Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq

Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.     

Reciprocal inhibition of G-protein signaling is induced by CB(1) cannabinoid and GABA(B) receptor interactions in rat hippocampal membranes

Cannabinoid CB(1) and the metabotropic GABA(B) receptors have been shown to display similar pharmacological effects and co-localization in certain brain regions. Previous studies have reported a functional link between the two systems. As a first step to investigate the underlying molecular mechanism, here we show cross-inhibition of G-protein signaling between GABA(B) and CB(1) receptors in rat hippocampal membranes. The CB(1) agonist R-Win55,212-2 displayed high potency and efficacy in stimulating guanosine-5'-O-(3-[(35)S]thio)triphosphate, [(35)S]GTPgammaS binding. Its effect was completely blocked by the specific CB(1) antagonist AM251 suggesting that the signaling was via CB(1) receptors. The GABA(B) agonists baclofen and SKF97541 also elevated [(35)S]GTPgammaS binding by about 60%, with potency values in the micromolar range. Phaclofen behaved as a low potency antagonist with an ED(50) approximately 1mM. However, phaclofen at low doses (1 and 10nM) slightly but significantly attenuated maximal stimulation of [(35)S]GTPgammaS binding by the CB(1) agonist R-Win55,212-2. The observation that higher concentrations of phaclofen had no such effect rule out the possibility of its direct action on CB(1) receptors. The pharmacologically inactive stereoisomer S-Win55,212-3 had no effect either alone or in combination with phaclofen establishing that the interaction is stereospecific in hippocampus. The specific CB(1) antagonist AM251 at a low dose (1 nM) also inhibited the efficacy of G-protein signaling of the GABA(B) receptor agonist SKF97541. Cross-talk of the two receptor systems was not detected in either spinal cord or cerebral cortex membranes. It is speculated that the interaction might occur via an allosteric interaction between a subset of GABA(B) and CB(1) receptors in rat hippocampal membranes. Although the exact molecular mechanism of the reciprocal inhibition between CB(1) and GABA(B) receptors will have to be explored by future studies it is intriguing that the cross-talk might be involved in balance tuning the endocannabinoid and GABAergic signaling in hippocampus.     

BC-N102 suppress breast cancer tumorigenesis by interfering with cell cycle regulatory proteins and hormonal signaling,and induction of time-course arrest of cell cycle at G1/G0 phase

Mechanisms of breast cancer progression and invasion, often involve alteration of hormonal signaling, and upregulation and/or activation of signal transduction pathways that input to cell cycle regulation. Herein, we describe a rationally designed first-in-class novel small molecule inhibitor for targeting oncogenic and hormonal signaling in ER-positive breast cancer. BC-N102 treatment exhibits dose-dependent cytotoxic effects against ER+ breast cancer cell lines. BC-N102 exhibited time course- and dose-dependent cell cycle arrest via downregulation of the estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR), phosphatidylinositol 3-kinase (PI3K), phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-Akt, CDK2, and CDK4 while increasing p38 mitogen-activated protein kinase (MAPK), and mineralocorticoid receptor (MR) signaling in breast cancer cell line. In addition, we found that BC-N102 suppressed breast cancer tumorigenesis in vivo and prolonged the survival of animals. Our results suggest that the proper application of BC-N102 may be a beneficial chemotherapeutic strategy for ER+ breast cancer patients.     

Antitumor effects of a novel benzonaphthofurandione derivative (8e) on the human colon cancer cells in vitro and in vivo through cell cycle arrest accompanied with the modulation of EGFR and mTOR signaling

Benzonaphthofurandione has been considered as an important class of naturally occurring and synthetic compounds having a variety of biological functions. In this study, we evaluated the antitumor effects of 3-[2-(dimethylamino)isopropoxy]-1-hydroxybenzo[b]naphtho[2,3-d]furan-6,11-dione (8e), a novel benzonaphthofurandione derivative, on the growth of colorectal cancer HCT 116 cells both in vitro culture and an in vivo animal model.Compound 8e exhibited the potential growth inhibition of the colon cancer cells in a concentration-dependent manner. The anti-proliferative activity of 8e was also associated with the induction of cell cycle arrest in the G₀/G₁ phase. The 8e-induced cell cycle arrest was well correlated with the suppression of cyclin-dependent kinase 2 (CDK2), CDK4, cyclin D1, cyclin E, c-Myc, and phosphorylated retinoblastoma protein (pRb). The tumor growth in xenograft nude mice bearing HCT 116 cells by compound 8e (10 mg/kg) also significantly inhibited without any overt toxicity. In addition, the down-regulation of epidermal growth factor receptor (EGFR), Akt, and mTOR signalings were associated with the anti-proliferative activity of compound 8e in colon cancer cells. Taken together, these findings suggested that cell cycle arrest and modulation of cell signal transduction pathways might be the plausible mechanisms of actions for the anti-proliferative activity of 8e, and thus 8e might be used as an effective chemotherapeutic agent in human colon cancer.     

