5-alpha-reductase: 2012-08-26

2012年9月1日星期六

Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1.

Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1.

Sci Rep. 2011;1:172

Authors: Corin K, Baaske P, Geissler S, Wienken CJ, Duhr S, Braun D, Zhang S

Abstract
The vomeronasal system is one of several fine-tuned scent-detecting signaling systems in mammals. However, despite significant efforts, how these receptors detect scent remains an enigma. One reason is the lack of sufficient purified receptors to perform detailed biochemical, biophysical and structural analyses. Here we report the ability to express and purify milligrams of purified, functional human vomeronasal receptor hVN1R1. Circular dichroism showed that purified hVN1R1 had an alpha-helical structure, similar to that of other GPCRs. Microscale thermophoresis showed that hVN1R1 bound its known ligand myrtenal with an EC(50) approximately 1 �M. This expression system can enable structural and functional analyses towards understanding how mammalian scent detection works.

PMID: 22355687 [PubMed]

NF-kB signaling NF-kappaB signaling pathway read more

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

PLoS One. 2012;7(4):e35304

Authors: Liu YC, Lin IH, Chung WJ, Hu WS, Ng WV, Lu CY, Huang TY, Shu HW, Hsiao KJ, Tsai SF, Chang CH, Lin CH

Abstract
Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.

PMID: 22536369 [PubMed - indexed for MEDLINE]

Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C.

Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C.

Plant J. 2007 Aug;51(4):656-69

Authors: Misra S, Wu Y, Venkataraman G, Sopory SK, Tuteja N

Abstract
Heterotrimeric G-proteins transduce signals from activated G-protein-coupled receptors (GPCR) to appropriate downstream effectors and thereby play an important role in signaling. A role of G-proteins in salinity and heat stress tolerance has not heretofore been described. We report isolation of cDNAs of two isoforms of Galpha (Galpha1, 1152 bp; Galpha2, 1152 bp), one Gbeta (1134 bp), two isoforms of Ggamma (Ggamma1, 345 bp; Ggamma2, 303 bp) and a GPCR (1008 bp) from Pisum sativum, and purification of all the encoded recombinant proteins (Galpha, 44 kDa; Gbeta, 41 kDa; Ggamma, 14 kDa; GPCR, 35 kDa). The transcript levels of Galpha and Gbeta were upregulated following NaCl, heat and H(2)O(2) treatments. Protein-protein interaction studies using an in vitro yeast two-hybrid system and in planta co-immunoprecipitation showed that the Galpha subunit interacted with the pea Gbeta subunit and pea phospholipase C (PLCdelta) at the calcium-binding domain (fn1). The GTPase activity of the Galpha subunit increased after interaction with PLCdelta. The GPCR protein interacted with all the subunits of G-proteins and with itself. Transgenic tobacco plants (T(0) and T(1)) constitutively over-expressing Galpha showed tolerance to salinity and heat, while Gbeta-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and germination/growth of T(1) seeds/seedlings. These findings provide direct evidence for a novel role of Galpha and Gbeta subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling pathways.

PMID: 17587233 [PubMed - indexed for MEDLINE]

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

Bihelix: Towards de novo structure prediction of an ensemble of G-protein coupled receptor conformations.

Bihelix: Towards de novo structure prediction of an ensemble of G-protein coupled receptor conformations.

Proteins. 2011 Oct 12;

Authors: Abrol R, Bray JK, Goddard WA

Abstract
G-Protein Coupled Receptors (GPCRs) play a critical role in cellular signal transduction pathways and are prominent therapeutic targets. Recently there has been major progress in obtaining experimental structures for a few GPCRs. Each GPCR, however, exhibits multiple conformations that play a role in their function and we have been developing methods aimed at predicting structures for all these conformations. Analysis of available structures shows that these conformations differ in relative helix tilts and rotations. The essential issue is, determining how to orient each of the seven helices about its axis since this determines how it interacts with the other six helices. Considering all possible helix rotations to ensure that no important packings are overlooked, and using rotation angle increments of 30� about the helical axis would still lead to 12(7) or 35 million possible conformations each with optimal residue positions. We show in this paper how to accomplish this. The fundamental idea is to optimize the interactions between each pair of contacting helices while ignoring the other 5 and then to estimate the energies of all 35 million combinations using these pair-wise interactions. This BiHelix approach dramatically reduces the effort to examine the complete set of conformations and correctly identifies the crystal packing for the experimental structures plus other near-native packings we believe may play an important role in activation. This approach also enables a detailed structural analysis of functionally distinct conformations using helix-helix interaction energy landscapes and should be useful for other helical transmembrane proteins as well. Proteins 2011; � 2011 Wiley Periodicals, Inc.

