Yung Hou Wong

Regulation of Multiple Effectors by the Cloned δ-Opioid Receptor: Stimulation of Phospholipase C and Type II Adenylyl Cyclase

Abstract: The δ‐opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ‐opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ‐selective agonist [d‐Pen2,d‐Pen5]enkephalin. The inhibitory response of [d‐Pen2,d‐Pen5]enkephalin was dependent on the expression of the δ‐opioid receptor and exhibited an EC50 of 1 nM. The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ‐opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ‐opioid receptor with type II adenylyl cyclase and an activated mutant of αs converted the δ‐opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk− fibroblasts, the cloned δ‐opioid receptor was able to stimulate the formation of inositol phosphates (EC50 = 8 nM). This response was sensitive to pertussis toxin. The opioid‐mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ‐opioid receptor to couple to G proteins other than Gi was also examined. Cotransfection studies revealed that the δ‐opioid receptor can utilize Gz to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.

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.

Molecular mechanisms mediating the G protein-coupled receptor regulation of cell cycle progression

G protein-coupled receptors are key regulators of cellular communication, mediating the efficient coordination of a cells responses to extracellular stimuli. When stimulated these receptors modulate the activity of a wide range of intracellular signalling pathways that facilitate the ordered development, growth and reproduction of the organism. There is now a growing body of evidence examining the mechanisms by which G protein-coupled receptors are able to regulate the expression, activity, localization and stability of cell cycle regulatory proteins that either promote or inhibit the initiation of DNA synthesis. In this review, we will detail the intracellular pathways that mediate the G protein-coupled receptor regulation of cellular proliferation, specifically the progression from the G1 phase to the S phase of the cell cycle.

Constitutively active Gα16 stimulates STAT3 via a c-Src/JAK-and ERK-dependent mechanism

The hematopoietic-specific Gα16 protein has recently been shown to mediate receptor-induced activation of the signal transducer and activator of transcription 3 (STAT3). In the present study, we have delineated the mechanism by which Gα16 stimulates STAT3 in human embryonic kidney 293 cells. A constitutively active Gα16 mutant, Gα16QL, stimulated STAT3-dependent luciferase activity as well as the phosphorylation of STAT3 at both Tyr705 and Ser727. Gα16QL-induced STAT3 activation was enhanced by overexpression of extracellular signal-regulated kinase 1 (ERK1), but was inhibited by U0126, a Raf-1 inhibitor, and coexpression of the dominant negative mutants of Ras and Rac1. Inhibition of phospholipase Cβ, protein kinase C, and calmodulin-dependent kinase II by their respective inhibitors also suppressed Gα16QL-induced STAT3 activation. The involvement of tyrosine kinases such as c-Src and Janus kinase 2 and 3 (JAK2 and JAK3) in Gα16QL-induced activation of STAT3 was illustrated by the combined use of selective inhibitors and dominant negative mutants. In contrast, c-Jun N-terminal kinase, p38 MAPK, RhoA, Cdc42, phosphatidylinositol 3-kinase, and the epidermal growth factor receptor did not appear to be required. Similar observations were obtained with human erythroleukemia cells, where STAT3 phosphorylation was stimulated by C5a in a PTX-insensitive manner. Collectively, these results highlight the important regulatory roles of the Ras/Raf/MEK/ERK and c-Src/JAK pathways on the stimulation of STAT3 by activated Gα16. Demonstration of the involvement of different kinases in Gα16QL-induced STAT3 activation supports the involvement of multiple signaling pathways in the regulation of transcription by G proteins.

Characterization and channel coupling of the P2Y(12) nucleotide receptor of brain capillary endothelial cells.

Rat brain capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y12 ADP receptor (“P2Y T ”) of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC50, 2 nm), ADP, and adenosine 5′-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-d-β,γ-difluoromethylene-ATP was a competitive antagonist (K B , 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC50, 0.4 nm), ATP (1.9 μm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC50, 0.1 nm) GIRK1/GIRK2 inward rectifier K+ channels when co-expressed with P2Y12 receptors in sympathetic neurons. Surprisingly, P2Y1 receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y12 receptors. A new P2Y12-mediated transduction was found, the closing of native N-type Ca2+channels; again both 2-MeSATP and 2-MeSADP are agonists (EC50, 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca2+ channel inhibition and K+ channel activation are mediated by βγ subunit release from a heterotrimeric G-protein. Gα subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y12 receptor is a significant extension of its functional range.

