Showing papers on "GTP-Binding Protein alpha Subunits published in 2007"

GTPase acceleration as the rate-limiting step in Arabidopsis G protein-coupled sugar signaling

Abstract: Heterotrimeric G protein signaling is important for cell-proliferative and glucose-sensing signal transduction pathways in the model plant organism Arabidopsis thaliana. AtRGS1 is a seven-transmembrane, RGS domain-containing protein that is a putative membrane receptor for d-glucose. Here we show, by using FRET, that d-glucose alters the interaction between the AtGPA1 and AtRGS1 in vivo. AtGPA1 is a unique heterotrimeric G protein α subunit that is constitutively GTP-bound given its high spontaneous nucleotide exchange coupled with slow GTP hydrolysis. Analysis of a point mutation in AtRGS1 that abrogates GTPase-accelerating activity demonstrates that the regulation of AtGPA1 GTP hydrolysis mediates sugar signal transduction during Arabidopsis development, in contrast to animals where nucleotide exchange is the limiting step in the heterotrimeric G protein nucleotide cycle.

Rgs1 regulates multiple Gα subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism

Abstract: Regulators of G-protein signaling (RGS proteins) negatively regulate heterotrimeric G-protein cascades that enable eukaryotic cells to perceive and respond to external stimuli. The rice-blast fungus Magnaporthe grisea forms specialized infection structures called appressoria in response to inductive surface cues. We isolated Magnaporthe RGS1 in a screen for mutants that form precocious appressoria on non-inductive surfaces. We report that a thigmotropic cue is necessary for initiating appressoria and for accumulating cAMP. Similar to an RGS1-deletion strain, magAG187S (RGS-insensitive Gαs) and magAQ208L (GTPase-dead) mutants accumulated excessive cAMP and elaborated appressoria on non-inductive surfaces, suggesting that Rgs1 regulates MagA during pathogenesis. Rgs1 was also found to negatively regulate the Gαi subunit MagB during asexual development. Deficiency of MAGB suppressed the hyper-conidiation defect in RGS1-deletion strain, whereas magBG183S and magBQ204L mutants produced more conidia, similar to the RGS1-deletion strain. Rgs1 physically interacted with GDP·AlF4−-activated forms of MagA, MagB and MagC (a GαII subunit). Thus, Rgs1 serves as a negative regulator of all Gα subunits in Magnaporthe and controls important developmental events during asexual and pathogenic development.

Direct Interactions with Gαi and Gβγ Mediate Nongenomic Signaling by Estrogen Receptor α

Abstract: Estrogen induces G protein-dependent nongenomic signaling in a variety of cell types via the activation of a plasma membrane-associated subpopulation of estrogen receptor alpha (ER alpha). Using pull-down experiments with purified recombinant proteins, we now demonstrate that ER alpha binds directly to G alpha i and G betagamma. Mutagenesis and the addition of blocking peptide reveals that this occurs via amino acids 251-260 and 271-595 of ER alpha, respectively. Studies of ER alpha complexed with heterotrimeric G proteins further show that estradiol causes the release of both G alpha i and G betagamma without stimulating GTP binding to G alpha i. Moreover, in COS-7 cells, the disruption of ER alpha-G alpha i interaction by deletion mutagenesis of ER alpha or expression of blocking peptide, as well as G betagamma sequestration with beta-adrenergic receptor kinase C terminus, prevents nongenomic responses to estradiol including src and erk activation. In endothelial cells, the disruption of ER alpha-G alpha i interaction prevents estradiol-induced nitric oxide synthase activation and the resulting attenuation of monocyte adhesion that contributes to estrogen-related cardiovascular protection. Thus, through direct interactions, ER alpha mediates a novel mechanism of G protein activation that provides greater diversity of function of both the steroid hormone receptor and G proteins.

An obligatory requirement for the heterotrimeric G protein Gi3 in the antiautophagic action of insulin in the liver.

