Differential Regulation of CXCL8 Production by Different G Protein Subunits with Synergistic Stimulation by Gi- and Gq-Regulated Pathways.

CXCL8 (also known as interleukin-8 or IL-8) is a proinflammatory chemokine that not only modulates the inflammatory and immune responses, but whose upregulation is often associated with diseases including various types of cancer. Although numerous ligands for G protein-coupled receptors (GPCRs) have been shown to stimulate the production of CXCL8, the specificity of the G protein signal remains undefined. By expressing the constitutively active Gα subunits in HEK293 cells, CXCL8 production was herein demonstrated to be most effectively stimulated by Gαq family members, while those of Gαs and Gα12 elicited much weaker activities, and Gαi being totally ineffective. However, in cell lines such as HepG2, HeLa, and MCF-7 that endogenously express Gßγ-responsive phospholipase Cß isoforms (PLCß2/3), activation of the Gi-coupled α2-adrenoceptor significantly stimulated CXCL8 production. This Gi-induced CXCL8 production was apparently mediated via specific Gßγ dimers and required the presence of PLCß2/3. Co-activation of Gi-coupled α2-adrenoceptor and Gq-coupled bradykinin receptor resulted in a synergistic CXCL8 production, with Gßγ-responsive PLCß2/3, Src, ERK, and STAT3 serving as critical signaling intermediates. The treatment of HepG2 and B-10 endothelial cells with bradykinin stimulated CXCL8 production and cell proliferation. Interestingly, the latter response was driven by CXCL8 autocrine signaling because it was abolished by SB225002, an antagonist that prevents CXCL8 from binding to CXCR2. Collectively, our results provide a mechanistic basis for various G protein subfamilies to regulate the production of CXCL8, which may then lead to paracrine and/or autocrine signaling with major implications in both normal physiology and pathophysiological conditions.

An intact helical domain is required for Gα14 to stimulate phospholipase Cß.

BACKGROUND: Stimulation of phospholipase Cß (PLCß) by the activated α-subunit of Gq (Gαq) constitutes a major signaling pathway for cellular regulation, and structural studies have recently revealed the molecular interactions between PLCß and Gαq. Yet, most of the PLCß-interacting residues identified on Gαq are not unique to members of the Gαq family. Molecular modeling predicts that the core PLCß-interacting residues located on the switch regions of Gαq are similarly positioned in Gαz which does not stimulate PLCß. Using wild-type and constitutively active chimeras constructed between Gαz and Gα14, a member of the Gαq family, we examined if the PLCß-interacting residues identified in Gαq are indeed essential. RESULTS: Four chimeras with the core PLCß-interacting residues composed of Gαz sequences were capable of binding PLCß2 and stimulating the formation of inositol trisphosphate. Surprisingly, all chimeras with a Gαz N-terminal half failed to functionally associate with PLCß2, despite the fact that many of them contained the core PLCß-interacting residues from Gα14. Further analyses revealed that the non-PLCß2 interacting chimeras were capable of interacting with other effector molecules such as adenylyl cyclase and tetratricopeptide repeat 1, indicating that they could adopt a GTP-bound active conformation. CONCLUSION: Collectively, our study suggests that the previously identified PLCß-interacting residues are insufficient to ensure productive interaction of Gα14 with PLCß, while an intact N-terminal half of Gα14 is apparently required for PLCß interaction.

Activation of Gαq subunits up-regulates the expression of the tumor suppressor Fhit.

