Showing papers on "GTP-Binding Protein alpha Subunits published in 2000"

Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes

Abstract: Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.

Gq protein alpha subunits Galphaq and Galpha11 are localized at postsynaptic extra-junctional membrane of cerebellar Purkinje cells and hippocampal pyramidal cells.

Abstract: Following cell surface receptor activation, the alpha subunit of the Gq subclass of GTP-binding proteins activates the phosphoinositide signalling pathway. Here we examined the expression and localization of Gq protein alpha subunits in the adult mouse brain by in situ hybridization and immunohistochemistry. Of the four members of the Gq protein alpha subunits, Galphaq and Galpha11 were transcribed predominantly in the brain. The highest transcriptional level of Galphaq was observed in cerebellar Purkinje cells (PCs) and hippocampal pyramidal cells, while that of Galpha11 was noted in hippocampal pyramidal cells. Antibody against the C-terminal peptide common to Galphaq and Galpha11 strongly labelled the cerebellar molecular layer and hippocampal neuropil layers. In these regions, immunogold preferentially labelled the cytoplasmic face of postsynaptic cell membrane of PCs and pyramidal cells. Immunoparticles were distributed along the extra-junctional cell membrane of spines, dendrites and somata, but were almost excluded from the junctional membrane. By double immunofluorescence, Galphaq/Galpha11 was extensively colocalized with metabotropic glutamate receptor mGluR1alpha in dendritic spines of PCs and with mGluR5 in those of hippocampal pyramidal cells. Together with concentrated localization of mGluR1alpha and mGluR5 in a peri-junctional annulus on PC and pyramidal cell synapses (Baude et al. 1993, Neuron, 11, 771-787; Lujan et al. 1996, Eur. J. Neurosci., 8, 1488-1500), the present molecular-anatomical findings suggest that peri-junctional stimulation of the group I metabotropic glutamate receptors is mediated by Galphaq and/or Galpha11, leading to the activation of the intracellular effector, phospholipase Cbeta.

Signal transduction by a nondissociable heterotrimeric yeast G protein.

Abstract: Many signal transduction pathways involve heterotrimeric G proteins. The accepted model for activation of heterotrimeric G proteins states that the protein dissociates to the free Gα (GTP)-bound subunit and free Gβγ dimer. On GTP hydrolysis, Gα (GDP) then reassociates with Gβγ [Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615–649]. We reexamined this hypothesis, by using the mating G protein of the yeast Saccharomyces cerevisiae encoded by the genes GPA1, STE4, and STE18. In the absence of mating pheromone, the Gα (Gpa1) subunit represses the mating pathway. On activation by binding of pheromone to a serpentine receptor, the Gβγ (Ste4, Ste18) dimer transmits the signal to a mitogen-activated protein kinase cascade, leading to gene activation, arrest in the G1 stage of the cell cycle, production of shmoos (mating projections), and cell fusion. We found that a Ste4-Gpa1 fusion protein transmitted the pheromone signal and activated the mating pathway as effectively as when Ste4 (Gβ) and Gpa1 (Gα) were coexpressed as separate proteins. Hence, dissociation of this G protein is not required for its activation. Rather, a conformational change in the heterotrimeric complex is likely to be involved in signal transduction.

Dual Lipid Modification Motifs in Gα and Gγ Subunits Are Required for Full Activity of the Pheromone Response Pathway in Saccharomyces cerevisiae

Abstract: To establish the biological function of thioacylation (palmitoylation), we have studied the heterotrimeric guanine nucleotide-binding protein (G protein) subunits of the pheromone response pathway of Saccharomyces cerevisiae. The yeast G protein gamma subunit (Ste18p) is unusual among G(gamma) subunits because it is farnesylated at cysteine 107 and has the potential to be thioacylated at cysteine 106. Substitution of either cysteine results in a strong signaling defect. In this study, we found that Ste18p is thioacylated at cysteine 106, which depended on prenylation of cysteine 107. Ste18p was targeted to the plasma membrane even in the absence of prenylation or thioacylation. However, G protein activation released prenylation- or thioacylation-defective Ste18p into the cytoplasm. Hence, lipid modifications of the G(gamma) subunit are dispensable for G protein activation by receptor, but they are required to maintain the plasma membrane association of G(betagamma) after receptor-stimulated release from G(alpha). The G protein alpha subunit (Gpa1p) is tandemly modified at its N terminus with amide- and thioester-linked fatty acids. Here we show that Gpa1p was thioacylated in vivo with a mixture of radioactive myristate and palmitate. Mutation of the thioacylation site in Gpa1p resulted in yeast cells that displayed partial activation of the pathway in the absence of pheromone. Thus, dual lipidation motifs on Gpa1p and Ste18p are required for a fully functional pheromone response pathway.

