Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.

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Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase.

The Journal of Cell Biology ◽

10.1083/jcb.121.3.579 ◽

1993 ◽

Vol 121 (3) ◽

pp. 579-586 ◽

Cited By ~ 155

Author(s):

R W Mercer ◽

D Biemesderfer ◽

D P Bliss ◽

J H Collins ◽

B Forbush

Keyword(s):

Northern Blot Analysis ◽

Beta Subunits ◽

Gamma Subunit ◽

Hydrophobic Domain ◽

Subunit Mrna ◽

Nephron Segments ◽

Gamma Subunits ◽

Specific Association ◽

Hydropathy Analysis

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.

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Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein.

The Journal of Cell Biology ◽

10.1083/jcb.121.4.775 ◽

1993 ◽

Vol 121 (4) ◽

pp. 775-783 ◽

Cited By ~ 68

Author(s):

L A Jaffe ◽

C J Gallo ◽

R H Lee ◽

Y K Ho ◽

T L Jones

Keyword(s):

G Protein ◽

G Proteins ◽

Oocyte Maturation ◽

Alpha Subunit ◽

Meiotic Maturation ◽

Amino Terminus ◽

Hormone Response ◽

Gamma Subunit ◽

Gamma Subunits ◽

Stimulation Of

The stimulation of meiotic maturation of starfish oocytes by the hormone 1-methyladenine is mimicked by injection of beta gamma subunits of G-proteins from either retina or brain. Conversely, the hormone response is inhibited by injection of the GDP-bound forms of alpha i1 or alpha t subunits, or by injection of phosducin; all of these proteins should bind free beta gamma. alpha-subunit forms with reduced affinity for beta gamma (alpha i1 or alpha t bound to hydrolysis-resistant GTP analogs, or alpha i1-GMPPCP treated with trypsin to remove the amino terminus of the protein) are less effective inhibitors of 1-methyladenine action. These results indicate that the beta gamma subunit of a G-protein mediates 1-methyladenine stimulation of oocyte maturation.

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Isolation and characterization of a genomic sequence encoding the maizeCat3 catalase gene

Plant Molecular Biology ◽

10.1007/bf00028975 ◽

1993 ◽

Vol 22 (6) ◽

pp. 1031-1038 ◽

Cited By ~ 16

Author(s):

Michael L. Abler ◽

John G. Scandalios

Keyword(s):

Genomic Sequence ◽

Catalase Gene ◽

Isolation And Characterization ◽

Sequence Encoding

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G Protein Signaling in Plants: Characterization of Alpha and Gamma Subunits

Biophysical Journal ◽

10.1016/j.bpj.2016.11.1052 ◽

2017 ◽

Vol 112 (3) ◽

pp. 189a-190a

Author(s):

Zehra Sayers ◽

Bihter Avsar ◽

Ines Karmous ◽

Ersoy Cholak

Keyword(s):

G Protein ◽

G Protein Signaling ◽

Protein Signaling ◽

Gamma Subunits

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Localization of G alpha 0 to growth cones in PC12 cells: role of G alpha 0 association with receptors and G beta gamma

Journal of Cell Science ◽

10.1242/jcs.109.1.221 ◽

1996 ◽

Vol 109 (1) ◽

pp. 221-228

Author(s):

O. Nusse ◽

E.J. Neer

Keyword(s):

G Protein ◽

Pc12 Cells ◽

Pertussis Toxin ◽

Growth Cones ◽

Neonatal Rat ◽

Gamma Subunit ◽

Adp Ribosylation ◽

Gamma Subunits ◽

Membrane Attachment

The heterotrimeric G protein G0 is highly enriched in the growth cones of neuronal cells and makes up 10% of the membrane protein of growth cones from neonatal rat brain. We have used PC12 cells, a cell line that differentiates to a neuron-like phenotype, as a model with which to study the mechanism of G protein localization. First, the role of the beta gamma-subunit was investigated. The attachment of the beta gamma-subunit to the membrane depends on the isoprenylation of the gamma-subunit. The drug lovastatin blocks isoprenylation by inhibiting a key enzyme in the biosynthetic pathway. After treatment of PC12 cells with 10 microM lovastatin for 48 hours 50% of the beta gamma-subunits were cytosolic compared with 100% membrane bound beta gamma in control cells, as determined by cell fractionation, gel electrophoresis and western blot. Addition of 200 microM mevalonic acid reverses this effect. However, lovastatin affects neither the membrane attachment of alpha 0 nor its localization to the growth cones as determined by immunohistochemistry. This suggests that the localization and retention of alpha 0 are independent of the membrane attachment of the full complement of beta gamma-subunits. Second, pertussis toxin was used to block the interaction between alpha 0 and receptors. PC12 cells were treated with 0.1 microgram/ml pertussis toxin prior to and during nerve growth factor-induced differentiation. In vitro [32P]ADP-ribosylation confirmed that alpha 0 and alpha i were completely ADP-ribosylated by this treatment. The ADP-ribosylation by pertussis toxin did not interfere with neurite outgrowth. The localization of alpha 0 to the growth cones was indistinguishable from that in untreated cells. We conclude that G protein-receptor interaction is not necessary for the distribution of alpha 0 to growth cones.

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The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein Gαt

Biochemical Journal ◽

10.1042/bj3110987 ◽

1995 ◽

Vol 311 (3) ◽

pp. 987-993 ◽

Cited By ~ 9

Author(s):

R R Vaillancourt ◽

N Dhanasekaran ◽

A E Ruoho

Keyword(s):

G Protein ◽

Molecular Interactions ◽

Molecular Mapping ◽

Intramolecular Interactions ◽

Alpha Subunit ◽

Gamma Subunit ◽

Terminal Domain ◽

C Terminus ◽

Gamma Subunits ◽

Close Proximity

Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to ‘tether’ [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the ‘acceptor’ domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2′-nitrophenylsulphenyl)-3-methyl-3′- bromoindolenine (‘BNPS-skatole’) and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.

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