Abstract: Heterotrimeric G proteins of the Gi class have been implicated in signaling pathways regulating growth and metabolism under physiological and pathophysiological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed Gαi class genes, Gαi2 and Gαi3, demonstrate shared as well as gene-specific functions. The presence of a single active allele of Gαi3 is sufficient for embryonic development, whereas at least one allele of Gαi2 is required for extrauterine life. Mice lacking both Gαi2 and Gαi3 are massively growth-retarded and die in utero. We have used biochemical and cell biological methods together with in situ liver perfusion experiments to study Gαi isoform-specific functions in Gαi2- and Gαi3-deficient mice. The subcellular localization of Gαi3 in isolated mouse hepatocytes depends on the cellular metabolic status. Gαi3 localizes to autophagosomes upon starvation-induced autophagy and distributes to the plasma membrane upon insulin stimulation. Analysis of autophagic proteolysis in perfused mouse livers showed that mice lacking Gαi3 are deficient in the inhibitory action of insulin. These data indicate that Gαi3 is crucial for the antiautophagic action of insulin and suggest an as-yet-unrecognized function for Gαi3 on autophagosomal membranes.

G protein signaling governing cell fate decisions involves opposing Gα subunits in Cryptococcus neoformans

Abstract: Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Gα subunits, most filamentous ascomycetes and basidiomycetes have three Gα subunits. Previous studies have defined the Gα subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Gα subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Gα subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Gα into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development.

Mechanisms of dominant negative G-protein α subunits

Abstract: G-protein-coupled receptors (GPCRs) represent the largest class of membrane proteins and are the targets of 25-50% of drugs currently on the market. Dominant negative mutant Galpha subunits of heterotrimeric G-proteins have been extensively utilized to delineate G-protein signaling pathways and represent a promising new tool to study GPCR-dependent signaling in the CNS. There are different regions in various types of Galpha subunits in which mutations can give rise to a dominant negative phenotype. Such a mutant Galpha would compete with wild-type Galpha for binding to other proteins involved in the G-protein cycle and either block or reduce the response caused by wild-type Galpha. To date, there are three different mechanisms described for dominant negative Galpha subunits: sequestration of the Gbetagamma subunits, sequestration of the activated GPCR by the heterotrimeric complex, and sequestration of the activated GPCR by nucleotide-free Galpha. This review focuses on the development of dominant negative Galpha subunits, the different mechanisms used by various mutant Galpha subunits, and potential structural changes underlying the dominant negative effects.

The G-protein coupling properties of the human sweet and amino acid taste receptors.

Abstract: The human T1R taste receptors are family C G-protein-coupled receptors (GPCRs) that act as heterodimers to mediate sweet (hT1R2 + hT1R3) and umami (hT1R1 + hT1R3) taste modalities. Each T1R has a large extracellular ligand-binding domain linked to a seven transmembrane-spanning core domain (7TMD). We demonstrate that the 7TMDs of hT1R1 and hT1R2 display robust ligand-independent constitutive activity, efficiently catalyzing the exchange of GDP for GTP on Galpha subunits. In contrast, relative to the 7TMDs of hT1R1 and hT1R2, the 7TMD of hT1R3 couples poorly to G-proteins, suggesting that in vivo signaling may proceed primarily through hT1R1 and hT1R2. In addition, we provide direct evidence that the hT1Rs selectively signal through Galpha(i/o) pathways, coupling to multiple Galpha(i/o) subunits as well as the taste cell specific Gbeta(1)gamma(13) dimer.

Gα–Gβγ dissociation may be due to retraction of a buried lysine and disruption of an aromatic cluster by a GTP-sensing Arg–Trp pair

