5-alpha-reductase: 2012-09-16

2012年9月22日星期六

Hyperthermia promotes apoptosis and suppresses invasion in c6 rat glioma cells.

Hyperthermia promotes apoptosis and suppresses invasion in c6 rat glioma cells.

Asian Pac J Cancer Prev. 2012;13(7):3239-45

Authors: Wang DC, Zhang Y, Chen HY, Li XL, Qin LJ, Li YJ, Zhang HY, Wang S

Abstract
Gliomas are a group of heterogeneous primary central nervous system tumors. Hyperthermia has proven to be a potential therapeutic tool for cancers in the clinic. However, the molecular mechanisms of hyperthermia remain unclear. The objective of this study was to investigate the effects of hyperthermia on the invasiveness in C6 glioma cells and related molecular pathways. Here our data show hyperthermia stimulated the release of tumor necrosis factor-alpha (TNF-?) and decreased C6 glioma cell migration and invasive capability at 30, 60, 120 and 180 min; with increased spontaneous apoptosis in C6 glioma cells at 120 min. We also found mitogen- activated protein kinase (P38 MAPK) protein expression to be increased and nuclear factor-kappa B (NF-?B) protein expression decreased. Based on the results, we conclude that hyperthermia alone reduced invasion of C6 glioma cells through stimulating TNF-?signaling to activate apoptosis, enhancing P38 MAPK expression and inhibiting the NF-?B pathway, a first report in C6 rat glioma cells.

PMID: 22994741 [PubMed - in process]

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An expression and purification system for the biosynthesis of adenosine receptor peptides for biophysical and structural characterization.

An expression and purification system for the biosynthesis of adenosine receptor peptides for biophysical and structural characterization.

Protein Expr Purif. 2012 Aug;84(2):224-35

Authors: Britton ZT, Hanle EI, Robinson AS

Abstract
Biophysical and structural characterization of G protein-coupled receptors (GPCRs) has been limited due to difficulties in expression, purification, and vitro stability of the full-length receptors. "Divide and conquer" approaches aimed at the NMR characterization of peptides corresponding to specific regions of the receptor have yielded insights into the structure and dynamics of GPCR activation and signaling. Though significant progress has been made in the generation of peptides that are composed of GPCR transmembrane domains, current methods utilize fusion protein strategies that require chemical cleavage and peptide separation via chromatographic means. We have developed an expression and purification system based on fusion to ketosteroid isomerase, thrombin cleavage, and tandem affinity chromatography that enables the solubilization, cleavage, and characterization in a single detergent system relevant for biophysical and structural characterization. We have applied this expression and purification system to the production and characterization of peptides of the adenosine receptor family of GPCRs in Escherichia coli. Herein, we demonstrate using a model peptide that includes extracellular loop 3, transmembrane domain 7, and a portion of the carboxy-terminus of the adenosine A(2)a receptor that the peptide is sufficiently pure for biophysical characterization, where it adopts ?-helical structure. Furthermore, we demonstrate the utility of this system by optimizing the construct for thrombin processing and apply the system to peptides with more complex structures.

PMID: 22722102 [PubMed - in process]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

Receptor binding kinetics and cellular responses of six N-formyl peptide agonists in human neutrophils.

Biochemistry. 2004 Jun 29;43(25):8204-16

Authors: Waller A, Sutton KL, Kinzer-Ursem TL, Absood A, Traynor JR, Linderman JJ, Omann GM

Abstract
The goal of this study was to elucidate the relationships between early ligand binding/receptor processing events and cellular responses for the N-formyl peptide receptor system on human neutrophils as a model of a GPCR system in a physiologically relevant context. Binding kinetics of N-formyl-methionyl-leucyl-phenylalanyl-phenylalanyl-lysine-fluorescein and N-formyl-valyl-leucyl-phenylalanyl-lysine-fluorescein to the N-formyl peptide receptor on human neutrophils were characterized and combined with previously published binding data for four other ligands. Binding was best fit by an interconverting two-receptor state model that included a low affinity receptor state that converted to a high affinity state. Response behaviors elicited at 37 degrees C by the six different agonists for the N-formyl peptide receptor were measured. Dose response curves for oxidant production, actin polymerization, and G-protein activation were obtained for each ligand; whereas all ligands showed equal efficacy for all three responses, the ED(50) values varied as much as 7000-fold. The level of agonism and rank order of potencies of ligands for actin and oxidant responses were the same as for the G-protein activation assay, suggesting that the differences in abilities of ligands to mediate responses were determined upstream of G-protein activation at the level of ligand-receptor interactions. The rate constants governing ligand binding and receptor affinity conversion were ligand-dependent. Analysis of the forward and reverse rate constants governing binding to the proposed signaling receptor state showed that it was of a similar energy for all six ligands, suggesting the hypothesis that ligand efficacy is dictated by the energy state of this ligand-receptor complex. However, the interconverting two-receptor state model was not sufficient to predict response potency, suggesting the presence of receptor states not discriminated by the binding data.

PMID: 15209517 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

Evolutionary Analysis of Functional Divergence among Chemokine Receptors, Decoy Receptors, and Viral Receptors.