Rho-dependent, Rho kinase-independent inhibitory regulation of Rac and cell migration by LPA1 receptor in Gi-inactivated CHO cells

Lysophosphatidic acid (LPA) is a major serum lysophospholipid that stimulates cell migration in diverse cell types including ovarian cancer cells. We report here that in the absence of Gi function, LPA induces inhibition, rather than stimulation, of cellular Rac activity, lamellipodium formation, and cell migration in response to insulin like growth factor I (IGF-I) in Chinese hamster ovary (CHO) cells, which solely express LPA1 as a LPA receptor. The inhibitory effects of LPA are abrogated by the expression of either Galpha13 C-terminal peptide or C3 toxin pretreatment, but not a Rho kinase inhibitor. Without PTX pretreatment, LPA stimulates Rac and cell migration yet similarly activates Rho, indicating that Rho activation by itself is not sufficient for inhibition of cell migration. Conversely, the expression of a dominant negative Rac mutant sufficiently mimics the LPA inhibition of cell migration. LPA inhibits IGF I-induced Akt activation by only 40% in a manner dependent on Rho kinase. These results demonstrate that inhibition of Gi function converts LPA regulation on Rac and cell migration to an inhibitory mode, which is mediated by G13 and Rho but not Rho kinase, and raise a possibility of Gi as a new therapeutic target for LPA-dependent tumor progression.     

Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-β-cyclodextrin (MβCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C_m), fluctuations of which reflect exocytosis and endocytosis. Treatment with MβCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca²⁺-evoked increase in C_m (IC₅₀ = 5.3 mM) vs. untreated cells. Cytosol dialysis of MβCD enhanced the attenuation of C_m increase (IC₅₀ = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MβCD resulted in an immediate C_m decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in C_m was three-fold slower than MβCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MβCD had no effect on the specific C_m and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MβCD-induced C_m decline. Thus acute exposure to MβCD evokes a C_m decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.     

Polarized signaling: basolateral receptor localization in epithelial cells by PDZ-containing proteins

Extracellular signals are normally presented to one surface of epithelial cells and to one end of neurons, and so neuronal and epithelial cell signaling is inherently polarized. Another aspect of signaling polarity is that receptors are often asymmetrically distributed on the surfaces of polarized cells. Recent evidence from studies of Caenorhabditis elegans shows that signaling polarity plays an important role in development. The underlying mesoderm induces the overlying ectoderm to form the vulva, and asymmetric distribution of the signal receptor on the basolateral surface of the epithelium is crucial for this signaling. In neurons, the localization of neurotransmitter receptors and ion channels at synapses allows neurons to be exquisitely sensitive to synaptic inputs. Exciting recent reports suggest that receptor localization to neuronal synapses and the basolateral membrane domains of epithelia may involve a common molecular mechanism involving localization by PDZ-containing proteins.     

The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.     

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.     

Evidence of Antagonistic Regulation of Restart from G1 Delay in Response to Osmotic Stress by the Hog1 and Whi3 in Budding Yeast

Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G₁, but the mechanism by which cells restart from G₁ delay remains elusive. We found that Whi3, a negative regulator of G₁ cyclin, counteracted Hog1 in the restart from G₁ delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G₁ delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G₁ arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G₁ arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism.     

Galphaq/11 signaling induces apoptosis through two pathways involving reduction of Akt phosphorylation and activation of RhoA in HeLa cells

We have previously reported that expression of the constitutively active mutant of Galpha11 or stimulation of m1 muscarinic acetylcholine receptor induced proteolytic activation of Rho-associated kinase (ROCK-I) by caspase and apoptosis in HeLa cells. In this study, we investigate the molecular mechanisms of Galphaq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of Bcl-2 inhibited carbachol-induced ROCK-I cleavage, indicating a mitochondrial apoptotic pathway. Overexpression of the constitutively active mutant of Akt that delivers an anti-apoptotic survival signal had a similar influence. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitors, orthovanadate and pervanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results demonstrate that Gq/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.     

Modelling cellular signal communication mediated by phosphorylation dependent interaction with 14-3-3 proteins

The 14-3-3 proteins are important effectors of Ser/Thr phosphorylation in eukaryotic cells. Using mathematical modelling we investigated the roles of these proteins as effectors in signalling pathways that involve multi-phosphorylation events. We defined optimal conditions for positive and negative cross-talk. Particularly, synergistic signal interaction was evident at very different sets of binding affinities and phosphorylation kinetics. We identified three classes of 14-3-3 targets that all have two binding sites, but displayed synergistic interaction between converging signalling pathways for different ranges of parameter values. Consequently, these protein targets will respond differently to interventions that affect 14-3-3 binding affinities or phosphorylation kinetics.     