Abstract: The heterotrimeric G protein α subunit (Gα) functions as a molecular switch by cycling between inactive GDP-bound and active GTP-bound states. When bound to GDP, Gα interacts with high affinity to a complex of the β and γ subunits (Gβγ), but when bound to GTP, Gα dissociates from this complex to activate downstream signaling pathways. Gα's state is communicated to other cellular components via conformational changes within its switch I and II regions. To identify key determinants of Gα's function as a signaling pathway molecular switch, a Bayesian approach was used to infer the selective constraints that most distinguish Gα and closely related Arf family GTPases from distantly related translational and metabolic GTPases. The strongest of these constraints are imposed on seven residues within or near the switch II region. Likewise, constraints imposed on Gα but not on other, closely related molecular switches correspond to four nearby residues. These constraints are explained by a proposed mechanism for GTP-induced dissociation of Gα from Gβγ where an Arg–Trp pair senses the presence of bound GTP leading to conformational retraction of a nearby lysine and to disruption of an aromatic cluster. Within a complex of Giα, Giβγ, and GDP, this lysine establishes greater surface contact with Giβ than does any other residue in Giα, whereas the aromatic cluster packs against a highly conserved tryptophan in Giβ that establishes greater surface contact with Giα than does any other residue in Giβ. Other structural features associated with Gα functional divergence further support the proposed mechanism.

Gα12/13 Basally Regulates p53 through Mdm4 Expression

Abstract: G alpha(12/13), which belongs to the G alpha(12) family, participates in the regulation of diverse physiologic processes. In view of the control of G alpha(12/13) in cell proliferation, this study investigated the role of G alpha(12/13) in the regulation of p53 and mdm4. Immunoblotting and immunocytochemistry revealed that p53 was expressed in control embryonic fibroblasts and was largely localized in the nuclei. G alpha(12) deficiency decreased p53 levels and its DNA binding activity, accompanying p21 repression with Bcl(2) induction, whereas G alpha(13) deficiency exerted weak effects. G alpha(12) or G alpha(13) deficiency did not change p53 mRNA expression. ERK1/2 or Akt was not responsible for p53 repression due to G alpha(12) deficiency. Mdm4, a p53-stabilizing protein, was repressed by G alpha(12) deficiency and to a lesser extent by G alpha(13) deficiency, whereas mdm2, PTEN, beta-catenin, ATM, and Chk2 were unaffected. p53 accumulation by proteasomal inhibition during G alpha(12) deficiency suggested the role of G alpha(12) in p53 stabilization. Constitutively active G alpha(12) (G alpha(12)QL) or G alpha(13) (G alpha(13)QL) promoted p53 accumulation with mdm4 induction in MCF10A cells. p53 accumulation by mdm4 overexpression, but no mdm4 induction by p53 overexpression, and small interfering RNA knockdown verified the regulatory role of mdm4 for p53 downstream of G alpha(12/13). In control or G alpha(12)/G alpha(13)-deficient cells, genotoxic stress led to p53 accumulation. At concentrations increasing the flow cytometric pre-G(1) phase, doxorubicin or etoposide treatment caused serine phosphorylations in G alpha(12)-/- or G alpha(12/13)-/- cells, but did not induce mdm4. G alpha(12/13)QL transfection failed to phosphorylate p53 at serines. Our results indicate that G alpha(12/13) regulate basal p53 levels via mdm4, which constitutes a cell signaling pathway distinct from p53 phosphorylations elicited by genotoxic stress.

An inhibitory compound against the interaction between G alpha(s) and the third intracellular loop region of serotonin receptor subtype 6 (5-HT6) disrupts the signaling pathway of 5-HT6.

Abstract: Serotonin receptor subtype 6 (5-HT(6)) is a neurotransmitter receptor, which is involved in various brain functions such as memory and mood. It mediates signaling via the interaction with a stimulatory G-protein. Especially, the third intracellular loop (iL3) of 5-HT(6) and the alpha subunit of stimulatory G protein (G alpha(s)) are responsible for the signaling process of 5-HT(6). Chemical compounds that could inhibit the interaction between the iL3 region of 5-HT(6) and G alpha(s) were screened from a chemical library consisted of 5,600 synthetic compounds. One of the identified compounds bound to G alpha(s) and effectively blocked the interaction between G alpha(s) and the iL3 region of 5-HT(6). The identified compound was further shown to reduce the serotonin-induced accumulation of cAMP in 293T cells transformed with 5-HT(6) cDNA. It also lowered the Ca(2+) efflux induced by serotonin in cells expressing 5-HT(6) and chimeric G alpha(s5/q). These results indicate that the interaction between the iL3 of 5-HT(6) and G alpha(s) can be exploited for screening of regulatory compounds against the signaling pathway of 5-HT(6).