Mutations at the dimer interface and surface residues of Nm23-H1 metastasis suppressor affect its expression and function.

The Nm23 metastasis suppressor family is involved in a variety of physiological and pathological processes including cell proliferation, differentiation, tumorigenesis, and metastasis. Given that Nm23 proteins may function as hexamers composed of different members of the family, especially Nm23-H1 and H2 isoforms, it is pertinent to assess the importance of interface and surface residues in defining the functional characteristics of Nm23 proteins. Using molecular modeling to identify clusters of residues that may affect dimer formation and isoform specificity, mutants of Nm23-H1 were constructed and assayed for their ability to modulate cell migration. Mutations of dimer interface residues Gly22 and Lys39 affected the expression level of Nm23-H1, without altering the transcript level. The reduced protein expression was not due to increased protein degradation or altered subcellular distribution. Substitution of the surface residues of Nm23-H1 with Nm23-H2-specific Ser131 and/or Lys124/135 affected the electrophoretic mobility of the protein. Moreover, in cell migration assays, several mutants with altered surface residues exhibited impaired ability to suppress the mobility of MDA-MB-231 cells. Collectively, the study suggests that disrupting the dimer interface may affect the expression of Nm23-H1, while the residues at α-helix and ß-sheet on the surface of Nm23-H1 may contribute to its metastasis suppressive function.

AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

Role of G Protein-Coupled Receptors in the Regulation of Structural Plasticity and Cognitive Function.

Cognition and other higher brain functions are known to be intricately associated with the capacity of neural circuits to undergo structural reorganization. Structural remodelling of neural circuits, or structural plasticity, in the hippocampus plays a major role in learning and memory. Dynamic modifications of neuronal connectivity in the form of dendritic spine morphology alteration, as well as synapse formation and elimination, often result in the strengthening or weakening of specific neural circuits that determine synaptic plasticity. Changes in dendritic complexity and synapse number are mediated by cellular processes that are regulated by extracellular signals such as neurotransmitters and neurotrophic factors. As many neurotransmitters act on G protein-coupled receptors (GPCRs), it has become increasingly apparent that GPCRs can regulate structural plasticity through a myriad of G protein-dependent pathways and non-canonical signals. A thorough understanding of how GPCRs exert their regulatory influence on dendritic spine morphogenesis may provide new insights for treating cognitive impairment and decline in various age-related diseases. In this article, we review the evidence of GPCR-mediated regulation of structural plasticity, with a special emphasis on the involvement of common as well as distinct signalling pathways that are regulated by major neurotransmitters.

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.

Association of activated Gαq to the tumor suppressor Fhit is enhanced by phospholipase Cß.

BACKGROUND: G proteins are known to modulate various growth signals and are implicated in the regulation of tumorigenesis. The tumor suppressor Fhit is a newly identified interaction partner of Gq proteins that typically stimulate the phospholipase C pathway. Activated Gαq subunits have been shown to interact directly with Fhit, up-regulate Fhit expression and enhance its suppressive effect on cell growth and migration. Other signaling molecules may be involved in modulating Gαq/Fhit interaction. METHODS: To test the relationship of PLCß with the interaction between Gαq and Fhit, co-immunoprecipication assay was performed on HEK293 cells co-transfected with different combinations of Flag-Fhit, Gα16, Gα16QL, pcDNA3 vector, and PLCß isoforms. Possible associations of Fhit with other effectors of Gαq were also demonstrated by co-immunoprecipitation. The regions of Gαq for Fhit interaction and PLCß stimulation were further evaluated by inositol phosphates accumulation assay using a series of Gα16/z chimeras with discrete regions of Gα16 replaced by those of Gαz. RESULTS: PLCß1, 2 and 3 interacted with Fhit regardless of the expression of Gαq. Expression of PLCß increased the affinities of Fhit for both wild-type and activated Gαq. Swapping of the Fhit-interacting α2-ß4 region of Gαq with Gαi eliminated the association of Gαq with Fhit without affecting the ability of the mutant to stimulate PLCß. Other effectors of Gαq including RGS2 and p63RhoGEF were unable to interact with Fhit. CONCLUSIONS: PLCß may participate in the regulation of Fhit by Gq in a unique way. PLCß interacts with Fhit and increases the interaction between Gαq and Fhit. The Gαq/PLCß/Fhit complex formation points to a novel signaling pathway that may negatively regulate tumor cell growth.

