Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean

Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants.     

Arabidopsis Heterotrimeric G-Proteins Play a Critical Role in Host and Nonhost Resistance against Pseudomonas syringae Pathogens

Heterotrimeric G-proteins have been proposed to be involved in many aspects of plant disease resistance but their precise role in mediating nonhost disease resistance is not well understood. We evaluated the roles of specific subunits of heterotrimeric G-proteins using knock-out mutants of Arabidopsis Gα, Gβ and Gγ subunits in response to host and nonhost Pseudomonas pathogens. Plants lacking functional Gα, Gβ and Gγ1Gγ2 proteins displayed enhanced bacterial growth and disease susceptibility in response to host and nonhost pathogens. Mutations of single Gγ subunits Gγ1, Gγ2 and Gγ3 did not alter bacterial disease resistance. Some specificity of subunit usage was observed when comparing host pathogen versus nonhost pathogen. Overexpression of both Gα and Gβ led to reduced bacterial multiplication of nonhost pathogen P. syringae pv. tabaci whereas overexpression of Gβ, but not of Gα, resulted in reduced bacterial growth of host pathogen P. syringae pv. maculicola, compared to wild-type Col-0. Moreover, the regulation of stomatal aperture by bacterial pathogens was altered in Gα and Gβ mutants but not in any of the single or double Gγ mutants. Taken together, these data substantiate the critical role of heterotrimeric G-proteins in plant innate immunity and stomatal modulation in response to P. syringae.     

Evolution,Expression Differentiation and Interaction Specificity of Heterotrimeric G-Protein Subunit Gene Family in the Mesohexaploid Brassica rapa

Heterotrimeric G-proteins, comprising of Gα, Gβ, and Gγ subunits, are important signal transducers which regulate many aspects of fundamental growth and developmental processes in all eukaryotes. Initial studies in model plants Arabidopsis and rice suggest that the repertoire of plant G-protein is much simpler than that observed in metazoans. In order to assess the consequence of whole genome triplication events within Brassicaceae family, we investigated the multiplicity of G-protein subunit genes in mesohexaploid Brassica rapa, a globally important vegetable and oilseed crop. We identified one Gα (BraA.Gα1), three Gβ (BraA.Gβ1, BraA.Gβ2, and BraA.Gβ3), and five Gγ (BraA.Gγ1, BraA.Gγ2, BraA.Gγ3, BraA.Gγ4, and BraA.Gγ5) genes from B. rapa, with a possibility of 15 Gαβγ heterotrimer combinations. Our analysis suggested that the process of genome triplication coupled with gene-loss (gene-fractionation) phenomenon have shaped the quantitative and sequence diversity of G-protein subunit genes in the extant B. rapa genome. Detailed expression analysis using qRT-PCR assays revealed that the G-protein genes have retained ubiquitous but distinct expression profiles across plant development. The expression of multiple G-protein genes was differentially regulated during seed-maturation and germination stages, and in response to various phytohormone treatments and stress conditions. Yeast-based interaction analysis showed that G-protein subunits interacted in most of the possible combinations, with some degree of subunit-specific interaction specificity, to control the functional selectivity of G-protein heterotrimer in different cell and tissue-types or in response to different environmental conditions. Taken together, this research identifies a highly diverse G-protein signaling network known to date from B. rapa, and provides a clue about the possible complexity of G-protein signaling networks present across globally important Brassica species.     

Canonical and Noncanonical G-Protein Signaling Helps Coordinate Actin Dynamics To Promote Macrophage Phagocytosis of Zymosan

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gα_i nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gα_i proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca²⁺ that was partially sensitive to Gα_i nucleotide exchange inhibition and expression of GTP-bound Gα_i recruited Elmo1 to the plasma membrane. Reducing GDP-Gα_i nucleotide exchange, decreasing Gα_i expression, pharmacologically interrupting Gβγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gα_i remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.     