Evolutionary Analysis of Functional Divergence among Chemokine Receptors, Decoy Receptors, and Viral Receptors.

Front Microbiol. 2012;3:264

Authors: Daiyasu H, Nemoto W, Toh H

Abstract
Chemokine receptors (CKRs) function in the inflammatory response and in vertebrate homeostasis. Decoy and viral receptors are two types of CKR homologs with modified functions from those of the typical CKRs. The decoy receptors are able to bind ligands without signaling. On the other hand, the viral receptors show constitutive signaling without ligands. We examined the sites related to the functional difference. At first, the decoy and viral receptors were each classified into five groups, based on the molecular phylogenetic analysis. A multiple amino acid sequence alignment between each group and the CKRs was then constructed. The difference in the amino acid composition between the group and the CKRs was evaluated as the Kullback-Leibler (KL) information value at each alignment site. The KL information value is considered to reflect the difference in the functional constraints at the site. The sites with the top 5% of KL information values were selected and mapped on the structure of a CKR. The comparisons with decoy receptor groups revealed that the detected sites were biased on the intracellular side. In contrast, the sites detected from the comparisons with viral receptor groups were found on both the extracellular and intracellular sides. More sites were found in the ligand binding pocket in the analyses of the viral receptor groups, as compared to the decoy receptor groups. Some of the detected sites were located in the GPCR motifs. For example, the DRY motif of the decoy receptors was often degraded, although the motif of the viral receptors was basically conserved. The observations for the viral receptor groups suggested that the constraints in the pocket region are loose and that the sites on the intracellular side are different from those for the decoy receptors, which may be related to the constitutive signaling activity of the viral receptors.

PMID: 22855685 [PubMed - in process]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function.

Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function.

Pharmacol Ther. 2004 Jul;103(1):21-80

Authors: Kristiansen K

Abstract
The superfamily of G-protein-coupled receptors (GPCRs) could be subclassified into 7 families (A, B, large N-terminal family B-7 transmembrane helix, C, Frizzled/Smoothened, taste 2, and vomeronasal 1 receptors) among mammalian species. Cloning and functional studies of GPCRs have revealed that the superfamily of GPCRs comprises receptors for chemically diverse native ligands including (1) endogenous compounds like amines, peptides, and Wnt proteins (i.e., secreted proteins activating Frizzled receptors); (2) endogenous cell surface adhesion molecules; and (3) photons and exogenous compounds like odorants. The combined use of site-directed mutagenesis and molecular modeling approaches have provided detailed insight into molecular mechanisms of ligand binding, receptor folding, receptor activation, G-protein coupling, and regulation of GPCRs. The vast majority of family A, B, C, vomeronasal 1, and taste 2 receptors are able to transduce signals into cells through G-protein coupling. However, G-protein-independent signaling mechanisms have also been reported for many GPCRs. Specific interaction motifs in the intracellular parts of these receptors allow them to interact with scaffold proteins. Protein engineering techniques have provided information on molecular mechanisms of GPCR-accessory protein, GPCR-GPCR, and GPCR-scaffold protein interactions. Site-directed mutagenesis and molecular dynamics simulations have revealed that the inactive state conformations are stabilized by specific interhelical and intrahelical salt bridge interactions and hydrophobic-type interactions. Constitutively activating mutations or agonist binding disrupts such constraining interactions leading to receptor conformations that associates with and activate G-proteins.

PMID: 15251227 [PubMed - indexed for MEDLINE]

NF-kB signaling NF-kappaB signaling pathway read more

2012年9月21日星期五

G-protein-coupled receptor prediction using pseudo-amino-acid composition and multiscale energy representation of different physiochemical properties.

G-protein-coupled receptor prediction using pseudo-amino-acid composition and multiscale energy representation of different physiochemical properties.

Anal Biochem. 2011 May 15;412(2):173-82

Authors: Ur-Rehman Z, Khan A

Abstract
G-protein-coupled receptors (GPCRs) are the largest family of cell surface receptors that, via trimetric guanine nucleotide-binding proteins (G-proteins), initiate some signaling pathways in the eukaryotic cell. Many diseases involve malfunction of GPCRs making their role evident in drug discovery. Thus, the automatic prediction of GPCRs can be very helpful in the pharmaceutical industry. However, prediction of GPCRs, their families, and their subfamilies is a challenging task. In this article, GPCRs are classified into families, subfamilies, and sub-subfamilies using pseudo-amino-acid composition and multiscale energy representation of different physiochemical properties of amino acids. The aim of the current research is to assess different feature extraction strategies and to develop a hybrid feature extraction strategy that can exploit the discrimination capability in both the spatial and transform domains for GPCR classification. Support vector machine, nearest neighbor, and probabilistic neural network are used for classification purposes. The overall performance of each classifier is computed individually for each feature extraction strategy. It is observed that using the jackknife test the proposed GPCR-hybrid method provides the best results reported so far. The GPCR-hybrid web predictor to help researchers working on GPCRs in the field of biochemistry and bioinformatics is available at http://111.68.99.218/GPCR.