Activator of G protein signaling 3 forms a complex with resistance to inhibitors of cholinesterase-8A without promoting nucleotide exchange on Gα(i3).

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) which stabilizes the Gα(i/o) subunits as an AGS3/Gα(i/o)-GDP complex. It has recently been demonstrated in reconstitution experiments that the AGS3/Gα(i/o)-GDP complex may act as a substrate of resistance to inhibitors of cholinesterase 8A (Ric-8A), a guanine exchange factor (GEF) for heterotrimeric Gα proteins. Since the ability of Ric-8A to activate Gα(i/o) subunits that are bound to AGS3 in a cellular environment has not been confirmed, we thus examined the effect of Ric-8A on cAMP accumulation in HEK293 cells expressing different forms of AGS3 and Gα(i3). Co-immunoprecipitation assays indicate that full-length AGS3 and its N- and C-terminal truncated mutants can interact with Ric-8A in HEK293 cells. Yeast two-hybrid assay further confirmed that Ric-8A can directly bind to AGS3S, a short form of AGS3 which is endogenously expressed in heart. However, Ric-8A failed to facilitate Gα(i)-induced suppression of adenylyl cyclase, suggesting that it may not serve as a GEF for AGS3/Gα(i/o)-GDP complex in a cellular environment.

CCR1-mediated STAT3 tyrosine phosphorylation and CXCL8 expression in THP-1 macrophage-like cells involve pertussis toxin-insensitive Gα(14/16) signaling and IL-6 release.

Agonists of CCR1 contribute to hypersensitivity reactions and atherosclerotic lesions, possibly via the regulation of the transcription factor STAT3. CCR1 was demonstrated to use pertussis toxin-insensitive Gα(14/16) to stimulate phospholipase Cß and NF-κB, whereas both Gα(14) and Gα(16) are also capable of activating STAT3. The coexpression of CCR1 and Gα(14/16) in human THP-1 macrophage-like cells suggests that CCR1 may use Gα(14/16) to induce STAT3 activation. In this study, we demonstrated that a CCR1 agonist, leukotactin-1 (CCL15), could indeed stimulate STAT3 Tyr(705) and Ser(727) phosphorylation via pertussis toxin-insensitive G proteins in PMA-differentiated THP-1 cells, human erythroleukemia cells, and HEK293 cells overexpressing CCR1 and Gα(14/16). The STAT3 Tyr(705) and Ser(727) phosphorylations were independent of each other and temporally distinct. Subcellular fractionation and confocal microscopy illustrated that Tyr(705)-phosphorylated STAT3 translocated to the nucleus, whereas Ser(727)-phosphorylated STAT3 was retained in the cytosol after CCR1/Gα(14) activation. CCL15 was capable of inducing IL-6 and IL-8 (CXCL8) production in both THP-1 macrophage-like cells and HEK293 cells overexpressing CCR1 and Gα(14/16). Neutralizing Ab to IL-6 inhibited CCL15-mediated STAT3 Tyr(705) phosphorylation, whereas inhibition of STAT3 activity abolished CCL15-activated CXCL8 release. The ability of CCR1 to signal through Gα(14/16) provides a linkage for CCL15 to regulate IL-6/STAT3-signaling cascades, leading to expression of CXCL8, a cytokine that is involved in inflammation and the rupture of atherosclerotic plaque.

Rapid uptake and degradation of CXCL12 depend on CXCR7 carboxyl-terminal serine/threonine residues.

CXCL12 signaling through G protein-coupled CXCR4 regulates cell migration during ontogenesis and disease states including cancer and inflammation. The second CXCL12-receptor CXCR7 modulates the CXCL12/CXCR4 pathway by acting as a CXCL12 scavenger and exerts G protein-independent functions. Given the distinct properties of CXCR4 and CXCR7, we hypothesized that the distinct C-terminal domains differently regulate receptor trafficking and stability. Here, we examined epitope-tagged wild type and C-terminal mutant receptors in human embryonic kidney cells (HEK293) with respect to trafficking, stability, (125)I-CXCL12 degradation, and G protein-coupling. The 24 CXCR7 C-terminal residues were sufficient to promote rapid spontaneous internalization. Replacement of the CXCR7 C terminus with that of CXCR4 (CXCR7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization and phosphorylation at the heterologous domain. The reverse tail-swap caused ligand-independent internalization of the resulting CXCR4-7tail mutant. Receptor-mediated (125)I-CXCL12 uptake and release of (125)I-CXCL12 degradation products were accelerated with receptors bearing the CXCR7 C terminus and impaired after conversion of CXCR7 C-terminal serine/threonine residues into alanines. C-terminal lysine residues were dispensable for plasma membrane targeting and the CXCL12 scavenger function but involved in constitutive degradation of CXCR7. Although the CXCR7 C terminus abolished G protein coupling in the CXCR4-7tail mutant, replacement of the CXCR7 C terminus, CXCR7 second intracellular loop, or both domains with the corresponding CXCR4 domain did not result in a G protein-coupled CXCR7 chimera. Taken together, we provide evidence that the CXCR7 C terminus influences the ligand-uptake/degradation rate, G protein coupling, and receptor stability. Regulatory pathways targeting CXCR7 C-terminal serine/threonine sites may control the CXCL12 scavenger activity of CXCR7.