Self-activating G protein α subunits engage seven-transmembrane regulator of G protein signaling (RGS) proteins and a Rho guanine nucleotide exchange factor effector in the amoeba Naegleria fowleri

The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein–centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein–coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., “self-activating” behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα_12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.     

Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor

G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind Gα_i free of Gβγ providing an unusual scaffold for the “G-switch” and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3·Gα_i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα_i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of α₂-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gα_i1-YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by Gβγ sequestration with the carboxyl terminus of GRK2. Gα_i-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα_i and/or Gα_o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both Gαβγ and GPR-Gα_i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.     

Entamoeba histolytica Cysteine Proteinase 5 Binds Integrin on Colonic Cells and Stimulates NFκB-mediated Pro-inflammatory Responses

Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 10⁵ deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to α_Vβ₃ integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to α_Vβ₃ integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2^−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for α_Vβ₃ integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.     

Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.In all known eukaryotes, cellular signaling involves heterotrimeric GTP-binding proteins (G-proteins), which consist of Gα, Gβ, and Gγ subunits (Cabrera-Vera et al., 2003). According to the established paradigm, when Gα is GDP-bound, it forms a trimeric complex with the Gβγ dimer and remains associated with a G-protein coupled receptor. Signal perception by the receptor facilitates GDP to GTP exchange on Gα. GTP-Gα dissociates from the Gβγ dimer, and both these entities can transduce the signal by interacting with different effectors. The duration of the active state is determined by the intrinsic GTPase activity of Gα, which hydrolyzes bound GTP into GDP and inorganic phosphate (Pi), followed by the reassociation of the inactive, trimeric complex (Siderovski and Willard, 2005).In plants, G-protein signaling has been studied in only a few angiosperms to date at the functional level, although the proteins exist in the entire plant lineage (Hackenberg and Pandey, 2014; Urano and Jones, 2014; Hackenberg et al., 2016). Interestingly, while the overall biochemistry of the individual G-protein components and the interactions between them are conserved between plant and metazoan systems, deviations from the established norm are also obvious. For example, the repertoire of canonical G-proteins is significantly limited in plants; the human genome codes for 23 Gα, 5 Gβ, and 12 Gγ proteins, whereas most plant genomes, including those of basal plants, typically encode 1 canonical Gα, 1 Gβ, and three to five Gγ proteins (Urano and Jones, 2014). The only exceptions are some polyploid species, such as soybean, which have retained most of the duplicated G-protein genes (Bisht et al., 2011; Choudhury et al., 2011). Moreover, even in plants that possess only a single canonical Gα and Gβ protein, for example Arabidopsis (Arabidopsis thaliana) and rice, the phenotypes of plants lacking either one or both proteins are relatively subtle. The mutant plants exhibit multiple developmental and signaling defects but are able to complete the life cycle without any major consequences. These observations have questioned the significance of G-protein mediated signaling pathways in plants.Interestingly, plants also possess certain unique variants of the classical G-protein components such as the type III Cys-rich Gγ proteins and extra-large GTP-binding (XLG) proteins, which add to the diversity and expanse of the G-protein signaling networks (Roy Choudhury et al., 2011; Chakravorty et al., 2015; Maruta et al., 2015). The XLG proteins are almost twice the size of typical Gα proteins, with the C-terminal region that codes for Gα-like domain and an extended N-terminal region without any distinctive features. Plant XLGs are encoded by entirely