RTKs), the signalling steps leading to Ras/MAP kinase activation by G-protein-coupled receptors (GPCRs) are still poorly characterized but appear to include beta gamma subunits of heterotrimeric G-proteins and as-yet unidentified tyrosine kinases. We report here that the epidermal growth factor receptor (EGFR) and the neu oncoprotein become rapidly tyrosine-phosphorylated upon stimulation of Rat-1 cells with the GPCR agonists endothelin-1, lysophosphatic acid and thrombin, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by either the selective tyrphostin AG1478 or a dominant-negative EGFR mutant suppressed MAP kinase activation and strongly inhibited induction of fos gene expression and DNA synthesis. Our results demonstrate a role for RTKs as downstream mediators in GPCR mitogenic signalling and suggest a ligand-independent mechanism of RTK activation through intracellular signal crosstalk.
Stimulation of a variety of cell surface receptors enhances the enzymatic activity of mitogen-activated protein kinases (MAPKs). MAPKs have been classified in three subfamilies: extracellular signal-regulated kinases (ERKs), stress-activated protein kinases or c-Jun NH2-terminal kinases (SAPKs/JNKs), and p38 kinase. Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated, the mechanism of activation of JNKs is still poorly understood. Recently, we have shown that stimulation of G protein-coupled receptors can effectively induce JNK in NIH 3T3 cells (Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J., and Gutkind, J. S. (1995) J. Biol. Chem. 270, 5620-5624). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the signaling pathway linking G protein-coupled receptors to JNK. We show that stimulation of either muscarinic receptor subtype leads to JNK activation; however, this effect was not mimicked by expression of activated forms of alphas, alphai2, alphaq, or alpha13 G protein alpha subunits. In contrast, overexpression of Gbetagamma subunits potently induced JNK activity. Furthermore, we show that signaling from m1 and m2 mAChRs to JNK involves betagamma subunits of heterotrimeric G proteins, acting on a Ras and Rac1-dependent pathway.
Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.
Cell adhesion to extracellular matrix proteins such as fibronectin (FN) triggers a number of intracellular signaling events including the increased tyrosine phosphorylation of the cytoplasmic focal adhesion protein-tyrosine kinase (PTK) and also the stimulation of the mitogen-activated protein kinase ERK2. Focal adhesion kinase (FAK) associates with integrin receptors, and FN-stimulated phosphorylation of FAK at Tyr-397 and Tyr-925 promotes the binding of Src family PTKs and Grb2, respectively. To investigate the mechanisms by which FAK, c-Src, and Grb2 function in FN-stimulated signaling events to ERK2, we expressed wild type and mutant forms of FAK in human 293 epithelial cells by transient transfection. FAK overexpression enhanced FN-stimulated activation of ERK2 approximately 4-fold. This was blocked by co-expression of the dominant negative Asn-17 mutant Ras, indicating that FN stimulation of ERK2 was Ras-dependent. FN-stimulated c-Src PTK activity was enhanced by wild type FAK expression, whereas FN-stimulated activation of ERK2 was blocked by expression of the c-Src binding site Phe-397 mutant of FAK. Expression of the Grb2 binding site Phe-925 mutant of FAK enhanced activation of ERK2, whereas a kinase-inactive Arg-454 mutant FAK did not. Expression of wild type and Phe-925 FAK, but not Phe-397 FAK, enhanced p130(Cas) association with FAK, Shc tyrosine phosphorylation, and Grb2 binding to Shc after FN stimulation. FN-induced Grb2-Shc association is another pathway leading to activation of ERK2 via Ras. The inhibitory effects of Tyr-397 FAK expression show that FAK-mediated association and activation of c-Src is essential for maximal signaling to ERK2. Moreover, multiple signaling pathways are activated upon the formation of an FAK.c-Src complex, and several of these can lead to Ras-dependent ERK2 mitogen-activated protein kinase activation.
The Son of Sevenless (Sos) proteins control receptor-mediated activation of Ras by catalyzing the exchange of guanosine diphosphate for guanosine triphosphate on Ras. The NH2-terminal region of Sos contains a Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. In COS-1 cells, the DH domain of Sos stimulated guanine nucleotide exchange on Rac but not Cdc42 in vitro and in vivo. The tandem DH-PH domain of Sos (DH-PH-Sos) was defective in Rac activation but regained Rac stimulating activity when it was coexpressed with activated Ras. Ras-mediated activation of DH-PH-Sos did not require activation of mitogen-activated protein kinase but it was dependent on activation of phosphoinositide 3-kinase. These results reveal a potential mechanism for coupling of Ras and Rac signaling pathways.
Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a betagamma scavenger, CD8- beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8- beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and betagamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.
The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to Gi/Go proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of beta-adrenergic receptor kinase (betaARKct), which specifically blocks Gbetagamma-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21ras, also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing mu-opioid receptor, [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO; a mu-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, betaARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and mu-opioid receptors mediate MAP kinase activation via a signaling pathway using the betagamma-subunit of Gi, a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and mu-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate mu-opioid receptor signaling in a cellular system.