Determination of TJ0711 hydrochloride in rat plasma by high performance liquid chromatography with fluorescence detection and its application to pharmacokinetics

In the present study, a simple and sensitive high performance liquid chromatography with fluorescence detection (HPLC-FD) method was developed to determine TJ0711 hydrochloride, a novel α- and β-receptor blocker. TJ0711 hydrochloride and verapamil hydrochloride (the internal standard) were separated on Knauer Eurospher C₁₈ (250 mm × 4.0 mm i.d., 5 μm) column at 50 °C. The mobile phase was methanol:perchloric acid (12 nM, aq) (56:44, v:v), with a flow rate of 1.0 mL/min. The wavelengths of FD were set at 246 nm for excitation and 300 nm for emission. For plasma samples of rats, the analytes were extracted with acetic ether from alkalinized plasma, and then back-extracted into 10 mM dilute sulfuric acid. The linearity was over a concentration range of 20–10,000 ng/mL. The intra- and inter-day precisions referred by relative standard deviation were less than 2.0% and 4.3%, respectively. The mean analytical recoveries of TJ0711 hydrochloride at different concentrations (50, 1000 and 8000 ng/mL) ranged from 88.3% to 92.9%. The lower limit of quantification (LLOQ) was 20 ng/mL. Finally, this method was successfully applied to the estimation of pharmacokinetic parameters of TJ0711 hydrochloride after intravenous doses of 4, 8 and 16 mg/kg in rats.     

Proterguride, a highly potent dopamine receptor agonist promising for transdermal administration in Parkinson's disease: interactions with alpha(1)-, 5-HT(2)- and H(1)-receptors

Dopamine receptor agonists play an important role in the treatment of Parkinson's disease and hyperprolactinemic conditions. Proterguride (n-propyldihydrolisuride) was already reported to be a highly potent dopamine receptor agonist, thus its action at different non-dopaminergic monoamine receptors, alpha(1A/1B/1D), 5-HT(2A/2B)- and histamine H(1), was investigated using different functional in vitro assays. The drug behaved as an antagonist at alpha(1)-adrenoceptors without the ability to discriminate between the subtypes (pA(2) values: alpha(1A) 7.31; alpha(1B) 7.37; alpha(1D) 7.35) and showed antagonistic properties at the histamine H(1) receptor. In contrast, at serotonergic receptors (5-HT(2A), 5-HT(2B)) proterguride acted as a partial agonist. The drug stimulated 5-HT(2A) receptors of rat tail artery in lower concentrations than 5-HT itself but failed to evoke comparable efficacy (proterguride: pEC(50) 8.34, E(max) 53% related to the maximum response to 5-HT; 5-HT: pEC(50) 7.03). Agonism at 5-HT(2B) receptors is presently considered to be involved in drug-induced valvular heart disease. Activation of 5-HT(2B) receptors in porcine pulmonary arteries by proterguride (pEC(50) 7.13, E(max) 49%; E(max) (5-HT) 69%), however, occurred at concentrations much higher than plasma concentrations achieving dopaminergic efficacy in humans. The results are discussed focussing on the relevance of action at 5-HT(2B) receptors as well as their significance for a transdermal administration of proterguride. Since it is well accepted that pulsatile dopaminergic stimulation is associated with treatment-related motor complications in the dopaminergic therapy of Parkinson's disease, the transdermal route of administration is of great clinical interest due to the possibility to achieve constant plasma concentrations.     

The targeting of the atToc159 preprotein receptor to the chloroplast outer membrane is mediated by its GTPase domain and is regulated by GTP

The multimeric translocon at the outer envelope membrane of chloroplasts (Toc) initiates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two Toc GTPases, Toc159 and Toc33/34, mediate preprotein recognition and regulate preprotein translocation. Although these two proteins account for the requirement of GTP hydrolysis for import, the functional significance of GTP binding and hydrolysis by either GTPase has not been defined. A recent study indicates that Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, raising the possibility that it might cycle between the cytoplasm and chloroplast as a soluble preprotein receptor. In the present study, we examined the mechanism of targeting and insertion of the Arabidopsis thaliana orthologue of Toc159, atToc159, to chloroplasts. Targeting of atToc159 to the outer envelope membrane is strictly dependent only on guanine nucleotides. Although GTP is not required for initial binding, the productive insertion and assembly of atToc159 into the Toc complex requires its intrinsic GTPase activity. Targeting is mediated by direct binding between the GTPase domain of atToc159 and the homologous GTPase domain of atToc33, the Arabidopsis Toc33/34 orthologue. Our findings demonstrate a role for the coordinate action of the Toc GTPases in assembly of the functional Toc complex at the chloroplast outer envelope membrane.     

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin_1A receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin_1A receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin_1A receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin_1A receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.