independent genes and therefore are different from the mammalian extra-long versions of Gα proteins such as XLαs and XXLαs, which are expressed due to the use of alternate exons (Abramowitz et al., 2004). Three to five copies of XLG proteins are present in the genome of most angiosperms. At the functional level, the XLG proteins have been characterized only from Arabidopsis, to date, where recent studies suggest that the proteins compete with canonical Gα for binding with the Gβγ dimers and may form functional trimeric complexes (Chakravorty et al., 2015; Maruta et al., 2015). The XLG and Gβγ mutants of Arabidopsis seem to function in the same pathways during the regulation of a subset of plant responses, for example primary root length and its regulation by abscisic acid (ABA); the root waving and skewing responses; sensitivity to Glc, salt, and tunicamycin; and sensitivity to certain bacterial and fungal pathogens (Ding et al., 2008; Pandey et al., 2008; Chakravorty et al., 2015; Maruta et al., 2015). However, many of the phenotypes of Arabidopsis Gα and Gβγ mutants are also distinct from that of the xlg triple mutants. For example, compared to the wild-type plants, the canonical G-protein mutants exhibit altered response to gibberellic acid, brassinosteroids, and auxin and show changes in leaf shape, branching, flowering time, and stomatal densities (Ullah et al., 2003; Chen et al., 2004; Pandey et al., 2006; Zhang et al., 2008; Nilson and Assmann, 2010). The xlg triple mutants behave similarly to wild-type plants in all these aspects of development and signaling. Moreover, whether the XLG proteins are authentic GTP-binding and -hydrolyzing proteins and the extent to which they directly participate in G-protein-mediated signaling pathways remains confounding (Chakravorty et al., 2015; Maruta et al., 2015). Even in plants with a limited number of G-protein subunits such as Arabidopsis, one Gα and three XLGs potentially compete for a single Gβ protein, and the analysis of null mutants is not straightforward, that is, it is not possible to delineate whether the phenotypes seen in the Gα null mutants are truly due to the lack of Gα and/or because of an altered stoichiometry or availability of Gβ for the XLG proteins.As a bryophyte, Physcomitrella patens occupies a unique position in the evolutionary history of plants. It lacks vasculature but exhibits alteration between generations, which is dominated by a gametophytic (haploid) phase and a short sporophytic (diploid) phase (Cove et al., 2009). Many of the pathways related to hormone signaling, stress responses, and development are conserved between angiosperms and P. patens (Cove et al., 2009; Sun, 2011; Komatsu et al., 2013; Yasumura et al., 2015). It is also an intriguing example in the context of the G-protein signaling, because its fully sequenced genome does not encode a canonical Gα gene, although genes coding for the Gβ and Gγ proteins exist. A single gene for a potential XLG homolog also exists in the P. patens genome. This unique assortment of proteins predicts several alternative scenarios for G-protein signaling in P. patens. For example, the P. patens Gβγ proteins might be nonfunctional due to the loss of canonical Gα and are left in the genome as evolutionary artifacts. Alternatively, the Gβγ proteins of P. patens might maintain functionality regardless of the existence of a canonical Gα protein in pathways not regulated via classic G-protein signaling modes. Finally, a more likely scenario could be that the potential XLG protein can substitute for the Gα function in P. patens.To explore these possibilities and understand better the conserved and unique mechanisms of G-protein signaling pathways in plants and their significance, we examined the role of G-protein subunits in P. patens. We provide unambiguous evidence for the genetic coupling of XLG and Gβ proteins in controlling P. patens development. In contrast to all other plant species analyzed to date, where G-proteins are not essential for growth and survival, the XLG or one of the Gβ proteins is required for the sporophyte formation and life cycle completion in P. patens. Furthermore, one of the Arabidopsis XLG proteins, XLG2, and the canonical Gβ protein AGB1 can functionally complement the P. patens mutant phenotypes. These data provide new insights in the evolutionary breadth and the spectrum of signaling pathways regulated by G-proteins in plants.     

A sweet cycle for Arabidopsis G-proteins: Recent discoveries and controversies in plant G-protein signal transduction