Regulation of NHE3 activity by G protein subunits in renal brush-border membranes.

Abstract: NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na+/H+ exchanger (NHE) can also be regulated by G proteins independent...

Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits.

Abstract: Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.

G protein beta 5 subunit interactions with alpha subunits and effectors.

Abstract: When the beta(5) (short form) and gamma(2) subunits of heterotrimeric G proteins were expressed with hexahistidine-tagged alpha(i) in insect cells, a heterotrimeric complex was formed that bound to a Ni-NTA-agarose affinity matrix. Binding to the Ni-NTA-agarose column was dependent on expression of hexahistidine-tagged alpha(i) and resulted in purification of beta(5)gamma(2) to near homogeneity. Subsequent anion-exchange chromatography of beta(5)gamma(2) resulted in resolution of beta(5) from gamma(2) and further purification of beta(5). The purified beta(5) eluted as a monomer from a size-exclusion column and was resistant to trypsin digestion suggesting that it was stably folded in the absence of gamma. beta(5) monomer could be assembled with partially purified hexahistidine-tagged gamma(2) in vitro to form a functional dimer that could selectively activate PLC beta2 but not PLC beta3. alpha(o)-GDP inhibited activation of PLC beta2 by beta(5)gamma(2) supporting the idea that beta(5)gamma(2) can bind to alpha(o). beta(5) monomer and beta(5)gamma(2) only supported a small degree of ADP ribosylation of alpha(i) by pertussis toxin (PTX), but beta(5) monomer was able to compete for beta(1)gamma(2) binding to alpha(i) and alpha(o) to inhibit PTX-catalyzed ADP ribosylation. These data indicate that beta(5) functionally interacts with PTX-sensitive GDP alpha subunits and that beta(5) subunits can be assembled with gamma subunits in vitro to reconstitute activity and also support the idea that there are determinants on beta subunits that are selective for even very closely related effectors.

Nicotiana tabacum cDNAs encoding α and β subunits of a heterotrimeric GTP-binding protein isolated from hairy root tissues

Abstract: Heterotrimeric GTP-binding proteins (G-proteins) play important roles in signal transduction pathways in eukaryotic cells. Through differential screening of a hairy root cDNA library of tobacco (Nicotiana tabacum L.) against transcripts from non-root tissues of normal cuttings, we obtained a partial cDNA clone that showed abundant expression and high homology to the alpha subunit gene of plant G-protein. After RACE-PCR, a full-length cDNA clone was obtained, which was 1,677-bp in length and contained an open reading frame encoding a protein of 384 amino acids. A cDNA clone encoding a beta subunit of G-protein was also isolated from the same cDNA library based on PCR amplification and library screening. The clone was 1,600-bp in length and contained an open reading frame encoding 377 amino acids. The deduced amino acid sequences of these clones showed high homology (75.5 to 99.8% amino acid identity) with alpha and beta subunits of other plant G-proteins. Genomic Southern blot analysis showed that the amphidiploid tobacco genome possessed two major copies of both alpha and beta subunit genes and some minor homologous copies. Northern blot analysis showed that the transcript of alpha subunit gene was abundant in the root tissues, particularly in the hairy root tissues. In contrast, the level of expression of the beta subunit gene was equivalent in all the tissues studied. Possible function of tobacco G-protein was discussed.

The GTP hydrolysis defect of the Saccharomyces cerevisiae mutant G-protein Gpa1(G50V).

Abstract: The Saccharomyces cerevisiae haploid cell response to pheromone involves two seven-transmembrane-domain pheromone receptors that couple to a heterotrimeric G protein. The G50V mutation in the G protein alpha subunit (G(alpha)), Gpa1p, is analogous to the p21(ras) transforming mutation Gly-->Val 12, and has been extensively examined for the phenotypes it produces in yeast cells. Here we have characterized the Gpa1(G50V) mutant protein in vitro by examining GTPgammaS binding, GDP exchange, GTP occupancy and guanosine triphosphatase (GTPase) activity. Compared to wild-type (WT) Gpa1p, Gpa1(G50V)p was found to have a moderately reduced GTPase activity and increased GTP occupancy, while GTPgammaS binding and GDP exchange were not significantly altered. The yeast regulator of G protein Signalling (RGS) protein, Sst2p, was also expressed and purified, and found to have a significantly reduced ability to stimulate the initial rate of GTP hydrolysis of Gpa1(G50V)p compared to its effect on WT Gpa1p. Probing conformational transitions by a protease sensitivity assay suggested that Gpa1(G50V)p did not bind the transition state mimetic GDP/AlF(4)(-) as efficiently as the WT Gpa1p. These biochemical results can explain many of the known gpa1(G50V) yeast cell phenotypes.