It has been reported that expression of the active mutant of heterotrimeric GTP-binding protein alpha subunit G alpha i2 transforms Rat-1 cells. However, the G alpha i2-mediated mitogenic signaling pathways remain to be elucidated. Here, we demonstrate that inducible expression of the active mutant of G alpha i2 (G alpha i2(Q205L)) activates Ras and c-Jun N-terminal kinase (JNK) in addition to extracellular signal-regulated kinase (ERK) in Rat-1 cells. Our findings suggest that Ras may play a critical role in the G alpha i2-induced transformation and G alpha i2 can transduce signals from the Gi-coupled receptor to JNK and ERK in certain types of mammalian cells.
Pleckstrin homology (PH) domains, comprised of rather weakly conserved sequences of about 100 amino acid residues, are a protein motif found in many signaling and cytoskeletal proteins. PH domains have been shown to bind to the betagamma subunits of heterotrimeric GTP-binding proteins (Gbetagamma), but the affinity of PH domains for Gbetagamma has not been quantitatively estimated in detail. To characterize the nature of the interaction between PH domains and Gbetagamma its kinetic parameters were analyzed using a BIAcore instrument. All PH domains tested (PH domains of ras-specific guanine nucleotide exchange factor (ras-GRF), phospholipase (PLC) gamma1, and Son of sevenless protein (Sos)) appeared to bind to Gbeta1gamma2 with affinity constants K(D) of 0.108, 0.318, and 0.208 microM, respectively. The binding of PH domains to Gbetagamma was inhibited by preincubation of Gbetagamma with the GDP-bound but not the GTP-bound form of Gialpha. This study showed a high affinity interaction between PH domains and Gbetagamma, and suggests a potential role of PH domains in Gbetagamma-mediated signal transduction in intact cells.
Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK-) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK- cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.
As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous kappa-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the kappa-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gbetagamma subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa-opioid receptor in C6 glioma cells.
It has been well known that immune function is modulated by endogenous opioid peptides: beta-endorphin and enkephalins. However, the effect of dynorphin A on the immune function has not been well documented. In this study, we investigated dynorphin A in the regulation of mitogen-induced proliferation and and interleukin-2 (IL-2) production of rat splenocytes. The results showed that dynorphin A 1-13 as well as dynorphin A 1-17 enhanced concanavalin A-stimulated [(3)H] thymidine uptake 46-112% and IL-2 production in a dose-dependent fashion. These effects were reversed by naloxone and norBNI, a selective kappa-receptor antagonist. Dynorphin A reduced cyclic AMP contents in spenocytes in naloxone and norBNI reversible fashion. The data suggest that dynorphin A enhanced mitogen-stumulated lymphocyte proliferation and IL-2 production via kappa-opioid receptor and cAMP pathway.
1. Opioid (μ, δ, κ) and cannabinoid (CB1, CB2) receptors are coupled mainly toGi/Go GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins.
2. The stimulation of [35S]GTPγS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of μ- and δ-opioid receptors led to stimulation of [35S]GTPγS binding by either μ-selective (DAMGO) or δ-selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes.
3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either μ- or δ-opioid receptors.
4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and δ-opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress μ- and δ-opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptorsubtype.
5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.
Endogenous and exogenous kappa-opioid agonists have been widely reported to modulate the immune response. We have published results that show that the superantigen-induced proliferative response of thymocytes is inhibited by the selective kappa-opioid agonist trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl] benzeneaceamide methanesulfonate (U50,488H). Previous work has established that the kappa-opioid receptor is widely expressed within the thymus; however, little is known about the role of the kappa-opioid receptor in the function of thymocytes. In the present report, we have examined the impact of U50,488H administration on the expression of cytokines in superantigen-stimulated thymocytes by RNase protection analysis. We have measured detectable levels of the cytokines IL-2, IL-4, IL-5, IL-13, and IFN-gamma, and the chemokines lymphotactin and RANTES, in stimulated thymocyte cultures; however, addition of U50,488H did not alter the expression of these cytokines. Examination of cytokine receptor expression by these thymocytes revealed a significant inhibition in the expression of the transcript for the IL-7 receptor alpha-chain (IL-7Ralpha), and these results were confirmed by flow cytometry. Surprisingly, the expression of several other cytokine receptor chains including the common gamma-chain, IL-2Rbeta, or the IL-2Ralpha, IL-4Ralpha, and IL-15Ralpha chains, was not altered. In contrast to these results, a significant elevation in the expression of the chemokine receptor CCR2 was observed in U50,488H-treated cultures. These results suggest that the kappa-opioid receptor may function to promote cellular migration at the expense of the sensitivity to the growth-promoting/maturation activity of IL-7.