Heterotrimeric G-proteins are a class of signal transduction proteins highly conserved throughout evolution that serve as dynamic molecular switches regulating the intracellular communication initiated by extracellular signals including sensory information. This property is achieved by a guanine nucleotide cycle wherein the inactive, signaling-incompetent Gα subunit is normally bound to GDP; activation to signaling-competent Gα occurs through the exchange of GDP for GTP (typically catalyzed via seven-transmembrane domain G-protein coupled receptors [GPCRs]), which dissociates the Gβγ dimer from Gα-GTP and initiates signal transduction. The hydrolysis of GTP, greatly accelerated by “Regulator of G-protein Signaling” (RGS) proteins, returns Gα to its inactive GDP-bound form and terminates signaling. Through extensive characterization of mammalian Gα isoforms, the rate-limiting step in this cycle is currently considered to be the GDP/GTP exchange rate, which can be orders of magnitude slower than the GTP hydrolysis rate. However, we have recently demonstrated that, in Arabidopsis, the guanine nucleotide cycle appears to be limited by the rate of GTP hydrolysis rather than nucleotide exchange. This finding has important implications for the mechanism of sugar sensing in Arabidopsis. We also discuss these data on Arabidopsis G-protein nucleotide cycling in relation to recent reports of putative plant GPCRs and heterotrimeric G-protein effectors in Arabidopsis.Key words: Arabidopsis, glucose, G-protein, nucleotide exchange, RGS protein     

A New Generation of FRET Sensors for Robust Measurement of Gαi1, Gαi2 and Gαi3 Activation Kinetics in Single Cells

G-protein coupled receptors (GPCRs) can activate a heterotrimeric G-protein complex with subsecond kinetics. Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are ideally suited for the study of such fast signaling events in single living cells. Here we report on the construction and characterization of three FRET biosensors for the measurement of Gα_i1, Gα_i2 and Gα_i3 activation. To enable quantitative long-term imaging of FRET biosensors with high dynamic range, fluorescent proteins with enhanced photophysical properties are required. Therefore, we use the currently brightest and most photostable CFP variant, mTurquoise2, as donor fused to Gα_i subunit, and cp173Venus fused to the Gγ₂ subunit as acceptor. The Gα_i FRET biosensors constructs are expressed together with Gβ₁ from a single plasmid, providing preferred relative expression levels with reduced variation in mammalian cells. The Gα_i FRET sensors showed a robust response to activation of endogenous or over-expressed alpha-2A-adrenergic receptors, which was inhibited by pertussis toxin. Moreover, we observed activation of the Gα_i FRET sensor in single cells upon stimulation of several GPCRs, including the LPA₂, M₃ and BK₂ receptor. Furthermore, we show that the sensors are well suited to extract kinetic parameters from fast measurements in the millisecond time range. This new generation of FRET biosensors for Gα_i1, Gα_i2 and Gα_i3 activation will be valuable for live-cell measurements that probe Gα_i activation.     

Function of α subunit of heterotrimeric G protein in brassinosteroid response of rice plants

The α subunit of heterotrimeric G-proteins (Gα) is involved in a broad range of aspects of the brassinosteroid (BR) response, such as the enhancement of lamina bending. However, it has been suggested from epistatic analysis of d1 and d61, which are mutants deficient for Gα and the BR receptor BRI1, that Gα and BRI1 may function via distinct pathways in many cases. In this study, we investigated further the genetic interaction between Gα and BRI1. We report the analysis of transformants of T65d1 and T65d1/d61-7 into which were introduced a constitutively active form of Gα, Q223L. The application of 24-epi-brassinolide (24-epiBL) to T65d1 expressing Q223L still resulted in elongation of the coleoptile and, in fact, it was enhanced over the wild-type plant (WT) level in a concentration dependent manner. In T65d1/d61-7 expressing Q223L, the seed size was enlarged over that of d61-7 due to activation of Gα. These results suggest that Q223L is able to augment the BR response in response to 24-epiBL and also that Q223L functions independently of BRI1 in the process of determining seed morphology, given that Q223L was functional in the BRI1-deficient mutant, d61-7.Key words: brassinosteroid, BRASSINOSTEROID INSENSITIVE1 (BRI1), genetic interaction, G-protein α subunit, rice plants, seed morphology, transgenic plants     