PMID: 21295004 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

Caffeine Induces Apoptosis of Osteosarcoma Cells by Inhibiting AKT/mTOR/S6K, NF-?B and MAPK Pathways.

Caffeine Induces Apoptosis of Osteosarcoma Cells by Inhibiting AKT/mTOR/S6K, NF-?B and MAPK Pathways.

Anticancer Res. 2012 Sep;32(9):3643-9

Authors: Miwa S, Sugimoto N, Yamamoto N, Shirai T, Nishida H, Hayashi K, Kimura H, Takeuchi A, Igarashi K, Yachie A, Tsuchiya H

Abstract
We previously reported that caffeine-assisted chemotherapy improved the treatment of malignant bone and soft tissue tumours such as osteosarcoma. Caffeine affects tumour cells through various pathways, including phosphatase and tensin homolog deleted on chromosome 10 (PTEN), AKT, Bcl-2-associated X protein (BAX), caspase-3 and p53, and has therefore been indicated as being useful for the treatment of malignant tumours. Here, the effects of caffeine on the proliferation of HOS osteosarcoma cells were assessed by WST-8 assay, and the effects on the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B), mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways were assessed by western blot analyses. Caffeine inhibited proliferation of HOS cells and suppressed NF-?B, AKT, mTOR/S6K and ERK activities. Our results support those from previous studies relating to the use of caffeine in the treatment of osteosarcoma.

PMID: 22993301 [PubMed - in process]

G-protein Receptors gpcr pathway NF-κB

A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors.

A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors.

Biochem Biophys Res Commun. 2011 Apr 1;407(1):18-22

Authors: Wu C, Du L, Wang D, Wang L, Zhao L, Wang P

Abstract
Olfactory receptors, which are responsible for sensing odor molecules, form the largest G protein-coupled receptor (GPCR) family in mammalian animals. These proteins play an important role in the detection of chemical signals and signal transduction to the brain. Currently, only a limited number of olfactory receptors have been characterized, which is mainly due to the lack of sensitive and efficient tools for performing functional assays of these receptors. This paper describes a novel surface acoustic wave (SAW)-based biosensor for highly sensitive functional assays of olfactory receptors. An olfactory receptor of Caenorhabditis elegans, ODR-10, was expressed on the plasma membrane of human breast cancer MCF-7 cells, which was used as a model system for this study. For specific odorant response assays, the membrane fraction of MCF-7 cells containing ODR-10 was extracted and integrated with our SAW sensors. The response of ODR-10 to various odorants was monitored by recording the resonance frequency shifts of SAWs applied to the sensor. Our results show that heterologously expressed ODR-10 receptors can specifically respond to diacetyl, its natural ligand. Dose-dependent responses were obtained by performing measurements using various concentrations of diacetyl. The sensitivity of this biosensor is 2kHz/ng and can detect concentrations as low as 10(-10)mM, which is 10� lower than what has previously been reported. This biosensor can be used to characterize odorant response profiles of olfactory receptors and provide information rich data for functional assays of olfactory receptors. In addition to providing a greater understanding of the biological mechanisms of GPCRs, such data holds great potential in many other fields such as food industry, biomedicine, and environmental protection.

PMID: 21333624 [PubMed - indexed for MEDLINE]

NF-kB signaling NF-kappaB signaling pathway NF-kB signaling pathway

Molecular mechanisms of metabolic reprogramming in proliferating cells: implications for T-cell-mediated immunity.

Molecular mechanisms of metabolic reprogramming in proliferating cells: implications for T-cell-mediated immunity.

Immunology. 2012 Aug;136(4):363-9

Authors: Marelli-Berg FM, Fu H, Mauro C

Abstract
To engage in proliferation, cells need to increase their biomass and replicate their genome. This process presents a substantial bioenergetic challenge: proliferating cells must increase ATP production and acquire or synthesize raw materials, including lipids, proteins and nucleic acids. To do so, proliferating cells actively reprogramme their intracellular metabolism from catabolic mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis and other anabolic pathways. This metabolic reprogramming, which directs nutrient uptake and metabolism during cell activation and proliferation, is under the control of specific signal transduction pathways. The underlying molecular mechanisms of cell metabolism reprogramming and their relevance to physiology and disease are currently under intense study. Several reports have uncovered the mechanisms of metabolic reprogramming that drive high rates of cell proliferation in cancer. Some recent studies have elucidated the physiological role of metabolic reprogramming during T-cell activation, differentiation and trafficking, which are potentially relevant to inflammatory disorders. This review describes the impact of metabolic reprogramming on the pathogenesis of cancer and the physiology of T-cell-mediated immune responses, with an emphasis on the phosphatidyl inositol 3-kinase-serine/threonine kinase-mammalian target of rapamycin pathway and the recently discovered metabolic processes regulated by nuclear factor-?B. These discoveries will hopefully translate into a better understanding of the role of metabolic reprogramming as a key regulator of T-cell-mediated immune responses and offer novel, immune-based therapeutic approaches.

PMID: 22384794 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

2012年9月20日星期四

Latest development in drug discovery on G protein-coupled receptors.