PMID: 22173949 [PubMed - as supplied by publisher]

GPCR Signaling G-protein Receptors gpcr pathway

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]

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

2012年8月31日星期五

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-kappaB signaling pathway

Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.

Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.

Biotechnol Prog. 2006 Jul-Aug;22(4):954-60

Authors: Ishii J, Matsumura S, Kimura S, Tatematsu K, Kuroda S, Fukuda H, Kondo A

Abstract
The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods.

PMID: 16889369 [PubMed - indexed for MEDLINE]

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

Azidothymidine is Effective against Human Multiple Myeloma: A New Use for an Old Drug?

Azidothymidine is Effective against Human Multiple Myeloma: A New Use for an Old Drug?

Anticancer Agents Med Chem. 2012 Aug 27;

Authors: Pereira J, Levy D, Ruiz JL, Brocardo GA, Ferreira KA, Costa RO, Queiroz RG, Maria DA, Neto AE, Chamone DA, Bydlowski SP

Abstract
Azidothymidine(AZT)is an antiretroviral drug that affects cell proliferation, apoptosis, and the NF-?B pathway. As multiple myeloma (MM)presents with constitutive activation of NF-?B, we analyzed the effect of AZT on human MM cell lines. We evaluated the cytotoxic effect of AZT in human MM cell lines sensitive (8226/S)orresistant to doxorubicin (8226/DX5) and human T cell lymphoblast-like cells, uterine sarcoma cells, and HUVEC using MTT assay. Cytotoxicitywas also evaluated in vivo in nude mice xenografted with an8226/S tumor. The effect of AZT on the expression of genes involved in cell proliferation, apoptosis, angiogenesis, and the NF-?B pathway was analyzed in the xenografts usingreal-time polymerase chain reaction.AZT was effective against both 8226/S and 8226/DX5 cells in a dose and time-dependent manner(p = 0.02)in vitro and promoted cell cycle arrest in S phase in these cells. The tumor volume was lower in mice treated with AZT compared tountreated mice (p = 0.0003). AZT down-regulated the pro-proliferative genes encodingAKT1, MYC, STAT1, MAPK8, MAPK9, CCL-3, Bcl-3, and cyclin D2;pro-angiogenenic genes encodingVEGF and IL8; and genes involved in cell adhesion (ICAM1 and FN1) and the NF-?Bpathway. AZT up-regulated the expression of tumor suppressor gene FOXP1and the pro-apoptotic genes encoding BID, Bcl-10, and caspase-8. Thus, we demonstrated the cytotoxic effect of AZT in human MM cell lines for the first time. Our data may provide the rationale for future clinical trials of AZT for treating MM.

PMID: 22931421 [PubMed - as supplied by publisher]

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

2012年8月30日星期四

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

PMID: 15209517 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection.

A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection.

BMC Genomics. 2012 Aug 28;13(1):431

Authors: Hori TS, Gamperl AK, Booman M, Nash GW, Rise ML

Abstract
ABSTRACT: BACKGROUND: Atlantic cod (Gadus morhua) reared in sea-cages can experience large variations in temperature, and these have been shown to affect their immune function. We used the new 20 K Atlantic cod microarray to investigate how a water temperature change which simulates that seen in Newfoundland during the spring-summer (i.e. from 10[DEGREE SIGN]C to 16[DEGREE SIGN]C, 1[DEGREE SIGN]C increase every 5 days) impacted the cod spleen transcriptome response to the intraperitoneal injection of a viral mimic (polyriboinosinic polyribocytidylic acid, pIC). RESULTS: The temperature regime alone did not cause any significant increases in plasma cortisol levels and only minor changes in spleen gene transcription. However, it had a considerable impact on the fish spleen transcriptome response to pIC [290 and 339 significantly differentially expressed genes between 16[DEGREE SIGN]C and 10[DEGREE SIGN]C at 6 and 24 hours post-injection (HPI), respectively]. Seventeen microarray-identified transcripts were selected for QPCR validation based on immune-relevant functional annotations. Fifteen of these transcripts (i.e. 88%), including DHX58, STAT1, IRF7, ISG15, RSAD2 and IkappaBalpha, were shown by QPCR to be significantly induced by pIC. CONCLUSIONS: The temperature increase appeared to accelerate the spleen immune transcriptome response to pIC. We found 41 and 999 genes differentially expressed between fish injected with PBS vs. pIC at 10[DEGREE SIGN]C and sampled at 6HPI and 24HPI, respectively. In contrast, there were 656 and 246 genes differentially expressed between fish injected with PBS vs. pIC at 16[DEGREE SIGN]C and sampled at 6HPI and 24HPI, respectively. Our results indicate that the modulation of mRNA expression of genes belonging to the NF-kappaB and type I interferon signal transduction pathways may play a role in controlling temperature-induced changes in the spleen's transcript expression response to pIC. Moreover, interferon effector genes such as ISG15 and RSAD2 were differentially expressed between fish injected with pIC at 10[DEGREE SIGN]C vs. 16[DEGREE SIGN]C at 6HPI. These results substantially increase our understanding of the genes and molecular pathways involved in the negative impacts of elevated ambient temperature on fish health, and may also be valuable to our understanding of how accelerated global climate change could impact cold-water marine finfish species.