The tumor suppressor Fhit protein is defective or absent in many tumor cells due to methylation, mutation or deletion of the FHIT gene. Despite numerous attempts to unravel the functions of Fhit, the mechanisms by which the function and expression of Fhit are regulated remain poorly understood. We have recently shown that activated Gαq subunits interact directly with Fhit and enhance its inhibitory effect on cell growth. Here we investigated the regulation of Fhit expression by Gq. Our results showed that Fhit was up-regulated specifically by activating Gα subunits of the Gq subfamily but not by those of the other G protein subfamilies. This up-regulation effect was mediated by a PKC/MEK pathway independent of Src-mediated Fhit Tyr(114) phosphorylation. We further demonstrated that elevated Fhit expression was due to the specific regulation of Fhit protein synthesis in the ribosome by activated Gαq, where the regulations of cap-dependent protein synthesis were apparently not required. Moreover, we showed that activated Gαq could increase cell-cell adhesion through Fhit. These findings provide a possible handle to modulate the level of the Fhit tumor suppressor by manipulating the activity of Gq-coupled receptors.

Activation state-dependent interaction between Gαq subunits and the Fhit tumor suppressor.

BACKGROUND: The FHIT tumor suppressor gene is arguably the most commonly altered gene in cancer since it is inactivated in about 60% of human tumors. The Fhit protein is a member of the ubiquitous histidine triad proteins which hydrolyze dinucleoside polyphosphates such as Ap3A. Despite the fact that Fhit functions as a tumor suppressor, the pathway through which Fhit inhibits growth of cancer cells remains largely unknown. Phosphorylation by Src tyrosine kinases provides a linkage between Fhit and growth factor signaling. Since many G proteins can regulate cell proliferation through multiple signaling components including Src, we explored the relationship between Gα subunits and Fhit. RESULTS: Several members of the Gαq subfamily (Gα16, Gα14, and Gαq) were found to co-immunoprecipitate with Fhit in their GTP-bound active state in HEK293 cells. The binding of activated Gαq members to Fhit appeared to be direct and was detectable in native DLD-1 colon carcinoma cells. The use of Gα16/z chimeras further enabled the mapping of the Fhit-interacting domain to the α2-ß4 region of Gα16. However, Gαq/Fhit did not affect either Ap3A binding and hydrolysis by Fhit, or the ability of Gαq/16 to regulate downstream effectors including phospholipase Cß, Ras, ERK, STAT3, and IKK. Functional mutants of Fhit including the H96D, Y114F, L25W and L25W/I10W showed comparable abilities to associate with Gαq. Despite the lack of functional regulation of Gq signaling by Fhit, stimulation of Gq-coupled receptors in HEK293 and H1299 cells stably overexpressing Fhit led to reduced cell proliferation, as opposed to an enhanced cell proliferation typically seen with parental cells. CONCLUSIONS: Activated Gαq members interact with Fhit through their α2-ß4 region which may result in enhancement of the growth inhibitory effect of Fhit, thus providing a possible avenue for G protein-coupled receptors to modulate tumor suppression.

Interleukin-6 autocrine signaling mediates melatonin MT(1/2) receptor-induced STAT3 Tyr(705) phosphorylation.

Melatonin receptors have previously been shown to elicit cellular signaling through the hematopoietic-specific G protein, G(16) . In the present study, we show that this functional coupling elicited biphasic stimulatory phosphorylation on STAT3 in recombinant MT(1) /Gα(16) cells and native Jurkat T cells (endogenously expressing MT(1) and Gα(16) ), with maximal Ser(727) phosphorylation occurring at 15min, while marked Tyr(705) phosphorylation became detectable only upon agonist treatment for 4 hr or more. By employing signal transducer and activator of transcription 3 (STAT3) phosphorylation-resistant mutants (STAT3-Y705F and STAT3-S727A), we further showed that the receptor-mediated STAT3 phosphorylations at Ser(727) and Tyr(705) were independent of each other. Results obtained from fractionation of 2-IMT-induced cells revealed that the Ser(727) and Tyr(705) phosphorylations were spatially distinct, with the former mainly situated in mitochondria and cytosol, while the latter was predominantly located in the nucleus. Further experiments revealed that the agonist-induced STAT3 phosphorylation at Tyr(705) was significantly suppressed by pretreatment with cycloheximide (a ribosome inhibitor), suggesting that de novo protein synthesis might play a critical role for this response. Using conditioned media obtained from 2-IMT-treated MT(1) /Gα(16) cells, multiplex immunoassays revealed that prolonged agonist treatment led to elevated productions of IL-6, GM-CSF and CXCL-8. Antibody against IL-6, but not those for GM-CSF and CXCL-8, effectively abolished the agonist-induced STAT3 Tyr(705) phosphorylation, suggesting the involvement of IL-6 in melatonin receptor-mediated STAT3 activation. Our results demonstrate that melatonin receptor/Gα(16) coupling is capable of triggering the production of cytokines including IL-6, and this autocrine loop may account for the subsequent STAT3 phosphorylation at Tyr(705) .

