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

1995 ◽  
Vol 311 (3) ◽  
pp. 987-993 ◽  
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.

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

1993 ◽  
Vol 121 (4) ◽  
pp. 775-783 ◽  
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.

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

1992 ◽  
Vol 12 (4) ◽  
pp. 1585-1591 ◽  
Author(s):  
K J Fisher ◽  
N N Aronson
Keyword(s):  
G Protein ◽  
Genomic Sequence ◽  
Cdna Clones ◽  
Gamma Subunit ◽  
Bovine Gene ◽  
Gamma Subunits ◽  
Sequence Encoding ◽  
Rat Livers ◽  
Exon Structure

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.

scholarly journals Subcellular localization and quantitation of the major neutrophil pertussis toxin substrate, Gn.

1988 ◽  
Vol 106 (6) ◽  
pp. 1927-1936 ◽  
Author(s):  
G M Bokoch ◽  
K Bickford ◽  
B P Bohl
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Alpha Subunit ◽  
Human Neutrophils ◽  
Adp Ribosylation ◽  
Gamma Subunits ◽  
G Protein Alpha Subunit ◽  
Protein Alpha Subunit

The subcellular distribution of G protein subunits in the neutrophil was examined. Cells were nitrogen cavitated and subcellular organelles fractionated on discontinuous sucrose gradients. The presence of GTP-binding regulatory protein (G protein) alpha and beta/gamma subunits in each organelle was determined using three methods of analysis: specific binding of guanine nucleotide, ADP ribosylation by pertussis toxin, and immunoblot analysis with subunit-specific G protein antibodies. Both plasma membrane and cytosolic G protein components were detected. In contrast, neither the specific nor the azurophilic granules contained detectable G protein. Based on the ability of exogenous G protein beta/gamma subunits to increase the ADP ribosylation of the cytosolic form of G protein and upon the hydrodynamic behavior of the cytosolic protein, it is likely that this represents an uncomplexed G protein alpha subunit. Proteolytic mapping with Staphylococcus aureus V8 protease suggests the soluble alpha subunit is from Gn, the major pertussis toxin substrate of human neutrophils. Using quantitative analysis, the levels of the 40-kD G protein alpha subunit and of the 35/36-kD beta subunit in the neutrophil membrane were determined.

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

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.

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

1992 ◽  
Vol 12 (4) ◽  
pp. 1585-1591
Author(s):  
K J Fisher ◽  
N N Aronson
Keyword(s):  
G Protein ◽  
Genomic Sequence ◽  
Cdna Clones ◽  
Gamma Subunit ◽  
Bovine Gene ◽  
Gamma Subunits ◽  
Sequence Encoding ◽  
Rat Livers ◽  
Exon Structure

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.

scholarly journals Biosynthesis, assembly and secretion of fibrinogen in cultured rat hepatocytes

1988 ◽  
Vol 251 (2) ◽  
pp. 373-377 ◽  
Author(s):  
S Hirose ◽  
K Oda ◽  
Y Ikehara
Keyword(s):  
Rat Hepatocytes ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Gamma Subunit ◽  
Intracellular Pool ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Gamma Subunits ◽  
Cultured Rat Hepatocytes ◽  
Rate Limiting

The biosynthesis, assembly and secretion of fibrinogen were investigated in cultured rat hepatocytes which were incubated with [35S]methionine. When initial rates of the synthesis of three fibrinogen subunits were compared, the A alpha-subunit was found to be synthesized significantly slower than the B beta- and gamma-subunits. Pulse-chase experiments revealed that the secreted fibrinogen contained different proportions of the newly synthesized subunits, depending upon the chase times. Radioactivity in the A alpha subunit, which initially had the highest level of the three, was rapidly decreased in parallel with the chase time. The gamma-subunit had an increasing amount of the radioactivity in the secreted molecule during the chase periods, whereas that in the B beta-subunit was gradually decreased at the later stages of chase. Analysis of intracellular components of fibrinogen confirmed that the nascent A alpha-subunit was most rapidly exhausted, and the gamma-subunit occupied the largest proportion among the non-assembled subunits at later stages of chase. Taken together, these results suggest that the synthesis of A alpha-subunit, which has the lowest rate, could be the rate-limiting step in the production and secretion of fibrinogen in cultured rat hepatocytes, in contrast with what has been proposed for human and rabbit fibrinogen, namely that the synthesis of B beta-subunit is the rate-limiting step. The results also indicate that there is a large intracellular pool of gamma-subunit.

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