Neuronal functions of activators of G protein signaling.

G protein-coupled receptors (GPCRs) are one of the most important gateways for signal transduction across the plasma membrane. Over the past decade, several classes of alternative regulators of G protein signaling have been identified and reported to activate the G proteins independent of the GPCRs. One group of such regulators is the activator of G protein signaling (AGS) family which comprises of AGS1-10. They have entirely different activation mechanisms for G proteins as compared to the classic model of GPCR-mediated signaling and confer upon cells new avenues of signal transduction. As GPCRs are widely expressed in our nervous system, it is believed that the AGS family plays a major role in modulating the G protein signaling in neurons. In this article, we will review the current knowledge on AGS proteins in relation to their potential roles in neuronal regulations.

Gßγ-mediated activation of protein kinase D exhibits subunit specificity and requires Gßγ-responsive phospholipase Cß isoforms.

BACKGROUND: Protein kinase D (PKD) constitutes a novel family of serine/threonine protein kinases implicated in fundamental biological activities including cell proliferation, survival, migration, and immune responses. Activation of PKD in these cellular activities has been linked to many extracellular signals acting through antigen receptor engagement, receptor tyrosine kinases, as well as G protein-coupled receptors. In the latter case, it is generally believed that the Gα subunits of the Gq family are highly effective in mediating PKD activation, whereas little is known with regard to the ability of Gßγ dimers and other Gα subunits to stimulate PKD. It has been suggested that the interaction between Gßγ and the PH domain of PKD, or the Gßγ-induced PLCß/PKC activity is critical for the induction of PKD activation. However, the relative contribution of these two apparently independent events to Gßγ-mediated PKD activation has yet to be addressed. RESULTS: In this report, we demonstrate that among various members in the four G protein families, only the Gα subunits of the Gq family effectively activate all the three PKD isoforms (PKD1/2/3), while Gα subunits of other G protein families (Gs, Gi, and G12) are ineffective. Though the Gα subunits of Gi family are unable to stimulate PKD, receptors linked to Gi proteins are capable of triggering PKD activation in cell lines endogenously expressing (HeLa cells and Jurkat T-cells) or exogenously transfected with (HEK293 cells) Gßγ-sensitive PLCß2/3 isoforms. This indicates that the Gi-mediated PKD activation is dependent on the released Gßγ dimers upon stimulation. Further investigation on individual Gßγ combinations (i.e. Gß1 with Gγ1-13) revealed that, even if they can stimulate the PLCß activity in a comparable manner, only those Gß1γ dimers with γ2, γ3, γ4, γ5, γ7, and γ10 can serve as effective activators of PKD. We also demonstrated that Gi-mediated PKD activation is essential for the SDF-1α-induced chemotaxis on Jurkat T-cells. CONCLUSIONS: Our current report illustrates that Gßγ dimers from the Gi proteins may activate PKD in a PLCß2/3-dependent manner, and the specific identities of Gγ components within Gßγ dimers may determine this stimulatory action.

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.

Development of substituted N-[3-(3-methoxylphenyl)propyl] amides as MT(2)-selective melatonin agonists: improving metabolic stability.

A series of novel and selective N-[3-(6-benzyloxy-3-methoxyphenyl)propyl] amides has recently been shown to possess sub-nanomolar range binding affinity to the type 2 melatonin receptor (MT(2)). Pharmacokinetics studies suggested that these compounds were subject to vigorous CYP450-mediated metabolism, resulting in a series of metabolites with significantly decreased or diminished binding affinities toward MT(2) receptor. The ether bonds were found to be the major positions susceptible to metabolism. In this study, the benzyl ether bond was either removed or replaced with a carbon-carbon bond in an attempt to improve metabolic stability and enhance their resistance towards phase I oxidation. The synthesis, receptor binding affinity, intrinsic potency and metabolic stability of modified structures are reported in this article. By removal or replacement of metabolic labile ether linkerage with carbon linkers, a novel compound was identified with good potency and MT(2) selectivity, and with increased metabolic stability.