RGS19 enhances cell proliferation through its C-terminal PDZ motif.

Regulator of G protein signaling 19 (RGS19), also known as Galpha-interacting protein (GAIP), is a GTPase activating protein (GAP) for Galpha(i) subunits. Apart from its GAP function, RGS19 has been implicated in growth factor signaling through binding to GAIP-interacting protein C-terminus (GIPC) via its C-terminal PDZ-binding motif. To gain additional insight on its function, we have stably expressed RGS19 in a number of mammalian cell lines and examined its effect on cell proliferation. Interestingly, overexpression of RGS19 stimulated the growth of HEK293, PC12, Caco2, and NIH3T3 cells. This growth promoting effect was not shared by other RGS proteins including RGS4, RGS10 and RGS20. Despite its ability to stimulate cell proliferation, RGS19 failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft-agar assays, and it did not induce tumor growth in athymic nude mice. Deletion mutants of RGS19 lacking the PDZ-binding motif failed to complex with GIPC and did not exhibit any growth promoting effect. Overexpression of GIPC alone in HEK293 cells stimulated cell proliferation whereas its knockdown in H1299 non-small cell lung carcinomas suppressed cell proliferation. This study demonstrates that RGS19, in addition to acting as a GAP, is able to stimulate cell proliferation in a GIPC-dependent manner.

Activation of STAT3 by specific Galpha subunits and multiple Gbetagamma dimers.

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.

Gbeta3 forms distinct dimers with specific Ggamma subunits and preferentially activates the beta3 isoform of phospholipase C.

Heterotrimeric G proteins regulate multiple effectors of which some are mediated via the Gbetagamma dimers. There is evidence to suggest that the functions of Gbetagamma dimers are not shared by all possible permutations of Gbetagamma complexes. Here, we report our efforts in defining the formation of distinct Gbetagamma dimers and their functional differences in activating phospholipase Cbeta (PLCbeta) isoforms. Co-immunoprecipitation assays using Cos-7 cells transiently expressing 48 different combinations of Gbeta(1-4) and Ggamma(1-5, 7-13) subunits showed that Gbeta(1) and Gbeta(4) could form dimers with all known Ggamma subunits, whereas several dimers could not be observed for Gbeta(2) and Gbeta(3). All Gbeta(1)gamma and Gbeta(2)gamma dimers significantly stimulated PLCbeta isoforms (PLCbeta(2)> or =PLCbeta(3)>PLCbeta(1)), but Gbeta(3)gamma and Gbeta(4)gamma dimers were poor activators of PLCbeta(1) and exhibited preference for PLCbeta(3) and PLCbeta(2), respectively. All Gbeta subunits revealed to date contain the previously identified PLCbeta(2)-interacting residues, but their neighboring residues in the proposed 3-D structures are different. To test if differences in these neighboring residues affect the interactions with PLCbeta isoforms, we generated several Gbeta(3) mutants by replacing one or more of these residues with their Gbeta(1) counterparts. One of these mutants (M120I, S140A and A141G triple mutant) acquired enhanced PLCbeta(2)-activating functions when co-expressed with different Ggamma subunits, while the corresponding stimulation on PLCbeta(3) was not altered. Taken together, our results show that the exact composition of a Gbetagamma dimer can determine its selectivity for activating PLCbeta isoforms, and certain residues in Gbeta(3) may account for the preferential stimulation of PLCbeta(3) by Gbeta(3)gamma dimers.