PMID: 22928584 [PubMed - as supplied by publisher]

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

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

PMID: 20203671 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

Lipid raft-mediated regulation of G-protein coupled receptor signaling by ligands which influence receptor dimerization: a computational study.

Lipid raft-mediated regulation of G-protein coupled receptor signaling by ligands which influence receptor dimerization: a computational study.

PLoS One. 2009;4(8):e6604

Authors: Fallahi-Sichani M, Linderman JJ

Abstract
G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors; they activate heterotrimeric G-proteins in response to ligand stimulation. Although many GPCRs have been shown to form homo- and/or heterodimers on the cell membrane, the purpose of this dimerization is not known. Recent research has shown that receptor dimerization may have a role in organization of receptors on the cell surface. In addition, microdomains on the cell membrane termed lipid rafts have been shown to play a role in GPCR localization. Using a combination of stochastic (Monte Carlo) and deterministic modeling, we propose a novel mechanism for lipid raft partitioning of GPCRs based on reversible dimerization of receptors and then demonstrate that such localization can affect GPCR signaling. Modeling results are consistent with a variety of experimental data indicating that lipid rafts have a role in amplification or attenuation of G-protein signaling. Thus our work suggests a new mechanism by which dimerization-inducing or inhibiting characteristics of ligands can influence GPCR signaling by controlling receptor organization on the cell membrane.

PMID: 19668374 [PubMed - indexed for MEDLINE]

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

G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches.

G-protein-coupled receptors in drug discovery: nanosizing using cell-free technologies and molecular biology approaches.

J Biomol Screen. 2005 Dec;10(8):765-79

Authors: Leifert WR, Aloia AL, Bucco O, Glatz RV, McMurchie EJ

Abstract
Signal transduction by G-protein-coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. Signal transduction has been studied extensively with both cell-based systems and assays comprising isolated signaling components. Interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high throughput screening, biosensors, and so on will focus greater attention on assay development to allow for miniaturization, ultra-high throughput and, eventually, microarray/biochip assay formats. Although cell-based assays are adequate for many GPCRs, it is likely that these formats will limit the development of higher density GPCR assay platforms mandatory for other applications. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR assay platforms adaptable for such applications as microarrays. The authors review current cell-free GPCR assay technologies and molecular biological approaches for construction of novel, functional GPCR assays.

PMID: 16234342 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

2012年8月29日星期三

Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion.

Statins suppress apolipoprotein CIII-induced vascular endothelial cell activation and monocyte adhesion.

Eur Heart J. 2012 Aug 26;

Authors: Zheng C, Azcutia V, Aikawa E, Figueiredo JL, Croce K, Sonoki H, Sacks FM, Luscinskas FW, Aikawa M

Abstract
AimsActivation of vascular endothelial cells (ECs) contributes importantly to inflammation and atherogenesis. We previously reported that apolipoprotein CIII (apoCIII), found abundantly on circulating triglyceride-rich lipoproteins, enhances adhesion of human monocytes to ECs in vitro. Statins may exert lipid-independent anti-inflammatory effects. The present study examined whether statins suppress apoCIII-induced EC activation in vitro and in vivo.Methods and resultsPhysiologically relevant concentrations of purified human apoCIII enhanced attachment of the monocyte-like cell line THP-1 to human saphenous vein ECs (HSVECs) or human coronary artery ECs (HCAECs) under both static and laminar shear stress conditions. This process mainly depends on vascular cell adhesion molecule-1 (VCAM-1), as a blocking VCAM-1 antibody abolished apoCIII-induced monocyte adhesion. ApoCIII significantly increased VCAM-1 expression in HSVECs and HCAECs. Pre-treatment with statins suppressed apoCIII-induced VCAM-1 expression and monocyte adhesion, with two lipophilic statins (pitavastatin and atorvastatin) exhibiting inhibitory effects at lower concentration than those of hydrophilic pravastatin. Nuclear factor ?B (NF-?B) mediated apoCIII-induced VCAM-1 expression, as demonstrated via loss-of-function experiments, and pitavastatin treatment suppressed NF-?B activation. Furthermore, in the aorta of hypercholesterolaemic Ldlr(-/-) mice, pitavastatin administration in vivo suppressed VCAM-1 mRNA and protein, induced by apoCIII bolus injection. Similarly, in a subcutaneous dorsal air pouch mouse model of leucocyte recruitment, apoCIII injection induced F4/80+ monocyte and macrophage accumulation, whereas pitavastatin administration reduced this effect.ConclusionsThese findings further establish the direct role of apoCIII in atherogenesis and suggest that anti-inflammatory effects of statins could improve vascular disease in the population with elevated plasma apoCIII.