Activation of Ras-dependent signaling pathways by G(14) -coupled receptors requires the adaptor protein TPR1.

Many G(q) -coupled receptors mediate mitogenic signals by stimulating extracellular signal-regulated protein kinases (ERKs) that are typically regulated by the small GTPase Ras. Recent studies have revealed that members of the Gα(q) family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gα(q) family, the highly promiscuous Gα(14) can relay signals from numerous receptors. Here, we examined if Gα(14) interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα(14) QL mutant in HEK293 cells led to the formation of GTP-bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IκB kinase. Stimulation of endogenous G(14) -coupled somatostatin type 2 and α(2) -adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co-immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Gα(14) QL than Gα(14) , suggesting that TPR1 preferentially binds to the GTP-bound form of Gα(14) . Activated Gα(14) also interacted with the Ras guanine nucleotide exchange factors SOS1 and SOS2. Expression of a dominant negative mutant of TPR1 or siRNA-mediated knockdown of TPR1 effectively abolished the ability of Gα(14) to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα(14) QL-induced phosphorylations of ERK and IκB kinase, it did not affect Gα(14) QL-induced stimulation of phospholipase Cß or c-Jun N-terminal kinase. Our results suggest that TPR1 is required for Gα(14) to stimulate Ras-dependent signaling pathways, but not for the propagation of signals along Ras-independent pathways.

Elevated expression of RGS19 impairs the responsiveness of stress-activated protein kinases to serum.

Regulators of G protein signaling (RGS proteins) serve as GTPase activating proteins for the signal transducing Gα subunits. RGS19, also known as Gα-interacting protein (GAIP), has been shown to subserve other functions such as the regulation of macroautophagy and growth factor signaling. We have recently demonstrated that the expression of RGS19 in human embryonic kidney (HEK) 293 cells resulted in the disruption of serum-induced mitogenic response along the classical Ras/Raf/MEK/ERK pathway. Here, we further examined the effect of RGS19 expression on the stress-activated protein kinases (SAPKs). Both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) became non-responsive to serum in 293/RGS19 cells, yet the two SAPKs responded to UV irradiation or osmotic stress induced by sorbitol. Kinases upstream of JNK and p38 MAPK, including MKK3/6, MKK4, and MLK3, also failed to respond to serum stimulation in 293/RGS19 cells. Serum-induced activation of the small GTPases Rac1 and Cdc42 was similarly suppressed in these cells. Our results indicate that elevated expression of RGS19 can severely disrupt the regulation of MAPKs by small GTPases.

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) .

Pharmacokinetics, oral bioavailability and metabolism of a novel isoquinolinone-based melatonin receptor agonist in rats.

7-Methoxy-6-(3-methoxy-benzyloxy)-2-methylisoquinolin-1(2H)-one (named as IS0042) is a newly identified melatoninergic agonist which exhibits selectivity to the type 2 melatonin receptor. Here, we examined the in vitro and in vivo pharmacokinetics properties of IS0042 in rats. IS0042 was considerably lipophilic with a modest aqueous solubility of 27.3 µg/mL. It was stable in simulated gastrointestinal fluid, and readily penetrated across differentiated Caco-2 cell model of intestinal barrier, suggesting good oral absorption. IS0042 underwent metabolism in rat intestinal and liver microsomes with an in vitro half-life of 367.5 ± 36.6 and 17.5 ± 2.7 min, respectively. Metabolite identification suggested that the major biotransformation pathways included the cleavage of ether bond, hydroxylation and demethylation. The same metabolites were also present in blood circulation following oral administration, indicating a good correlation between in vitro and in vivo metabolism. The pharmacokinetics parameters of IS0042 were evaluated after intravenous administration (10 or 25 mg/kg) and oral administration (100 mg/kg) of the drug to rats. IS0042 showed moderate clearance (0.73-1.02 L/h/kg), large volume of distribution (1.76-3.16 L/kg) and long elimination half-life (3.11-6.04 h) after intravenous administration. The absolute oral bioavailability of IS0042 was relatively low (9.8-18.6%). Overall, these results provide important parameters for the further development of this novel class of melatoninergic ligands.

