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

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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

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

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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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An algebra of dimerization and its implications for G-protein coupled receptor signaling.

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An algebra of dimerization and its implications for G-protein coupled receptor signaling.

J Theor Biol. 2004 Jul 21;229(2):157-68

Authors: Woolf PJ, Linderman JJ

Abstract
Many species of receptors form dimers, but how can we use this information to make predictions about signal transduction? This problem is particularly difficult when receptors dimerize with many different species, leading to a combinatoric increase in the possible number of dimer pairs. As an example system, we focus on receptors in the G-protein coupled receptor (GPCR) family. GPCRs have been shown to reversibly form dimers, but this dimerization does not directly affect signal transduction. Here we present a new theoretical framework called a dimerization algebra. This algebra provides a systematic and rational way to represent, manipulate, and in some cases simplify large and often complicated networks of dimerization interactions. To compliment this algebra, Monte Carlo simulations are used to predict dimerization's effect on receptor organization on the membrane, signal transduction, and internalization. These simulation results are directly comparable to various experimental measures such as fluorescence resonance energy transfer (FRET), and as such provide a link between the dimerization algebra and experimental data. As an example, we show how the algebra and computational results can be used to predict the effects of dimerization on the dopamine D2 and somatastatin SSTR1 receptors. When these predictions were compared to experimental findings from the literature, good agreement was found, demonstrating the utility of our approach. Applications of this work to the development of a novel class of dimerization-modulating drugs are also discussed.

PMID: 15207471 [PubMed - indexed for MEDLINE]

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

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

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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]

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Regulation of cytochrome P450 4F11 by nuclear transcription factor-?B.

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Regulation of cytochrome P450 4F11 by nuclear transcription factor-?B.

Drug Metab Dispos. 2012 Jan;40(1):205-11

Authors: Bell JC, Strobel HW

Abstract
Although the mechanisms that regulate CYP4F genes have been and are currently being studied in a number of laboratories, the specific mechanisms for the regulation of these genes are not yet fully understood. This study shows that nuclear factor ?B of the light-chain-enhancer in activated B cells (NF-?B) can inhibit CYP4F11 expression in human liver carcinoma cell line (HepG2) as summarized below. Tumor necrosis factor-? (TNF-?), a proinflammatory cytokine, has been shown to activate NF-?B signaling while also activating the c-Jun NH(2)-terminal kinase (JNK) signaling pathway. Other studies have reported that JNK signaling can up-regulate CYP4F11 expression. The results of this study demonstrate that in the presence of TNF-? and the specific NF-?B translocation inhibitor N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD-0354), there is a greater increase in CYP4F11 expression than that elicited by TNF-? alone, indicating that NF-?B plays an inhibitory role. Moreover, NF-?B stimulation by overexpression of mitogen-activated protein kinase kinase kinase inhibited CYP4F11 promoter expression. CYP4F11 promoter inhibition can also be rescued in the presence of TNF-? when p65, a NF-?B protein, is knocked down. Thus, NF-?B signaling pathways negatively regulate the CYP4F11 gene.

PMID: 22011441 [PubMed - indexed for MEDLINE]

GPCR Signaling G-protein Receptors gpcr pathway

Engineering GPCR signaling pathways with RASSLs.

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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]

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

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

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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]

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