PMID: 22927557 [PubMed - as supplied by publisher]

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

The role of Raf kinase inhibitor protein in rheumatoid fibroblast-like synoviocytes invasiveness and cytokine and matrix metalloproteinase expression.

The role of Raf kinase inhibitor protein in rheumatoid fibroblast-like synoviocytes invasiveness and cytokine and matrix metalloproteinase expression.

Inflammation. 2012 Apr;35(2):474-83

Authors: Ahn JK, Hwang JW, Bae EK, Lee J, Jeon CH, Koh EM, Cha HS

Abstract
Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis. Raf kinase inhibitor protein (RKIP) negatively regulates the Raf/MEK/ERK and NF-?B pathway. The role of RKIP in rheumatoid FLS is unknown. The purpose of the present study was to investigate the function of RKIP in rheumatoid FLS. Rheumatoid FLS were transfected with either RKIP-expressing plasmids or RKIP small interfering RNA (siRNA). RKIP protein was detected in rheumatoid synovial tissue (ST) and FLS. RKIP overexpression significantly decreased IL-6 mRNA expression in TNF-?-stimulated rheumatoid FLS. RKIP overexpression also showed a decreased trend in IL-8, MMP-1, and MMP-3 mRNA expression in TNF-?-stimulated rheumatoid FLS. RKIP silencing resulted in significantly increased MMP-1 and MMP-3 mRNA expression in TNF-?-stimulated rheumatoid FLS. RKIP silencing also increased IL-6 and IL-8 mRNA expression in TNF-?-stimulated rheumatoid FLS, but this increase did not reach statistical significance. TNF-?-induced ERK and NF-?B activation was suppressed in FLS with RKIP overexpression. RKIP silencing resulted in a significantly higher invasion index in TNF-?-stimulated rheumatoid FLS compared to controls. These results suggest that RKIP might be a potential therapeutic target for rheumatoid arthritis.

PMID: 21556737 [PubMed - indexed for MEDLINE]

gpcr pathway NF-κB NF-kB signaling pathway

Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest.

Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest.

Front Immunol. 2012;3:188

Authors: Dixit N, Simon SI

Abstract
Leukocyte trafficking to acute sites of injury or infection requires spatial and temporal cues that fine tune precise sites of firm adhesion and guide migration to endothelial junctions where they undergo diapedesis to sites of insult. Many detailed studies on the location and gradient of chemokines such as IL-8 and other CXCR ligands reveal that their recognition shortly after selectin-mediated capture and rolling exerts acute effects on integrin activation and subsequent binding to their ligands on the endothelium, which directs firm adhesion, adhesion strengthening, and downstream migration. In this process, G-protein coupled receptor (GPCR) signaling has been found to play an integral role in activating and mobilizing intracellular stores of calcium, GTPases such as Rap-1 and Rho and cytokeletal proteins such as Talin and F-actin to facilitate cell polarity and directional pseudopod formation. A critical question remaining is how intracellular Ca(2+) flux from CRAC channels such as Orai1 synergizes with cytosolic stores to mediate a rapid flux which is critical to the onset of PMN arrest and polarization. Our review will highlight a specific role for calcium as a signaling messenger in activating focal clusters of integrins bound to the cytoskeleton which allows the cell to attain a migratory phenotype. The precise interplay between chemokines, selectins, and integrins binding under the ubiquitous presence of shear stress from blood flow provides an essential cooperative signaling mechanism for effective leukocyte recruitment.

PMID: 22787461 [PubMed - in process]

GPCR Signaling G-protein Receptors gpcr pathway

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-κB NF-kB signaling pathway NF-kB pathway

2012年8月28日星期二

[CYLD deubiquitinase as a recurrent target in oncogenic processes].

[CYLD deubiquitinase as a recurrent target in oncogenic processes].