Latest development in drug discovery on G protein-coupled receptors.

Curr Protein Pept Sci. 2006 Oct;7(5):465-70

Authors: Lundstrom K

Abstract
G protein-coupled receptors (GPCRs) represent the family of proteins with the highest impact from social, therapeutic and economic point of view. Today, more than 50% of drug targets are based on GPCRs and the annual worldwide sales exceeds 50 billion dollars. GPCRs are involved in all major disease areas such as cardiovascular, metabolic, neurodegenerative, psychiatric, cancer and infectious diseases. The classical drug discovery process has relied on screening compounds, which interact favorably with the GPCR of interest followed by further chemical engineering as a mean of improving efficacy and selectivity. In this review, methods for sophisticated chemical library screening procedures will be presented. Furthermore, development of cell-based assays for functional coupling of GPCRs to G proteins will be discussed. Finally, the possibility of applying structure-based drug design will be summarized. This includes the application of bioinformatics knowledge and molecular modeling approaches in drug development programs. The major efforts established through large networks of structural genomics on GPCRs, where recombinantly expressed GPCRs are subjected to purification and crystallization attempts with the intention of obtaining high-resolution structures, are presented as a promising future approach for tailor-made drug development.

PMID: 17073697 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

Chemokine receptor CCR2 undergoes transportin1-dependent nuclear translocation.

Chemokine receptor CCR2 undergoes transportin1-dependent nuclear translocation.

Proteomics. 2008 Nov;8(21):4560-76

Authors: Favre N, Camps M, Arod C, Chabert C, Rommel C, Pasquali C

Abstract
Chemokines (CCs) are small chemoattractant cytokines involved in a wide variety of biological and pathological processes. Released by cells in the milieu, and extracellular matrix and activating signalling cascades upon binding to specific G protein-coupled receptors (GPCRs), they trigger many cellular events. In various pathologies, CCs are directly responsible for excessive recruitment of leukocytes to inflammatory sites and recent studies using chemokine receptor (CCR) antagonists permitted these molecules to reach the market for medical use. While interaction of CCs with their receptors has been extensively documented, downstream GPCR signalling cascades triggered by CC are less well understood. Given the pivotal role of chemokine receptor 2 (CCR2) in monocyte recruitment, activation and differentiation and its implication in several autoimmune-inflammatory pathologies, we searched for potential new CCR2-interacting proteins by engineering a modified CCR2 that we used as bait. Herein, we show the direct interaction of CCR2 with transportin1 (TRN1), which we demonstrate is followed by CCR2 receptor internalization. Further characterization of this novel interaction revealed that TRN1-binding to CCR2 increased upon time in agonist treated cells and promotes its nuclear translocation in a TRN1-dependent manner. Finally, we provide evidence that following translocation, the receptor localizes at the outer edge of the nuclear envelope where it is finally released from TRN1.

PMID: 18846510 [PubMed - indexed for MEDLINE]

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Rice heterotrimeric G-protein Gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress.

Rice heterotrimeric G-protein Gamma subunits (RGG1 and RGG2) are differentially regulated under abiotic stress.

Plant Signal Behav. 2012 Jul 1;7(7)

Authors: Yadav DK, Islam SM, Tuteja N

Abstract
Heterotrimeric G-proteins (?, ? and ? subunits) are primarily involved in diverse signaling processes by transducing signals from an activated transmembrane G-protein coupled receptor (GPCR) to appropriate downstream effectors within cells. The role of ? and ? G-protein subunits in salinity and heat stress has been reported but the regulation of ? subunit of plant G-proteins in response to abiotic stress has not heretofore been described. In the present study we report the isolation of full-length cDNAs of two isoforms of G? [RGG1(I), 282 bp and RGG2(I), 453 bp] from rice (Oryza sativa cv Indica group Swarna) and described their transcript regulation in response to abiotic stresses. Protein sequence alignment and pairwise comparison of ? subunits of Indica rice [RGG(I)] with other known plant G-protein ? subunits demonstrated high homology to barley (HvGs) while soybean (GmG2) and Arabidopsis (AGG1) were least related. The numbers of the exons and introns were found to be similar between RGG1(I) and RGG2(I), but their sizes were different. Analyses of promoter sequences of RGG(I) confirmed the presence of stress-related cis-regulatory signature motifs suggesting their active and possible independent roles in abiotic stress signaling. The transcript levels of RGG1(I) and RGG2(I) were upregulated following NaCl, cold, heat and ABA treatments. However, in drought stress only RGG1(I) was upregulated. Strong support by transcript profiling suggests that ? subunits play a critical role via cross talk in signaling pathways. These findings provide first direct evidence for roles of G? subunits of rice G-proteins in regulation of abiotic stresses. These findings suggest the possible exploitation of ? subunits of G-protein machinery for promoting stress tolerance in plants.

PMID: 22751322 [PubMed - as supplied by publisher]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-?B, and MAPK activation in vitro and in vivo.

Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-?B, and MAPK activation in vitro and in vivo.

