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.

Related Articles

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.

Related Articles

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.

Related Articles

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

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

Related Articles

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.

Related Articles

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

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

Related Articles

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]

GPCR Signaling G-protein Receptors gpcr pathway