Anthony S.L. Chan

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.

Protocatechuic acid induces cell death in HepG2 hepatocellular carcinoma cells through a c-Jun N-terminal kinase-dependent mechanism

Protocatechuic acid (PCA), chlorogenic acid (CA) and luteolin (LT) are plant phenols found in Chinese medicinal herbs such as Lonicera japonica. Cytotoxicity assays showed that PCA, CA and LT (at 100 micromol/L) effectively killed the HepG2 hepatocellular carcinoma cells. Among these three naturally occurring compounds, only PCA was capable of stimulating the c-Jun N-terminal kinase (JNK) and p38 subgroups of the mitogen-activated protein kinase (MAPK) family. Coincidently, PCA-induced cell death was rescued by specific inhibitors for JNK and p38, while the cytotoxicities of CA and LT were partially eliminated by the antioxidant effect of N-acetyl-L-cysteine (NAC). Further investigation demonstrated that the aqueous extract of Lonicera japonica also triggered HepG2 cell death in a JNK-dependent manner, but the amount of PCA alone in this herbal extract was insufficient to contribute the subsequent cytotoxic effect. Collectively, our results suggest that PCA is a naturally occurring compound capable of inducing JNK-dependent hepatocellular carcinoma cell death.

Rac and Cdc42‐dependent regulation of c‐Jun N‐terminal kinases by the δ‐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 × 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.

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 (µ, δ and κ) are prototypical Gi‐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 δ‐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 µ‐opioid receptor could stimulate JNK in both SH‐SY5Y cells and transfected COS‐7 cells. The mechanism by which the µ‐opioid receptor stimulated JNK was delineated with the use of specific inhibitors and dominant‐negative mutants of signaling intermediates. Activation of JNK by the µ‐opioid receptor was mediated through Gβγ, Src kinase, son‐of‐sevenless (Sos), Rac and Cdc42. Interestingly, unlike the δ‐opioid receptors, the µ‐opioid receptor required phosphatidylinositol‐3 kinase (PI3K) to activate JNK. The µ‐opioid receptor‐induced JNK activation was effectively inhibited by wortmannin or the coexpression of a dominant negative mutant of PI3Kγ. Like the δ‐opioid receptor, activation of JNK by the κ‐opioid receptor occurred in a PI3K‐independent manner. These studies revealed that the µ‐opioid receptor utilize a distinct mechanism to regulate JNK.

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.

Gβ3 forms distinct dimers with specific Gγ subunits and preferentially activates the β3 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.

Interleukin-6 autocrine signaling mediates melatonin MT1/2 receptor-induced STAT3 Tyr705 phosphorylation

Abstract:  Melatonin receptors have previously been shown to elicit cellular signaling through the hematopoietic‐specific G protein, G16. In the present study, we show that this functional coupling elicited biphasic stimulatory phosphorylation on STAT3 in recombinant MT1/Gα16 cells and native Jurkat T cells (endogenously expressing MT1 and Gα16), with maximal Ser727 phosphorylation occurring at 15 min, while marked Tyr705 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 Ser727 and Tyr705 were independent of each other. Results obtained from fractionation of 2‐IMT‐induced cells revealed that the Ser727 and Tyr705 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 Tyr705 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 MT1/Gα16 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 Tyr705 phosphorylation, suggesting the involvement of IL‐6 in melatonin receptor‐mediated STAT3 activation. Our results demonstrate that melatonin receptor/Gα16 coupling is capable of triggering the production of cytokines including IL‐6, and this autocrine loop may account for the subsequent STAT3 phosphorylation at Tyr705.

Activation of STAT3 by specific Gα subunits and multiple Gβγ 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.

The first and third intracellular loops together with the carboxy terminal tail of the δ-opioid receptor contribute toward functional interaction with Gα16

Opioid peptides exert their regulatory effects on both central and pheripheral 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 μ‐opioid receptor, the δ‐opioid receptor can efficiently stimulate phospholipase C via G16. We used a series of μ/δ‐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 Gα16 and a μ/δ‐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 δ‐opioid receptor is necessary but insufficient for conferring coupling to Gα16, (ii) the third inner loop together with the carboxy terminal tail of the δ‐opioid receptor can provide sufficient contact domains for Gα16, and (iii) the first inner loop of the δ‐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 δ‐opioid receptor and Gα16. These results indicate that efficient coupling of the δ‐opioid receptor to Gα16 requires the participation of most of the intracellular regions, including the first intracellular loop.

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.