PLoS One. 2012;7(5):e35922

Authors: Huang GJ, Huang SS, Deng JS

Abstract
Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and ?-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)�[extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38], and nuclear factor-?B (NF-?B), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-?B, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4(th) and the 5(th) h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5(th) h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-?) levels on serum at the 5(th) h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-?B, and MMP-9 expressions at the 5(th) h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-?B, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-? and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.

PMID: 22590514 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

A dual receptor crosstalk model of G-protein-coupled signal transduction.

A dual receptor crosstalk model of G-protein-coupled signal transduction.

PLoS Comput Biol. 2008;4(9):e1000185

Authors: Flaherty P, Radhakrishnan ML, Dinh T, Rebres RA, Roach TI, Jordan MI, Arkin AP

Abstract
Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Galphai-coupled C5a receptor and the Galphaq-coupled UDP receptor signaling systems that results in synergistic calcium release.

PMID: 18818727 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

2012年9月19日星期三

Lipopolysaccharide/adenosine triphosphate-mediated signal transduction in the regulation of NLRP3 protein expression and caspase-1-mediated interleukin-1? secretion.

Lipopolysaccharide/adenosine triphosphate-mediated signal transduction in the regulation of NLRP3 protein expression and caspase-1-mediated interleukin-1? secretion.

Inflamm Res. 2012 Sep 18;

Authors: Liao PC, Chao LK, Chou JC, Dong WC, Lin CN, Lin CY, Chen A, Ka SM, Ho CL, Hua KF

Abstract
OBJECTIVE: Reactive oxygen species (ROS) plays a critical role in the regulation of NLRP3 inflammasome activation. However, the ROS-mediated signaling pathways controlling NLRP3 inflammasome activation are not well defined. METHODS: Using lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated murine macrophages as the testing model, cytokine release and protein expression were quantified by enzyme-linked immunosorbent assay and Western blot, respectively. ROS was scavenged by N-acetyl cysteine; NADPH oxidase, the major source of ROS, was inhibited by diphenyliodonium, apocynin or gp91-phox siRNA transfection; and protein kinase was inhibited by its specific inhibitor. RESULTS: LPS-induced NLRP3 protein expression was regulated through the NADPH oxidase/ROS/NF-?B-dependent, JAK2/PI3-kinase/AKT/NF-?B-dependent, and MAPK-dependent pathways, while ATP-induced caspase-1 activation was regulated through the NADPH oxidase/ROS-dependent pathway. CONCLUSIONS: These results demonstrate that ROS regulates not only the priming stage, but also the activation stage, of NLRP3 inflammasome activation in LPS�+�ATP-activated macrophages.

PMID: 22986467 [PubMed - as supplied by publisher]

GPCR Signaling G-protein Receptors gpcr pathway

Immunopathologic processes in sympathetic ophthalmia as signified by microRNA profiling.

Immunopathologic processes in sympathetic ophthalmia as signified by microRNA profiling.

Invest Ophthalmol Vis Sci. 2012;53(7):4197-204

Authors: Kaneko Y, Wu GS, Saraswathy S, Vasconcelos-Santos DV, Rao NA

Abstract
PURPOSE: Recent discovery of microRNAs and their negative gene regulation have provided new understanding in the pathogenesis of inflammatory diseases. This study demonstrated microRNA expression profiling and their likely role in sympathetic ophthalmia, using formalin-fixed, paraffin embedded samples.
METHODS: Two groups of four enucleated globes (total eight globes) from patients with clinical and histopathological diagnosis of SO (experimental samples) and one group of four age-matched, noninflamed enucleated globes (control samples) were used. Human genome-wide microRNA PCR array was performed and results were subjected to bioinformatics calculation and P values stringency tests. The targets were searched using the recently published and periodically updated miRWalk software. Quantitative real-time PCR and immunohistochemical staining were performed to confirm the validated targets in the mRNA and in the protein levels, respectively.
RESULTS: No microRNA was significantly upregulated in SO, but 27 microRNAs were significantly downregulated. Among these, four microRNAs (hsa-miR-1, hsa-let-7e, hsa-miR-9, and hsa-miR-182) were known to be associated with the inflammatory signaling pathway. Only hsa-miR-9 has the validated targets, tumor necrosis factor-?, and nuclear factor kappa B1, which have been previously shown to be associated with mitochondrial oxidative stress-mediated photoreceptor apoptosis in eyes with SO.
CONCLUSIONS: Identification of altered levels of microRNAs by microRNA expression profiling may yield new insights into the pathogenesis of SO by disclosing specific microRNA signatures. In the future these may be targeted by synthetic microRNA mimic-based therapeutic strategies.

PMID: 22589448 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Xylocoside G reduces amyloid-? induced neurotoxicity by inhibiting NF-?B signaling pathway in neuronal cells.

Xylocoside G reduces amyloid-? induced neurotoxicity by inhibiting NF-?B signaling pathway in neuronal cells.