Med Sci (Paris). 2011 Jun-Jul;27(6-7):626-31

Authors: Bonnet M, Courtois G

Abstract
CYLD deubiquitinase has been originally defined as a tumor suppressor based exclusively on genetic findings. Indeed, inactivation of CYLD in humans results in familial cylindromatosis and multiple trichoepithelioma, two pathologies characterized by the development of tumors originating specifically from the skin appendages. A set of recent publications has revealed that recurrent inactivation of CYLD occurs through diverse mechanisms in several forms of cancer, unequivocally confirming its tumor suppressor function. This property is associated with the critical role played by CYLD in negatively regulating several signaling pathway, among them the NF-?B signaling pathway.

PMID: 21718647 [PubMed - indexed for MEDLINE]

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MEKK1-MKK4-JNK-AP1 pathway negatively regulates Rgs4 expression in colonic smooth muscle cells.

MEKK1-MKK4-JNK-AP1 pathway negatively regulates Rgs4 expression in colonic smooth muscle cells.

PLoS One. 2012;7(4):e35646

Authors: Zhang Y, Li F, Liu S, Wang H, Mahavadi S, Murthy KS, Khalili K, Hu W

Abstract
BACKGROUND: Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1? is mediated by the activation of NF?B signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3 kinase. Here we investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs4 expression in rabbit colonic smooth muscle cells.
METHODOLOGY/PRINCIPAL FINDINGS: Cultured cells at first passage were treated with or without IL-1? (10 ng/ml) in the presence or absence of the selective JNK inhibitor (SP600125) or JNK small hairpin RNA (shRNA). The expression levels of Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1? stimulation. Overexpression of MEKK1, the key upstream kinase of JNK, inhibited Rgs4 expression, which was reversed by co-expression of JNK shRNA or dominant-negative mutants for MKK4 or JNK. Both constitutive and inducible upregulation of Rgs4 expression by SP600125 was significantly inhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1?. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confirmed that IL-1? treatment increased the phosphorylation of JNK, ATF-2 and c-Jun. Gel shift and chromatin immunoprecipitation assays validated that IL-1? increased the in vitro and ex vivo binding activities of AP1 within rabbit Rgs4 promoter.
CONCLUSION/SIGNIFICANCE: Activation of MEKK1-MKK4-JNK-AP1 signal pathway plays a tonic inhibitory role in regulating Rgs4 transcription in rabbit colonic smooth muscle cells. This negative regulation may aid in maintaining the transient level of RGS4 expression.

PMID: 22545125 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

Ginsenoside Rg1 protection against ?-amyloid peptide-induced neuronal apoptosis via estrogen receptor ? and glucocorticoid receptor-dependent anti-protein nitration pathway.

Ginsenoside Rg1 protection against ?-amyloid peptide-induced neuronal apoptosis via estrogen receptor ? and glucocorticoid receptor-dependent anti-protein nitration pathway.

Neuropharmacology. 2012 Sep;63(3):349-61

Authors: Wu J, Pan Z, Wang Z, Zhu W, Shen Y, Cui R, Lin J, Yu H, Wang Q, Qian J, Yu Y, Zhu D, Lou Y

Abstract
Ginsenoside Rg1 (Rg1) acts as a neuroprotective agent against various insults, however, the underlying mechanism has not been fully elucidated yet. Here, we report that Rg1 protects primary rat cerebrocortical neurons against ?-amyloid peptide????? (A??????) injury via estrogen receptor ? (ER?) and glucocorticoid receptor (GR)-dependent anti-protein nitration pathway. In primary rat cerebrocortical neuron cultures under basal conditions, Rg1 leads to nuclear translocation of ER? and GR, induces related responsive gene PR, pS? and MKP-1, SGK transcription. Meantime, Rg1 also increases the basal level of ERK1/2 phosphorylation. In the presence of toxic level of A??????, Rg1 maintains ERK1/2 phosphorylation, attenuates iNOS expression, NO production, and inhibits NF-?B nuclear translocation, protein nitration and cell death. The antiapoptotic effects of Rg1 via both ER? and GR were abolished by small interfering RNAs (siRNA). ERK1/2 phosphorylation inhibitor U0126 can block downstream iNOS expression and NO generation. Interestingly, the anti-protein nitration effect of Rg1 is well matched with ER? and GR activation, although its anti-ROS production effect is in an ER?- and GR-independent manner. These results suggest that Rg1 ameliorates A??????-induced neuronal apoptosis at least in part by two complementary ER?- and GR-dependent downstream pathways: (1) upregulation of ERK1/2 phosphorylation followed by inhibiting iNOS expression, NO generation and protein tyrosine nitration. (2) reduction NF-?B nuclear translocation. These data provide new understanding into the mechanisms of Rg1 anti-apoptotic functions after A?????? exposure, suggesting that ER? and GR-dependent anti-protein tyrosine nitration pathway might take an important role in the neuroprotective effect of Rg1.

PMID: 22534050 [PubMed - indexed for MEDLINE]

G-protein Receptors gpcr pathway NF-κB

The noncanonical NF-?B pathway.