Ste18p Is a Positive Control Element in the Mating Process of Candida albicans

Heterotrimeric G proteins are an important class of eukaryotic signaling molecules that have been identified as central elements in the pheromone response pathways of many fungi. In the fungal pathogen Candida albicans, the STE18 gene (ORF19.6551.1) encodes a potential γ subunit of a heterotrimeric G protein; this protein contains the C-terminal CAAX box characteristic of γ subunits and has sequence similarity to γ subunits implicated in the mating pathways of a variety of fungi. Disruption of this gene was shown to cause sterility of MTLa mating cells and to block pheromone-induced gene expression and shmoo formation; deletion of just the CAAX box residues is sufficient to inactivate Ste18 function in the mating process. Intriguingly, ectopic expression behind the strong ACT1 promoter of either the Gα or the Gβ subunit of the heterotrimeric G protein is able to suppress the mating defect caused by deletion of the Gγ subunit and restore both pheromone-induced gene expression and morphology changes.     

Translocation of Activator of G-protein Signaling 3 to the Golgi Apparatus in Response to Receptor Activation and Its Effect on the trans-Golgi Network

Group II activators of G-protein signaling play diverse functional roles through their interaction with Gα_i, Gα_t, and Gα_o via a G-protein regulatory (GPR) motif that serves as a docking site for Gα-GDP. We recently reported the regulation of the AGS3-Gα_i signaling module by a cell surface, seven-transmembrane receptor. Upon receptor activation, AGS3 reversibly dissociates from the cell cortex, suggesting that it may function as a signal transducer with downstream signaling implications, and this question is addressed in the current report. In HEK-293 and COS-7 cells expressing the α_2A/D-AR and Gα_i3, receptor activation resulted in the translocation of endogenous AGS3 and AGS3-GFP from the cell cortex to a juxtanuclear region, where it co-localized with markers of the Golgi apparatus (GA). The agonist-induced translocation of AGS3 was reversed by the α₂-AR antagonist rauwolscine. The TPR domain of AGS3 was required for agonist-induced translocation of AGS3 from the cell cortex to the GA, and the translocation was blocked by pertussis toxin pretreatment or by the phospholipase Cβ inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. Agonist-induced translocation of AGS3 to the GA altered the functional organization and protein sorting at the trans-Golgi network. The regulated movement of AGS3 between the cell cortex and the GA offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the trans-Golgi network.     

Effector Contributions to Gβγ-mediated Signaling as Revealed by Muscarinic Potassium Channel Gating

Receptor-mediated activation of heterotrimeric G proteins leading to dissociation of the Gα subunit from Gβγ is a highly conserved signaling strategy used by numerous extracellular stimuli. Although Gβγ subunits regulate a variety of effectors, including kinases, cyclases, phospholipases, and ion channels (Clapham, D.E., and E.J. Neer. 1993. Nature (Lond.). 365:403–406), few tools exist for probing instantaneous Gβγ-effector interactions, and little is known about the kinetic contributions of effectors to the signaling process. In this study, we used the atrial muscarinic K⁺ channel, which is activated by direct interactions with Gβγ subunits (Logothetis, D.E., Y. Kurachi, J. Galper, E.J. Neer, and D.E. Clap. 1987. Nature (Lond.). 325:321–326; Wickman, K., J.A. Iniguez-Liuhi, P.A. Davenport, R. Taussig, G.B. Krapivinsky, M.E. Linder, A.G. Gilman, and D.E. Clapham. 1994. Nature (Lond.). 366: 654–663; Huang, C.-L., P.A. Slesinger, P.J. Casey, Y.N. Jan, and L.Y. Jan. 1995. Neuron. 15:1133–1143), as a sensitive reporter of the dynamics of Gβγ-effector interactions. Muscarinic K⁺ channels exhibit bursting behavior upon G protein activation, shifting between three distinct functional modes, characterized by the frequency of channel openings during individual bursts. Acetylcholine concentration (and by inference, the concentration of activated Gβγ) controls the fraction of time spent in each mode without changing either the burst duration or channel gating within individual modes. The picture which emerges is of a Gβγ effector with allosteric regulation and an intrinsic “off” switch which serves to limit its own activation. These two features combine to establish exquisite channel sensitivity to changes in Gβγ concentration, and may be indicative of the factors regulating other Gβγ-modulated effectors.     