Differential coupling of μ-, δ-, and κ-opioid receptors to Gα16-Mediated stimulation of phospholipase C

Abstract: 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.  Transfusion 1993;33: 898–901.14. Butson MJ, Yu PK, Cheung T, et al. Dosimetry of blood irradiation with radiochromic film. Transfus Med 1999;9: 205–8.15. Nath R, Biggs PJ, Ling CC, et al. AAPM code of practice for radiotherapy accelerators: Report of AAPM Radiation Therapy Task Group No. 45. Med Phys

Activation of type II adenylyl cyclase by the cloned μ-opioid receptor: Coupling to multiple G proteins

Abstract: Opioid receptors are multifunctional receptors that utilize G proteins for signal transduction. The cloned δ‐opioid receptor has been shown recently to stimulate phospholipase C, as well as to inhibit or stimulate different isoforms of adenylyl cyclase. By using transient transfection studies, the ability of the cloned μ‐opioid receptor to stimulate type II adenylyl cyclase was examined. Coexpression of the μ‐opioid receptor with type II adenylyl cyclase in human embryonic kidney 293 cells allowed the μ‐selective agonist, [d‐Ala2, N‐Me‐Phe4,Gly5‐ol]enkephalin, to stimulate cyclic AMP accumulation in a dose‐dependent manner. The opioid‐induced stimulation of type II adenylyl cyclase was mediated via pertussis toxin‐sensitive Gi proteins, because it was abolished completely by the toxin. Possible coupling between the μ‐opioid receptor and various G protein α subunits was examined in the type II adenylyl cyclase system. The opioid‐induced response became pertussis toxin‐insensitive and was enhanced significantly upon co‐expression with the α subunit of Gz, whereas those of Gq, G12, or G13 inhibited the opioid response. When pertussis toxin‐sensitive G protein α subunits were tested under similar conditions, all three forms of αi and both forms of αo were able to enhance the opioid response to various extents. Enhancement of type II adenylyl cyclase responses by the co‐expression of α subunits reflects a functional coupling between α subunits and the μ‐opioid receptor, because such potentiations were not observed with the constitutively activated α subunit mutants. These results indicate that the μ‐opioid receptor can couple to Gi1–3, Go1–2, and Gz, but not to Gs, Gq, G12, G13, or Gt.

Naturally occurring phenylethanoid glycosides: potential leads for new therapeutics.

Natural products have long been regarded as excellent sources for drug discovery given their structure diversity and wide variety of biological activities. Phenylethanoid glycosides are naturally occurring compounds of plant origin and are structurally characterized with a hydroxyphenylethyl moiety to which a glucopyranose is linked through a glycosidic bond. To date several hundred compounds of this type have been isolated from medicinal plants and further pharmacological studies in vitro or in vivo have shown that these compounds possess a broad array of biological activities including antibacterial, antitumor, antiviral, anti-inflammatory, neuro-protective, antioxidant, hepatoprotective, immunomodulatory, and tyrosinase inhibitory actions. Given their extensive activity profile, structure-activity relationships analyses of these compounds have been performed in a number of studies to reveal potential leads for future drug design. This article will summarize the major developments in phenylethanoid glycosides-based research in the past decade. The progresses made in phytochemistry and biological activity studies of these compounds will be reviewed. Particular attention will be given to the novel structures identified to date and the prominent therapeutic values associated with these molecules.

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.

G z signaling: Emerging divergence from G i signaling

A large variety of neurotransmitters, hormones, and chemokines regulate cellular functions via cell surface receptors that are coupled to guanine nucleotide-binding regulatory proteins (G proteins) belonging to the Gi subfamily. All members of the Gi subfamily, with the sole exception of Gz, are substrates for the pertussis toxin ADP-ribosyl transferase. Gz also exhibits unique biochemical and regulatory properties. Initial portrayals of the cellular functions of Gz bear high resemblance to those of other Gi proteins both in terms of the receptors and effectors linked to Gz. However, recent discoveries have begun to insinuate a distinct role for Gz in cellular communication. Functional interactions of the α subunit of Gz (Gαz) with the NKR-P1 receptor, Gαz-specific regulator of G protein signaling, p21-activated kinase, G protein-regulated inducers of neurite outgrowth, and the Eya2 transcription cofactor have been demonstrated. These findings provide possible links for Gz to participate in cellular development, survival, proliferation, differentiation and even apoptosis. In this review, we have drawn a sketch of a signaling network with Gz as the centerpiece. The emerging picture is one that distinguishes Gz from other members of the Gi subfamily.