The noncanonical NF-?B pathway.

Immunol Rev. 2012 Mar;246(1):125-40

Authors: Sun SC

Abstract
The noncanonical nuclear factor-?B (NF-?B) signaling pathway mediates activation of the p52/RelB NF-?B complex and, thereby, regulates specific immunological processes. This NF-?B pathway relies on the inducible processing of NF-?B2 precursor protein, p100, as opposed to the degradation of I?B? in the canonical NF-?B pathway. A central signaling component of the noncanonical NF-?B pathway is NF-?B-inducing kinase (NIK), which functions together with a downstream kinase, IKK? (inhibitor of NF-?B kinase ?), to induce phosphorylation-dependent ubiquitination and processing of p100. Under normal conditions, NIK is targeted for continuous degradation by a tumor necrosis factor (TNF) receptor-associated factor-3 (TRAF3)-dependent E3 ubiquitin ligase. In response to signals mediated by a subset of TNF receptor superfamily members, NIK becomes stabilized as a result of TRAF3 degradation, leading to the activation of noncanonical NF-?B. This review discusses both the historical perspectives and the recent progress in the regulation and biological function of the noncanonical NF-?B pathway.

PMID: 22435551 [PubMed - indexed for MEDLINE]

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

Adhesion G Protein-Coupled Receptors: Signaling, Pharmacology & Mechanisms of Activation.

Adhesion G Protein-Coupled Receptors: Signaling, Pharmacology & Mechanisms of Activation.

Mol Pharmacol. 2012 Jul 20;

Authors: Paavola KJ, Hall RA

Abstract
The adhesion G protein-coupled receptors (GPCRs) are a distinct family of more than 30 receptors in vertebrate genomes. These receptors have been shown to play pivotal roles in a diverse range of biological functions and are characterized by extremely large N-termini featuring various adhesion domains capable of mediating cell-cell and cell-matrix interactions. The adhesion GPCR N-termini also contain GPCR proteolytic site (GPS) motifs that undergo autocatalytic cleavage during receptor processing to create mature GPCRs existing as non-covalently attached complexes between the N-terminus and transmembrane regions. There is mounting evidence that adhesion GPCRs can couple to G proteins to activate a variety of different downstream signaling pathways. Furthermore, recent studies have demonstrated that adhesion GPCR N-termini can bind to multiple ligands, which may differentially activate receptor signaling and/or mediate cell adhesion. Additionally, studies on several distinct adhesion GPCRs have revealed that truncations of the N-termini result in constitutively-active receptors, suggesting a model of receptor activation in which removal of the N-terminus may be a key event in stimulating receptor signaling. Since mutations to certain adhesion GPCRs cause human disease, and since many members of this receptor family exhibit highly discrete distribution patterns in different tissues, the adhesion GPCRs represent a class of potentially important drug targets that have not yet been exploited. For this reason, understanding the mechanisms of activation for these receptors and elucidating their downstream signaling pathways can provide insights with the potential to lead to novel therapeutics.

PMID: 22821233 [PubMed - as supplied by publisher]

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

2012年8月27日星期一

The extreme C-terminal region of G?s differentially couples to the luteinizing hormone and beta2-adrenergic receptors.

The extreme C-terminal region of G?s differentially couples to the luteinizing hormone and beta2-adrenergic receptors.

Mol Endocrinol. 2011 Aug;25(8):1416-30

Authors: DeMars G, Fanelli F, Puett D

Abstract
The mechanisms of G protein coupling to G protein-coupled receptors (GPCR) share general characteristics but may exhibit specific interactions unique for each GPCR/G protein partnership. The extreme C terminus (CT) of G protein ?-subunits has been shown to be important for association with GPCR. Hypothesizing that the extreme CT of G?(s) is an essential component of the molecular landscape of the GPCR, human LH receptor (LHR), and ?(2)-adrenergic receptor (?(2)-AR), a model cell system was created for the expression and manipulation of G?(s) subunits in LHR(+) s49 ck cells that lack endogenous G?(s). On the basis of studies involving truncations, mutations, and chain extensions of G?(s), the CT was found to be necessary for LHR and ?(2)-AR signaling. Some general similarities were found for the responses of the two receptors, but significant differences were also noted. Computational modeling was performed with a combination of comparative modeling, molecular dynamics simulations, and rigid body docking. The resulting models, focused on the G?(s) CT, are supported by the experimental observations and are characterized by the interaction of the four extreme CT amino acid residues of G?(s) with residues in LHR and ?(2)-AR helix 3, (including R of the DRY motif), helix 6, and intracellular loop 2. This portion of G?(s) recognizes the same regions of the two GPCR, although with differences in the details of selected interactions. The predicted longer cytosolic extensions of helices 5 and 6 of ?(2)-AR are expected to contribute significantly to differences in G?(s) recognition by the two receptors.