The hallmark of T-cell activation is the production of interleukin 2 (IL-2). c-Jun amino-terminal kinase (JNK), a MAP kinase that phosphorylates c-Jun and other components of the AP-1 group of transcription factors, has been implicated in the activation of IL-2 expression. Previously, we found that T cells from mice deficient in the Jnk1 or Jnk2 gene can be activated and produce IL-2 normally, but are deficient in functional differentiation into Th1 or Th2 subsets. However, studies of mice with compound mutations indicate that JNK1 and JNK2 are redundant during mouse development. Here we use three new mouse models in which peripheral T cells completely lack JNK proteins or signalling, to test whether the JNK signalling pathway is crucial for IL-2 expression and T-cell activation. Unexpectedly, these T cells made more IL-2 and proliferated better than wild-type cells. However, production of effector T-cell cytokines did require JNK. Thus, JNK is necessary for T-cell differentiation but not for naive T-cell activation.
MAP kinases are evolutionarily conserved proteins that are activated by a protein kinase cascade, including a MAP kinase kinase kinase, which phosphorylates a MAP kinase kinase, which in turn activates the MAP kinase by phosphorylation on Thr and Tyr residues. The primary sequence surrounding these phosphorylation sites serves to distinguish three major groups of mammalian MAP kinases. These include the Ras-activated ERK MAP kinases, which are characterized by the sequence TEY and the two stress-activated MAP kinases: p38 with the sequence TGY, and the c-Jun NH2-terminal kinases (JNK) with the sequence TPY. This review will focus on the JNK group of MAP kinases.
It is now clear that opioid receptors participate in the function of the cells of the immune system, and evidence suggests that opioids modulate both innate and acquired immune responses. We review literature here which establishes that mu-, kappa-, and delta-opioid compounds alter resistance to a variety of infectious agents, including the Human Immunodeficiency Virus (HIV). The nature of the immunomodulatory activity of the opioids has been the subject of a great deal of research over the last ten years. There is increasing evidence that effects of opioids on the immune response are mediated at several levels. Modulation of the inflammatory response appears to be a target of these compounds, including effects on phagocytic activity, as well as the response of cells to various chemoattractant molecules. Moreover, findings from several laboratories have demonstrated the impact of opioid treatment on antibody responses, and the molecular basis for this effect is likely due, at least in part, to the modulation of both cytokine and cytokine receptor expression. Future research should provide a clearer understanding of the cellular and molecular targets of opioid action within the immune system.
Opiate addicts are prone to recurrent infections. In the present study we evaluated the molecular mechanism of opiate-induced T cell apoptosis. Both morphine and DAGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin) enhanced T cell apoptosis. Morphine as well as DAGO activated c-Jun NH2
-terminal kinase (JNK) in T cells. Moreover, opiates increased the expression of ATF-2, a specific substrate for JNK and P38 mitogen activated kinases (MAPK). Furthermore, opiates attenuated extracellular signal related kinase (ERK) in T cells. Both morphine and DAGO cleaved pro-caspases 8, 9, and 10 and generated caspases 8, 9 and 10 (active products). Morphine as well as DAGO also cleaved poly- (ADP-ribose) polymerase (PARP) into 116 and 85 kD proteins indicating the activation of caspase-3. These results suggest that opiate-induced T cell apoptosis may be mediated through the JNK cascade and activation of caspases 8 and 3.
In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-beta promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-beta Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-beta Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-beta and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.
Cell migration is a complex phenomenon that is stimulated by chemoattractive factors such as chemokines, a family of ligands for G protein-coupled receptors (GPCRs). In contrast, factors that suppress cell migration, and the mechanism of their action, remain largely unknown. In this study, we show that endothelin, a GPCR ligand, inhibits cell motility in a manner dependent upon signaling through the c-Jun N-terminal kinase (JNK) pathway. We further demonstrate that this effect is dependent upon Src kinase and small GTPases Rac1 and Cdc42. These findings provide new insight into GPCR-mediated regulation of cell migration.
Opioid receptors are the therapeutic targets of narcotic analgesics. All three types of opioid receptors (mu, delta and kappa) are prototypical G(i)-coupled receptors with common signaling characteristics in their regulation of intracellular events. Nevertheless, numerous signaling processes are differentially regulated by the three receptors. We have recently demonstrated that stimulation of delta-opioid receptor can up-regulate the activity of the c-Jun N-terminal kinase (JNK) in a pertussis toxin-sensitive manner (Kam et al. 2003; J. Neurochem. 84, 503-513). The present study revealed that the mu-opioid receptor could stimulate JNK in both SH-SY5Y cells and transfected COS-7 cells. The mechanism by which the mu-opioid receptor stimulated JNK was delineated with the use of specific inhibitors and dominant-negative mutants of signaling intermediates. Activation of JNK by the mu-opioid receptor was mediated through G beta gamma, Src kinase, son-of-sevenless (Sos), Rac and Cdc42. Interestingly, unlike the delta-opioid receptors, the mu-opioid receptor required phosphatidylinositol-3 kinase (PI3K) to activate JNK. The mu-opioid receptor-induced JNK activation was effectively inhibited by wortmannin or the coexpression of a dominant negative mutant of PI3K gamma. Like the delta-opioid receptor, activation of JNK by the kappa-opioid receptor occurred in a PI3K-independent manner. These studies revealed that the mu-opioid receptor utilize a distinct mechanism to regulate JNK.