Astragaloside IV and cycloastragenol stimulate the phosphorylation of extracellular signal-regulated protein kinase in multiple cell types.

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

Gα16 interacts with tetratricopeptide repeat 1 (TPR1) through its ß3 region to activate Ras independently of phospholipase Cß signaling.

BACKGROUND: G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the Gα subunits are required to interact with multiple partners. The GTP-bound active state of many Gα subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the Gα subunit for binding. Using Gα16 as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase Cß (PLCß). RESULTS: We have examined the specificity of Gα16/TPR1 association by testing a series of chimeras between Gα16 and Gαz. TPR1 co-immunoprecipitated with Gα16 and more tightly with its constitutively active Gα16QL, but not Gαz. Progressive replacement of Gα16 sequence with the corresponding residues of Gαz eventually identified a stretch of six amino acids in the ß3 region of Gα16 which are responsible for TPR1 interaction and the subsequent Ras activation. Insertion of these six residues into Gαz allowed productive TPR1-interaction. Since the ß3 region only minimally contributes to interact with PLCß, several chimeras exhibited differential abilities to stimulate PLCß and Ras. The ability of the chimeras to activate downstream transcription factors such as signal transducer and activator of transcription 3 and nuclear factor κB appeared to be associated with PLCß signaling. CONCLUSIONS: Our results suggest that Gα16 can signal through TPR1/Ras and PLCß simultaneously and independently. The ß3 region of Gα16 is essential for interaction with TPR1 and the subsequent activation of Ras, but has relatively minor influence on the PLCß interaction. Gα16 may utilize different structural domains to bind TPR1 and PLCß.

3-Methoxylphenylpropyl amides as novel receptor subtype-selective melatoninergic ligands: characterization of physicochemical and pharmacokinetic properties.

Developing subtype-selective melatoninergic ligands has been a subject of considerable interest in drug discovery. A series of 3-methoxyphenylpropyl amide derivatives showing selective binding capacity to type 2 melatonin receptor with subnanomolar range of affinities has been identified recently by our laboratory. In the present study, their physicochemical properties, Caco-2 cell and mdr1-MDCK cell permeability, plasma protein binding, and metabolic stability were investigated. The selected compounds are lipophilic in nature, exhibiting aqueous solubility ranging from 40 to 200 microg/mL. Cell permeability studies on Caco-2 and mdr1-MDCK model revealed that they were readily transported through intestinal epithelium and possessed high penetration potential through blood-brain barrier, implying good oral absorption and central nervous system (CNS) distribution potential. They also showed substantial binding to human plasma protein ranging from 78.5% to 92.3%. These compounds were, however, subjected to rapid cytochrome P450-mediated degradation in rat and human liver microsomes with in vitro half-life of 9.5-31.9 min in rat and 5.5-66.7 min in human, which were much shorter than that of melatonin (approximately 73 min). Metabolite profiling unveiled that C6-ether linkage and methoxy substituents were likely the major metabolic soft spots in their structures, which provided important information for further improvement of their structural stability.

CTCF and EGR1 suppress breast cancer cell migration through transcriptional control of Nm23-H1.

Tumor metastasis remains an obstacle in cancer treatment and is responsible for most cancer-related deaths. Nm23-H1 is one of the first metastasis suppressor proteins discovered with the ability to inhibit metastasis of many cancers including breast, colon, and liver cancer. Although loss of Nm23-H1 is observed in aggressive cancers and correlated with metastatic potential, little is known regarding the mechanisms that regulate its cellular level. Here, we examined the mechanisms that control Nm23-H1 expression in breast cancer cells. Initial studies in aggressive MDA-MB-231 cells (expressing low Nm23-H1) and less invasive MCF-7 cells (expressing high Nm23-H1) revealed that mRNA levels correlated with protein expression, suggesting that transcriptional mechanisms may control Nm23-H1 expression. Truncational analysis of the Nm23-H1 promoter revealed a proximal and minimal promoter that harbor putative binding sites for transcription factors including CTCF and EGR1. CTCF and EGR1 induced Nm23-H1 expression and reduced cell migration of MDA-MB-231 cells. Moreover, CTCF and EGR1 were recruited to the Nm23-H1 promoter in MCF-7 cells and their expression correlated with Nm23-H1 levels. This study indicates that loss of Nm23-H1 in aggressive breast cancer is apparently caused by downregulation of CTCF and EGR1, which potentially drive Nm23-H1 expression to promote a less invasive phenotype.