J Alzheimers Dis. 2012;30(2):263-75

Authors: Yu Y, Zhou L, Sun M, Zhou T, Zhong K, Wang H, Liu Y, Liu X, Xiao R, Ge J, Tu P, Fan DS, Lan Y, Hui C, Chui D

Abstract
Amyloid-? (A?) peptide, which can invoke a cascade of inflammatory responses, is considered to play a causal role in the development and progress of Alzheimer's disease (AD). Xylocoside G (XG) is an active compound isolated from a traditional Chinese medicinal plant, Itoa orientalis. We have previously reported that XG has neuroprotective effects, of which the mechanism is yet unknown. In this study, we investigated the possible mechanisms underlying neuroprotection of XG against A?-induced toxicity in SH-SY5Y cells and primary neurons. Pretreatment with XG significantly attenuated the cell viability reduction induced by A? exposure in a dose dependent manner which was testified by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase release assay. In addition, pretreatment with XG countered the effect of A? on Bax and Bcl-2 expression and repressed A?-induced caspase-3 activation, suggesting that the neuroprotective effect of XG is associated with apoptosis regulation. Neuroinflammation has been implicated in A?-induced neuronal death. XG significantly attenuated A?-stimulated release of inflammatory factors such as tumor necrosis factor-?, interleukin-1?, and prostaglandin E2. It also downregulated the expression of cyclooxygenase-2 in SH-SY5Y cells. Further molecular mechanism studies demonstrated that XG inhibited A?-induced NF-?B p65 translocation, which was probably the result of inhibition of JNK phosphorylation but not ERK or p38 MAPK pathway by XG. This is the first study to demonstrate that XG protects SH-SY5Y cells against A?-induced inflammation and apoptosis by down-regulating NF-?B signaling pathways.

PMID: 22430528 [PubMed - indexed for MEDLINE]

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A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors.

A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors.

Biochem Biophys Res Commun. 2011 Apr 1;407(1):18-22

Authors: Wu C, Du L, Wang D, Wang L, Zhao L, Wang P

PMID: 21333624 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

Subtype-specific roles of phospholipase C-? via differential interactions with PDZ domain proteins.

Subtype-specific roles of phospholipase C-? via differential interactions with PDZ domain proteins.

Adv Enzyme Regul. 2011;51(1):138-51

Authors: Kim JK, Lim S, Kim J, Kim S, Kim JH, Ryu SH, Suh PG

Abstract
Since we first identified the PLC-? isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-? subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-? subtype exist. Most cells express multiple subtypes of PLC-? in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-? subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-? interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-? subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-? subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-? subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-? subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-? in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-? subtypes. It has been observed for a long time that the expression of certain PLC-? subtype fluctuates during diverse physiological conditions. For example, the expression of PLC-?1 is selectively increased during myoblast and adipocyte differentiation (Faenza et al., 2004; O'Carroll et al., 2009). Likewise, PLC-?2 is highly up-regulated during breast cancer progression and plays a critical role in cell migration and mitosis (Bertagnolo et al., 2007). Although PLC-?3 is selectively down-regulated in neuroendocrine tumors, the expression of PLC-?1 is increased in small cell lung carcinoma (Stalberg et al., 2003; Strassheim et al., 2000). In our hypothetical model, it is most likely that up- and down regulation of certain PLC-? subtypes are due to their selective coupling with specific GPCR-mediated signaling, implicated in these pathophysiologic conditions. Therefore, better understanding of selective coupling between PLC-? subtypes, PDZ domain proteins, and GPCRs will shed light on new prognosis and therapy of diverse diseases, and provide potential targets for drug development.

PMID: 21035486 [PubMed - indexed for MEDLINE]

discover more info here find out more info GPCR Signaling

2012年9月18日星期二

Soy isoflavones (daidzein & genistein) inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cutaneous inflammation via modulation of COX-2 and NF-?B in Swiss albino mice.

Soy isoflavones (daidzein & genistein) inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cutaneous inflammation via modulation of COX-2 and NF-?B in Swiss albino mice.

Toxicology. 2012 Sep 4;

Authors: Khan AQ, Khan R, Rehman MU, Lateef A, Tahir M, Ali F, Sultana S

Abstract
It is well established that aberrant production of inflammatory mediators has been associated with most the toxic manifestations and the genesis of different chronic diseases including cancer. The basic aim of the present study is to investigate the effects of soy isoflavones (SIF) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cutaneous inflammatory responses and to explore the underlying molecular mechanisms. We have studied the protective effects of SIF against TPA induced oxidative stress, pro-inflammatory cytokines level, activation of NF-?B, expression of COX-2 and ki-67 in mouse skin. Animals were divided into five groups I-V (n=6). Groups II, III and IV received topical application of TPA at the dose of 10nmol/0.2ml of acetone/animal/day, for 2 days. Animals of the groups III and IV were pre-treated with SIF 1.0?g (D1) and 2.0?g (D2) topically 30min prior to each TPA administration, while groups I and V were given acetone (0.2ml) and SIF (D2), respectively. We have found that SIF pretreatment significantly inhibited TPA induced oxidative stress, proinflammatory cytokines production and activation of NF-?B. SIF also inhibited the expression of COX-2 and ki-67. Histological findings further supported the protective effects of SIF against TPA-induced cutaneous damage. Thus, our results suggest that inhibitory effect of SIF on TPA-induced cutaneous inflammation includes inhibition of proinflammatory cytokines, attenuation of oxidative stress, activation of NF-?B and expression of COX-2.