Dopaminergic and adrenergic toxicities on SK-N-MC human neuroblastoma cells are mediated through G protein signaling and oxidative stress.

Dopamine and norepinephrine are neurotransmitters which participate in various regulatory functions of the human brain. These functions are lost in neurodegenerative diseases including Parkinson's disease and Alzheimer's disease. In this study, we used SK-N-MC neuroblastoma cells to investigate the cytotoxicities of high concentrations of dopamine and norepinephrine on neuronal cells. Dopamine, norepinephrine, as well as their corresponding synthetic agonists (SKF38393 and isoproterenol, respectively) triggered SK-N-MC cell death when applied at 50-100 muM persistently for 2 days. This catecholamine-induced cell death appears to be neuronal specific, as demonstrated by their inabilities of triggering apoptosis of A549 lung carcinoma cells and Cos-7 kidney fibroblasts. By pretreating SK-N-MC cells with target-specific inhibitors before administration of catecholamine, components of G protein signaling (i.e. G( s )/cAMP/PKA), monoamine oxidases, nitric oxide synthase, c-Jun N-terminal kinase and oxidative stress were found to be involved in this dopamine/norepinephrine-induced cytotoxicity, which subsequently led to caspase-dependent and -independent apoptotic responses as well as DNA degradation. In contrast, agonists of G( i )-coupled dopamine receptors and adrenergic receptors (quinpirole and UK14,304, respectively) were incapable of triggering apoptosis of SK-N-MC cells. Our results suggest that both G protein (G( s ))-mediated signaling cascade and oxidative stress participate in the dopamine/norepinephrine-induced neuronal apoptosis.

CKBM stimulates MAPKs but inhibits LPS-induced IFN-gamma in lymphocytes.

CKBM is an herbal formula composed of five Chinese medicinal herbs (Panax ginseng, Schisandra chinensis, Fructus crataegi, Ziziphus jujuba and Glycine max) supplemented with processed Saccharomyces cerevisiae. It has been demonstrated that CKBM is capable of triggering the release of IL-6 and TNFalpha from human peripheral blood mononuclear cells. In this report, T-lymphocytic Sup-T1 cells and B-lymphocytic Ramos cells were utilized as cellular models to investigate how CKBM regulates intracellular signaling as well as the production of cytokines. CKBM stimulated the three major subgroups of mitogen-activated protein kinase (i.e. ERK, JNK and p38) in Sup-T1 cells, but only triggered the activation of ERK and p38 in Ramos cells. The induction of mitogen-activated protein kinases (MAPK) activations varied with the duration of treatment, as well as with the dosage of CKBM. In terms of cytokine production, treatment of CKBM alone did not trigger the release of IL-1beta and IFNgamma, but it suppressed the LPS-induced IFNgamma production from both Sup-T1 cells and Ramos cells. In view of the therapeutic effects of traditional Chinese medicines in inflammatory and autoimmune disorders, the results suggest that CKBM may exhibit its immuno-modulatory effects by regulating intracellular signaling as well as cytokine production in different lymphocytic cell types.

The aqueous extract of Radix Glycyrrhizae stimulates mitogen-activated protein kinases and nuclear factor-kappaB in Jurkat T-cells and THP-1 monocytic cells.