PMID: 21622536 [PubMed - indexed for MEDLINE]

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

NF-?B: where did it come from and why?

NF-?B: where did it come from and why?

Immunol Rev. 2012 Mar;246(1):14-35

Authors: Gilmore TD, Wolenski FS

Abstract
The vast majority of research on nuclear factor ?B (NF-?B) signaling in the past 25 years has focused on its roles in normal and disease-related processes in vertebrates, especially mice and humans. Recent genome and transcriptome sequencing efforts have shown that homologs of NF-?B transcription factors, inhibitor of NF-?B (I?B) proteins, and I?B kinases are present in a variety of invertebrates, including several in phyla simpler than Arthropoda, the phylum containing insects such Drosophila. Moreover, many invertebrates also contain genes encoding homologs of upstream signaling proteins in the Toll-like receptor signaling pathway, which is well-known for its downstream activation of NF-?B for innate immunity. This review describes what we now know or can infer and speculate about the evolution of the core elements of NF-?B signaling as well as the biological processes controlled by NF-?B in invertebrates. Further research on NF-?B in invertebrates is likely to uncover information about the evolutionary origins of this key human signaling pathway and may have relevance to our management of the responses of ecologically and economically important organisms to environmental and adaptive pressures.

PMID: 22435545 [PubMed - indexed for MEDLINE]

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Molecular pharmacology of histamine H4 receptors.

Molecular pharmacology of histamine H4 receptors.

Front Biosci. 2012;17:2089-106

Authors: Nijmeijer S, de Graaf C, Leurs R, Vischer HF

Abstract
The histamine H4 receptor (H4R) is the youngest member of the histamine receptor family. Based on its predominant expression pattern in hematopoietic cells, the H4R is considered to be an interesting drug target for inflammatory disorders such as allergy and asthma. Since the identification and cloning of the H4R in 2000, drug discovery programs boosted the development of various H4R (specific) ligands. Differences between H4R orthologs in combination with available three-dimensional G protein-coupled receptor (GPCR) models have guided site-directed mutagenesis studies to gain insight in ligand binding and receptor activation. In addition, ongoing characterization of H4R-mediated signaling in transfected and native cells contributes to further unravel the (patho-) physiological functions of H4Rs.

PMID: 22652766 [PubMed - in process]

G-protein Receptors gpcr pathway NF-κB

An update of novel screening methods for GPCR in drug discovery.

An update of novel screening methods for GPCR in drug discovery.

Expert Opin Drug Discov. 2012 Jun 21;

Authors: Chen L, Jin L, Zhou N

Abstract
Introduction: G protein-coupled receptors (GPCRs) are the largest and most versatile group of cytomembrane receptors, comprising of approximately 300 non-sensory and druggable members. Traditional GPCR drug screening is based on radiometric competition binding assays, which are expensive and hazardous to human health. Furthermore, the paradox of high investment and low output, in terms of new drugs, highlights the need for more efficient and effective drug screening methods. Areas covered: This review summarizes non-radioactive assays assessing the ligand-receptor binding including: the fluorescence polarization assay, the TR-FRET assay and the surface plasmon resonance assay. It also looks at non-radioactive assays that assess receptor activation and signaling including: second messenger-based assays and ?-arrestin recruitment-based assays. This review also looks at assays based on cellular phenotypic change. Expert opinion: GPCR signaling pathways look to be more complicated than previously thought. The existence of receptor allosteric sites and multireceptor downstream effectors restricts the traditional assay methods. The emergence of novel drug screening methods such as those for assessing ?-arrestin recruitment and cellular phenotypic change may provide us with improved drug screening efficiency and effect.

PMID: 22716301 [PubMed - as supplied by publisher]

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

2012年8月26日星期日

Receptor-specific regulation of ERK1/2 activation by members of the "free fatty acid receptor" family.

Receptor-specific regulation of ERK1/2 activation by members of the "free fatty acid receptor" family.