PMID: 22981962 [PubMed - as supplied by publisher]

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G protein-coupled receptors serve as mechanosensors for fluid shear stress in neutrophils.

G protein-coupled receptors serve as mechanosensors for fluid shear stress in neutrophils.

Am J Physiol Cell Physiol. 2006 Jun;290(6):C1633-9

Authors: Makino A, Prossnitz ER, B�nemann M, Wang JM, Yao W, Schmid-Sch�nbein GW

Abstract
Many cells respond to fluid shear stress but in a cell type-specific fashion. Fluid shear stress applied to leukocytes serves to control pseudopod formation, migration, and other functions. Specifically, fresh neutrophils or neutrophilic leukocytes derived from differentiated HL60 cells respond to fluid shear stress by cytoplasmic pseudopod retraction. The membrane elements that sense fluid shear and induce such a specific response are still unknown, however. We hypothesized that membrane receptors may serve as fluid shear sensors. We found that fluid shear decreased the constitutive activity of G protein-coupled receptors (GPCRs). Inhibition of GPCR constitutive activity by inverse agonists abolished fluid shear stress-induced cell area reduction. Among the GPCRs in neutrophils, the formyl peptide receptor (FPR) exhibits relatively high constitutive activity. Undifferentiated HL60 cells that lacked FPR formed few pseudopods and showed no detectable response to fluid shear stress, whereas expression of FPR in undifferentiated HL60 cells caused pseudopod projection and robust pseudopod retraction during fluid shear. FPR small interfering RNA-transfected differentiated HL60 cells exhibited no response to fluid shear stress. These results suggest that GPCRs serve as mechanosensors for fluid shear stress in neutrophils by decreasing its constitutive activity and reducing pseudopod projection.

PMID: 16436471 [PubMed - indexed for MEDLINE]

NF-κB NF-kB signaling pathway NF-kB pathway

G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts.

G protein-coupled receptors and their signaling pathways: classical therapeutical targets susceptible to novel therapeutic concepts.

Curr Pharm Des. 2004;10(16):1937-58

Authors: Liebmann C

Abstract
In recent years, new strategies in cancer therapy have been developed targeting key signaling molecules in the receptor tyrosine kinase signal transduction pathway. In contrast, most therapeutical concepts to manipulate G protein-coupled receptors (GPCR)-mediated disorders are still limited to the use of receptor-specific agonists or antagonists. Visible progress in the understanding of GPCR signaling complexity, especially the detection of several families of highly target- and cell-specific regulator proteins of GPCRs, G proteins, and effector components may open new horizons to develop novel therapeutical concepts targeting GPCR signaling elements. Thus, this review will focus on different molecular levels that may be of particular interest in terms of new drug development such as: (i) GPCR subtypes, allosteric binding sites, dimerization and constitutive activity, the use of RAMPs (receptor-activity-modifying proteins) and RASSLs (receptor activated solely by synthetic ligands); (ii) AGS (activators of G protein signaling) and RGS (regulators of G protein signaling) proteins which modify G protein activity; (iii) the high diversity of isozymes involved in the generation, signal transmission, and degradation of second messenger molecules.

PMID: 15180530 [PubMed - indexed for MEDLINE]

NF-kB pathway NF-kB signaling NF-kappaB signaling pathway

Chimeric yeast G-protein ? subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae.

Chimeric yeast G-protein ? subunit harboring a 37-residue C-terminal gustducin-specific sequence is functional in Saccharomyces cerevisiae.

Biosci Biotechnol Biochem. 2012;76(3):512-6

Authors: Hara K, Inada Y, Ono T, Kuroda K, Yasuda-Kamatani Y, Ishiguro M, Tanaka T, Misaka T, Abe K, Ueda M

Abstract
Despite many recent studies of G-protein-coupled receptor (GPCR) structures, it is not yet well understood how these receptors activate G proteins. The GPCR assay using baker's yeast, Saccharomyces cerevisiae, is an effective experimental model for the characterization of GPCR-G? interactions. Here, using the yeast endogenous G? protein (Gpa1p) as template, we constructed various chimeric G? proteins with a region that is considered to be necessary for interaction with mammalian receptors. The signaling assay using the yeast pheromone receptor revealed that the chimeric G? protein harboring 37 gustducin-specific amino acid residues at its C-terminus (GPA1/gust37) maintained functionality in yeast. In contrast, GPA1/gust44, a variant routinely used in mammalian experimental systems, was not functional.

PMID: 22451393 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Engineered GPCRs as tools to modulate signal transduction.

Engineered GPCRs as tools to modulate signal transduction.