Radix Glycyrrhizae (RG) is a medicinal herb extensively utilized in numerous Chinese medical formulae for coordinating the actions of various components in the recipes and strengthening the body functions. In this report, we demonstrate that the aqueous extract of Radix Glycyrrhizae is capable of stimulating the c-Jun N-terminal kinase and p38 subgroups of mitogen-activated protein kinases (MAPKs), and the nuclear factor-kappaB (NFkappaB) in Jurkat T-lymphocytes. The activation magnitudes of MAPKs and NFkappaB were dose-dependent (EC(50) approximately 1 mg/ml) and time-dependent (maximal around 15-30 minutes). Stimulations of MAPKs and NFkappaB were not associated with changes in intracellular Ca(2+) mobilization. Similar activation profiles of MAPK and NFkappaB were obtained from THP-1 monocytes treated with the extract. In terms of chemotactic activity, the SDF-induced chemotaxis of Jurkat cells and THP-1 cells were inhibited by RG extract at 1-10 mg/ml, while a lower RG concentration (0.1-0.3 mg/ml) potentiated the SDF-induced chemotaxis for the former, but not the latter cell type. Given the fact that MAPKs and NFkappaB are important signaling intermediates for lymphocyte activities, our results suggest that Radix Glycyrrhizae may contain active constituents capable of modulating immuno-responses through various intracellular signaling pathways.

Gq-mediated activation of c-Jun N-terminal kinase by the gastrin-releasing peptide-preferring bombesin receptor is inhibited upon costimulation of the Gs-coupled dopamine D1 receptor in COS-7 cells.

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.

Integration of G protein signals by extracellular signal-regulated protein kinases in SK-N-MC neuroepithelioma cells.

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.

Epidermal growth factor differentially augments G(i)-mediated stimulation of c-Jun N-terminal kinase activity.

1. Signaling networks involving different receptor systems allow extracellular signals to be integrated and transformed into various biological activities. In this report, we studied the activity of the c-Jun N-terminal kinase (JNK) subgroup of mitogen-activated protein kinases (MAPKs), in response to stimulation by G protein-coupled receptors (GPCRs) and co-activation with epithermal growth factor receptor (EGFR). 2. Stimulation of exogenous GPCRs in Cos-7 cells induced JNK activation of different magnitudes depending on their G-protein coupling specificities (G(q)>G(i)>G(s)), and a moderate JNK activation was linked to stimulation of endogenous EGFR by EGF. 3. Co-stimulation with GPCR agonists and EGF resulted in differential augmentation of JNK activities, with G(i)-coupled receptors associated with a synergistic JNK activation upon co-stimulation with EGF, while G(q)- and G(s)-coupled receptors were incapable of triggering this effect. 4. This G(i)/EGF-induced synergistic JNK activation was inhibited by pertussis toxin and AG1478, and may involve Src family tyrosine kinases, PI3 K, Ca(2+)/calmodulin and small GTPases as important intermediates, while Ca(2+) mobilization was triggered by the stimulation of G(q)-coupled receptor or EGF treatment, but not by the G(i)- or G(s)-coupled receptors. 5. Transient expression of Gbetagamma subunits with EGF treatment, or co-activation of exogenous G(i)-coupled receptor with thapsigargin also resulted in a synergistic JNK activation. Activation of G(i)-coupled receptor accompanied with EGF treatment enhanced the expression level and activity of MAPK phosphatase type I, which occurred after the maximal synergistic JNK activation. 6. Our results support a mechanistic model where EGF signaling may differentially regulate the JNK activities triggered by GPCRs of different coupling specificities.

Gbetagamma signaling and Ca2+ mobilization co-operate synergistically in a Sos and Rac-dependent manner in the activation of JNK by Gq-coupled receptors.

The mechanism by which G(q)-coupled receptors stimulate the c-Jun N-terminal kinase (JNK) activity has not been fully delineated. Here, we showed that stimulation of endogenous G(q)-coupled receptors in human hepatocarcinoma HepG2 cells resulted in an Src family kinase- and Ca(2+)-dependent JNK activation. Cos-7 cells transfected with HA-tagged JNK and various G(q)-coupled receptors also exhibited similar characteristics and provided further evidence for the involvement of Gbetagamma, an upstream intermediate for Src family kinases. The Ca(2+) and Gbetagamma signals operate in a high degree of independence. Transient expression of Gbetagamma subunits and elevation of cytoplasmic Ca(2+) level by thapsigargin activated JNK in a synergistic fashion. JNK activities triggered by G(q)-coupled receptors, Gbetagamma and thapsigargin were all suppressed by dominant negative (DN) mutants of Son of sevenless (Sos) and Rac. We propose that the co-operative effect between Gbetagamma-mediated signaling and the increased intracellular Ca(2+) level represents a robust mechanism for the stimulation of JNK by G(q)-coupled receptors.