Competing Activities of Heterotrimeric G Proteins in Drosophila Wing Maturation

Drosophila genome encodes six alpha-subunits of heterotrimeric G proteins. The Gαs alpha-subunit is involved in the post-eclosion wing maturation, which consists of the epithelial-mesenchymal transition and cell death, accompanied by unfolding of the pupal wing into the firm adult flight organ. Here we show that another alpha-subunit Gαo can specifically antagonize the Gαs activities by competing for the Gβ13F/Gγ1 subunits of the heterotrimeric Gs protein complex. Loss of Gβ13F, Gγ1, or Gαs, but not any other G protein subunit, results in prevention of post-eclosion cell death and failure of the wing expansion. However, cell death prevention alone is not sufficient to induce the expansion defect, suggesting that the failure of epithelial-mesenchymal transition is key to the folded wing phenotypes. Overactivation of Gαs with cholera toxin mimics expression of constitutively activated Gαs and promotes wing blistering due to precocious cell death. In contrast, co-overexpression of Gβ13F and Gγ1 does not produce wing blistering, revealing the passive role of the Gβγ in the Gαs-mediated activation of apoptosis, but hinting at the possible function of Gβγ in the epithelial-mesenchymal transition. Our results provide a comprehensive functional analysis of the heterotrimeric G protein proteome in the late stages of Drosophila wing development.     

Signaling Specificity Provided by the Arabidopsis thaliana Heterotrimeric G-Protein γ Subunits AGG1 and AGG2 Is Partially but Not Exclusively Provided through Transcriptional Regulation

The heterotrimeric G-protein complex in Arabidopsis thaliana consists of one α, one ß and three γ subunits. While two of the γ subunits, AGG1 and AGG2 have been shown to provide functional selectivity to the Gßγ dimer in Arabidopsis, it is unclear if such selectivity is embedded in their molecular structures or conferred by the different expression patterns observed in both subunits. In order to study the molecular basis for such selectivity we tested genetic complementation of AGG1- and AGG2 driven by the respectively swapped gene promoters. When expressed in the same tissues as AGG1, AGG2 rescues some agg1 mutant phenotypes such as the hypersensitivity to Fusarium oxysporum and D-mannitol as well as the altered levels of lateral roots, but does not rescue the early flowering phenotype. Similarly, AGG1 when expressed in the same tissues as AGG2 rescues the osmotic stress and lateral-root phenotypes observed in agg2 mutants but failed to rescue the heat-stress induction of flowering. The fact that AGG1 and AGG2 are functionally interchangeable in some pathways implies that, at least for those pathways, signaling specificity resides in the distinctive spatiotemporal expression patterns exhibited by each γ subunit. On the other hand, the lack of complementation for some phenotypes indicates that there are pathways in which signaling specificity is provided by differences in the primary AGG1 and AGG2 amino acid sequences.     

The RGS proteins add to the diversity of soybean heterotrimeric G-protein signaling

Regulator of G-protein signaling (RGS) proteins are a family of highly diverse, multifunctional proteins that function primarily as GTPase accelerating proteins (GAPs). RGS proteins increase the rate of GTP hydrolysis by Gα proteins and essentially regulate the duration of active signaling. Recently, we have identified two chimeric RGS proteins from soybean and reported their distinct GAP activities on individual Gα proteins. A single amino acid substitution (Alanine 357 to Valine) of RGS2 is responsible for differential GAP activity. Surprisingly, most monocot plant genomes do not encode for a RGS protein homolog. Here we discuss the soybean RGS proteins in the context of their evolution in plants, their relatedness to non-plant RGS protein homologs and the effect they might have on the heterotrimeric G-protein signaling mechanisms. We also provide experimental evidence to show that the interaction interface between plant RGS and Gα proteins is different from what is predicted based on mammalian models.