J Recept Signal Transduct Res. 2012 Aug;32(4):196-201

Authors: Seljeset S, Siehler S

Abstract
Context: The "free fatty acid receptors" (FFARs) GPR40, GPR41, and GPR43 regulate various physiological homeostases, and are all linked to activation of extracellular signal-regulated kinases (ERK)1/2. Objective: Investigation of coupling of FFARs to two other mitogen-activated protein kinases (MAPKs) sometimes regulated by G protein-coupled receptors (GPCRs), c-Jun N-terminal kinase (JNK) and p38MAPK, and characterization of signaling proteins involved in the regulation of FFAR-mediated ERK1/2 activation. Methods: FFARs were recombinantly expressed, cells challenged with the respective agonist, and MAPK activation quantitatively determined using an AlphaScreen SureFire assay. Inhibitors for signaling proteins were utilized to characterize ERK1/2 pathways. Results: Propionate-stimulated GPR41 strongly coupled to ERK1/2 activation, while the coupling of linoleic acid-activated GPR40 and acetate-activated GPR43 was weaker. JNK and p38MAPK were weakly activated by FFARs. All three receptors activated ERK1/2 fully or partially via G(i/o) and Rac. PI3K was relevant for GPR40- and GPR41-mediated ERK1/2 activation, and Src was essential for GPR40- and GPR43-induced activation. Raf-1 was not involved in the GPR43-triggered activation. Conclusion: The results demonstrate a novel role of Rac in GPCR-mediated ERK1/2 signaling, and that GPCRs belonging to the same family can regulate ERK1/2 activation by different receptor-specific mechanisms.

PMID: 22712802 [PubMed - in process]

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Selection and characterization of DARPins specific for the neurotensin receptor 1.

Selection and characterization of DARPins specific for the neurotensin receptor 1.

Protein Eng Des Sel. 2009 Jun;22(6):357-66

Authors: Milovnik P, Ferrari D, Sarkar CA, Pl�ckthun A

Abstract
We describe here the selection and characterization of designed ankyrin repeat proteins (DARPins) that bind specifically to the rat neurotensin receptor 1 (NTR1), a G-protein coupled receptor (GPCR). The selection procedure using ribosome display and the initial clone analysis required <10 microg of detergent-solubilized, purified NTR1. Complex formation with solubilized GPCR was demonstrated by ELISA and size-exclusion chromatography; additionally, the GPCR could be detected in native membranes of mammalian cells using fluorescence microscopy. The main binding epitope in the GPCR lies within the 33 amino acids following the seventh transmembrane segment, which comprise the putative helix 8, and additional binding interactions are possibly contributed by the cytoplasmic loop 3, thus constituting a discontinuous epitope. Since the selected binders recognize the GPCR both in detergent-solubilized and in membrane-embedded forms, they will be potentially useful both in co-crystallization trials and for signal transduction experiments.

PMID: 19389717 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

Classification of G-protein coupled receptors at four levels.

Classification of G-protein coupled receptors at four levels.

Protein Eng Des Sel. 2006 Nov;19(11):511-6

Authors: Gao QB, Wang ZZ

Abstract
G-protein coupled receptors (GPCRs) are transmembrane proteins which via G-proteins initiate some of the important signaling pathways in a cell and are involved in various physiological processes. Thus, computational prediction and classification of GPCRs can supply significant information for the development of novel drugs in pharmaceutical industry. In this paper, a nearest neighbor method has been introduced to discriminate GPCRs from non-GPCRs and subsequently classify GPCRs at four levels on the basis of amino acid composition and dipeptide composition of proteins. Its performance is evaluated on a non-redundant dataset consisted of 1406 GPCRs for six families and 1406 globular proteins using the jackknife test. The present method based on amino acid composition achieved an overall accuracy of 96.4% and Matthew's correlation coefficient (MCC) of 0.930 for correctly picking out the GPCRs from globular proteins. The overall accuracy and MCC were further enhanced to 99.8% and 0.996 by dipeptide composition-based method. On the other hand, the present method has successfully classified 1406 GPCRs into six families with an overall accuracy of 89.6 and 98.8% using amino acid composition and dipeptide composition, respectively. For the subfamily prediction of 1181 GPCRs of rhodopsin-like family, the present method achieved an overall accuracy of 76.7 and 94.5% based on the amino acid composition and dipeptide composition, respectively. Finally, GPCRs belonging to the amine subfamily and olfactory subfamily of rhodopsin-like family were further analyzed at the type level. The overall accuracy of dipeptide composition-based method for the classification of amine type and olfactory type of GPCRs reached 94.5 and 86.9%, respectively, while the overall accuracy of amino acid composition-based method was very low for both subfamilies. In comparison with existing methods in the literature, the present method also displayed great competitiveness. These results demonstrate the effectiveness of our method on identifying and classifying GPCRs correctly. GPCRsIdentifier, a corresponding stand-alone executable program for GPCR identification and classification was also developed, which can be acquired freely on request from the authors for academic purposes.

PMID: 17032692 [PubMed - indexed for MEDLINE]

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The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

PLoS Biol. 2012 Jun;10(6):e1001337

Authors: Duvall LB, Taghert PH

Abstract
The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

PMID: 22679392 [PubMed - in process]

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