Phosphatidylinositol-3 kinase is distinctively required for mu-, but not kappa-opioid receptor-induced activation of c-Jun N-terminal kinase.

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.

Kappa-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.

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.

The first and third intracellular loops together with the carboxy terminal tail of the delta-opioid receptor contribute toward functional interaction with Galpha16.

Opioid peptides exert their regulatory effects on both central and peripheral nervous systems via multiple opioid receptors that are linked to seemingly identical sets of guanine nucleotide-binding regulatory proteins (G proteins). In contrast to the mu-opioid receptor, the delta-opioid receptor can efficiently stimulate phospholipase C via G16. We used a series of mu/delta-opioid receptor chimeras to examine the involvement of intracellular receptor domains in the recognition of G16. After ascertaining that the chimeras can bind opioid ligands with high affinity and elicit inhibition of adenylyl cyclase, COS-7 cells were cotransfected with cDNAs encoding Galpha16 and a mu/delta-opioid receptor chimera and assayed for [D-Ala2,D-Leu5]enkephalin-induced stimulation of phospholipase C. Our results indicate that (i) the carboxy terminal tail of the delta-opioid receptor is necessary but insufficient for conferring coupling to Galpha16, (ii) the third inner loop together with the carboxy terminal tail of the delta-opioid receptor can provide sufficient contact domains for Galpha16, and (iii) the first inner loop of the delta-opioid receptor, in particular Leu80, as well as the fifth transmembrane domain and/or the third extracellular loop may also contribute in defining the fidelity of interaction between the delta-opioid receptor and Galpha16. These results indicate that efficient coupling of the delta-opioid receptor to Galpha16 requires the participation of most of the intracellular regions, including the first intracellular loop.

Rac and Cdc42-dependent regulation of c-Jun N-terminal kinases by the delta-opioid receptor.

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 x glioma hybrid cells that endogenously express delta-opioid receptors, delta-agonist dose-dependently stimulated JNK activity in a pertussis toxin-sensitive manner. By using COS-7 cells transiently transfected with the cDNAs of delta-opioid receptor and hemagglutinin (HA)-tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gbetagamma 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 delta-opioid receptor, and this pathway requires Gbetagamma, Src kinases and the small GTPases Rac and Cdc42.

Melatonin mt1 and MT2 receptors stimulate c-Jun N-terminal kinase via pertussis toxin-sensitive and -insensitive G proteins.

Melatonin is a pineal hormone involved in neuroendocrine processes in mammals. It has been shown that melatonin inhibits the enzymatic activities of adenylyl cyclases and the transcriptional activities of CREB. In this report, we demonstrate that 2-iodomelatonin (2IMT) treatment on COS-7 cells transfected with melatonin receptors (mt1 and MT2) induces c-Jun N-terminal kinase (JNK) activation, which is pertussis toxin (PTX)-sensitive, Ras/Rac-dependent and may involve Src-family protein tyrosine kinases. Moreover, PTX-insensitive Gs, Gz and G16 are capable of linking activated melatonin receptors to the stimulation of JNK. Agonist stimulation on PTX-pretreated COS-7 cells overexpressing mt1 receptor, Galpha(s) and adenylyl cyclase VI led to increased cAMP accumulation. Stimulation of endogenous mt1 receptors in MCF-7 cells was associated with the activation of both JNK and extracellular signal-regulated kinase (ERK). This report demonstrates the stimulatory effect of melatonin receptors on JNK, and provides experimental evidence for a functional coupling between the G(i)-coupled melatonin receptor and Gs, in terms of adenylyl cyclase activation.