Physiology (Bethesda). 2008 Dec;23:313-21

Authors: Pei Y, Rogan SC, Yan F, Roth BL

Abstract
Different families of G-protein-coupled receptors (GPCRs) have been engineered to provide exclusive control over the activation of these receptors and thus to understand better the consequences of their signaling in vitro and in vivo. These engineered receptors, named RASSLs (receptors activated solely by synthetic ligands) and DREADDs (designer receptors exclusively activated by designer drugs), are insensitive to their endogenous ligands but can be activated by synthetic drug-like compounds. Currently, the existing RASSLs and DREADDs cover the Gi, Gq, and Gs signaling pathways. These modified GPCRs can be utilized as ideal tools to study GPCR functions selectively in specific cellular populations.

PMID: 19074739 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

2012年9月17日星期一

All p65-containing dimers are not equal.

All p65-containing dimers are not equal.

Cell Cycle. 2012 Feb 15;11(4):646-7

Authors: Saccani S

PMID: 22313733 [PubMed - indexed for MEDLINE]

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Signaling through G protein coupled receptors.

Signaling through G protein coupled receptors.

Plant Signal Behav. 2009 Oct;4(10):942-7

Authors: Tuteja N

Abstract
Heterotrimeric G proteins (Galpha, Gbeta/Ggamma subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its re-association with the Gbeta/Ggamma dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.

PMID: 19826234 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor.

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor.

PLoS One. 2007;2(12):e1317

Authors: Chang WC, Ng JK, Nguyen T, Pellissier L, Claeysen S, Hsiao EC, Conklin BR

Abstract
G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT(4b) receptor, a GPCR with high constitutive G(s) signaling and strong ligand-induced G-protein activation of the G(s) and G(s/q) pathways. The first receptor in this series, 5-HT(4)-D(100)A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G(s) signaling, but only a few (e.g., zacopride) also induced signaling via the G(q) pathway. Zacopride-induced G(q) signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT(2C) receptor. Additional point mutations (D(66)A and D(66)N) blocked constitutive G(s) signaling and lowered ligand-induced G(q) signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT(1A) conferred ligand-mediated G(i) signaling. This G(i)-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G(s) or G(q) pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.

PMID: 18338032 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

2012年9月16日星期日

All p65-containing dimers are not equal.

All p65-containing dimers are not equal.

Cell Cycle. 2012 Feb 15;11(4):646-7

Authors: Saccani S

PMID: 22313733 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

J Biol Chem. 2001 Aug 24;276(34):31596-601

Authors: Small KM, Brown KM, Forbes SL, Liggett SB

Abstract
Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the beta(2)-adrenergic receptor (beta(2)AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a beta-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 +/- 2.1 versus 41.4 +/- 8 pmol/min/mg for wild-type beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 +/- 5% versus 55 +/- 4% for wild-type beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other pathologic states.

PMID: 11402033 [PubMed - indexed for MEDLINE]

NF-kB signaling pathway NF-kB pathway NF-kB signaling

Engineering GPCR signaling pathways with RASSLs.

Engineering GPCR signaling pathways with RASSLs.

Nat Methods. 2008 Aug;5(8):673-8

Authors: Conklin BR, Hsiao EC, Claeysen S, Dumuis A, Srinivasan S, Forsayeth JR, Guettier JM, Chang WC, Pei Y, McCarthy KD, Nissenson RA, Wess J, Bockaert J, Roth BL

Abstract
We are creating families of designer G protein-coupled receptors (GPCRs) to allow for precise spatiotemporal control of GPCR signaling in vivo. These engineered GPCRs, called receptors activated solely by synthetic ligands (RASSLs), are unresponsive to endogenous ligands but can be activated by nanomolar concentrations of pharmacologically inert, drug-like small molecules. Currently, RASSLs exist for the three major GPCR signaling pathways (G(s), G(i) and G(q)). We review these advances here to facilitate the use of these powerful and diverse tools.

PMID: 18668035 [PubMed - indexed for MEDLINE]

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Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

Directed molecular evolution of DREADDs: a generic approach to creating next-generation RASSLs.

Nat Protoc. 2010 Mar;5(3):561-73

Authors: Dong S, Rogan SC, Roth BL

Abstract
G protein-coupled receptors (GPCRs) and their downstream signaling cascades contribute to most physiological processes and a variety of human diseases. Isolating the effects of GPCR activation in an in vivo experimental setting is challenging as exogenous ligands have off-target effects and endogenous ligands constantly modulate the activity of native receptors. Highly specific designer drug-designer receptor complexes are a valuable tool for elucidating the effects of activating particular receptors and signaling pathways within selected cell types in vivo. In this study, we describe a generic protocol for the directed molecular evolution of designer receptors exclusively activated by designer drugs (DREADDs). First, the yeast system is validated with the template receptor. Second, a mutant library is generated by error-prone PCR. Third, the library is screened by drug-dependent yeast growth assays. Mutants exhibiting the desired properties are selected for further rounds of mutagenesis or for characterization in mammalian systems. In total, these steps should take 6-8 weeks of experimentation and should result in the evolution of a receptor to be activated by the chosen ligand. This protocol should help improve the experimental targeting of select cell populations.

PMID: 20203671 [PubMed - indexed for MEDLINE]

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All p65-containing dimers are not equal.

All p65-containing dimers are not equal.

Cell Cycle. 2012 Feb 15;11(4):646-7

Authors: Saccani S

PMID: 22313733 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

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