<i>m</i>-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application

Zor, Tsaffrir; Halifa, I; Kleinhaus, S; Chorev, Michael; Selinger, Zvi

doi:10.1042/bj3060253

Cited by 12 publications

(16 citation statements)

References 22 publications

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“…Approximately 30 to 40% of the irradiated m-AcAGTP did not migrate from the origin (data not shown). Nonirradiated m-AcAGTP had a retardation factor value twice that of [␣-32 P]GTP, which agrees with that shown previously (Zor et al, 1995).…”

supporting

confidence: 82%

“…To investigate whether G olf is activated on stimulation with an A 2A receptor agonist, we used a photolabeling technique (Zor et al, 1995). We first established that G proteins present in the membranes can be photolabeled by [␣-32 P]m-AcAGTP and that activation of G proteins leads to an increase in this photolabeling.…”

Section: Resultsmentioning

confidence: 99%

“…The preparation of radioactive m-acetylanilido-GTP (m-AcAGTP) was performed as described by Zor et al (1995). Briefly, [␣-32 P]GTP (3000 Ci/mmol) was freeze-dried and redissolved in 50 l of 0.125 M 4-morpholineethanesulfonic acid buffer, pH 6.5.…”

mentioning

confidence: 99%

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Adenosine A_2AReceptors are Colocalized with and Activate G_olfin Rat Striatum

2000

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In situ hybridization with cRNA probes showed A 2A receptor and G olf mRNAs to be abundantly expressed in caudate putamen, nucleus accumbens, and olfactory tubercle, whereas G s mRNA shows a comparatively low expression in regions expressing A 2A receptors. In caudate putamen, 49% of the medium-sized neuron-like cells exhibited a strong signal for adenosine A 2A receptor mRNA, and 98% showed a strong signal for G olf mRNA. In contrast, G s mRNA was found in only 12% of the medium-sized neuron-like cells in caudate putamen. The coexpression of adenosine A 2A receptor mRNA with that of G olf or G s mRNAs was studied with double in situ hybridization. A large majority (91-95%) of the neurons in caudate-putamen that contained adenosine A 2A receptor mRNA also expressed G olf mRNA, whereas only 3 to 5% of the neurons with adenosine P]m-acetylanilido-GTP followed by immunoprecipitation with a specific antibody against G olf . Transfection of G olf cDNA into Chinese hamster ovary cells, which stably express human adenosine A 2A receptors, led to an increased efficacy of CGS 21680, as evidenced by a stronger cAMP response, indicating that activation of G olf by A 2A receptors leads to a biological signal. In conclusion, these results provide anatomical and biochemical evidence that adenosine A 2A receptors stimulate G olf rather than G s in striatum.

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supporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

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Adenosine A_2AReceptors are Colocalized with and Activate G_olfin Rat Striatum

2000

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“…These GTP analogues were stable for at least 3 months. The synthesis of GTP analogues l [12], 2 and 8 [11] was previously described. The identity of each purified GTP analogue was verified by acidic hydrolysis of the acid-labile phosphorus-nitrogen bond.…”

Section: Preparation Of Gtp Analoguesmentioning

confidence: 99%

“…Generally, GTP analogues with substituents attached to the y-phosphate, either through phosphonoamidate or ester linkage are not hydrolyzed by G-proteins [11][12][13][14][15]. We have recently shown, however, that the GTP analogue 3,4-diaminobenzophenone phosphonoamidate-GTP (DABP-GTP, analogue 8 in Fig.…”

Section: Introductionmentioning

confidence: 99%

GTP analogue hydrolysis by the Gs protein: implication for the role of catalytic glutamine in the GTPase reaction

et al. 1998

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Hydrolysis of GTP, bound to members of the Gprotein superfamily, terminates their downstream signaling activity. A conserved glutamine serves a critical role in this pivotal guanosine triphosphatase (GTPase) reaction. However, the role of the catalytic glutamine in GTP hydrolysis is still not well understood. We have employed substrate-assisted catalysis to probe the catalytic mechanism of Gscx using GTP analogues. These GTP analogues, each having different functional groups, were designed to support or refute particular putative GTPase mechanisms. We have found that a hydrogen donor group, in close proximity to the T-phosphate of GTP, is necessary and sufficient to substitute for the function of the catalytic glntamine in the GTPase reaction.

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Distribution of G-protein ? subunits and neurotransmitter activation of G?i and G?q in the brain of the lobsterHomarus americanus

McClintock

Bose

2000

J. Comp. Neurol.

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Immunocytochemistry using antisera specific for the G-protein alpha subunits G(alphai), G(alphaq), and G(alphas) revealed similar patterns of immunoreactivity in the lobster brain. Immunoreactivity was strongest in neuropil, especially the olfactory and accessory lobes, and was characterized by bundles of fine threads leading to dense concentrations of punctate staining in the glomeruli. This may reflect the concentration of G-protein alpha subunits at synapses. The major differences between the antisera were distinct patterns of staining intensity in subregions of glomeruli of the olfactory and accessory lobes. This result is potentially correlated with previous evidence that these subregions are neurochemically distinct. Neuronal cell bodies contained moderate levels of immunoreactivity at the plasma membrane and faint staining in the cytoplasm. The olfactory globular tract was moderately immunoreactive, but other fiber tracts were weakly immunoreactive. Immunoreactivity in the deutocerebral commissure consisted of small oval cell bodies and strands that formed a reticulated pattern, suggestive of glia. Photoaffinity labelling by using an analog of GTP demonstrated that histamine activated G(alphai) in brain homogenates. Further evidence of G-protein activation was obtained by showing that stimulation with a mixture of neuroactive substances increased the amount of phospholipase C-beta associated with membranes, G(alphaq), and G(beta). The lobster brain, especially in its neuropil regions, is richly endowed with neuromodulatory biochemical pathways involving G(alphai), G(alphaq), and G(alphas).

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m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application

Abstract: A novel photoaffinity label, .n-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In

Cited by 12 publications

References 22 publications

Adenosine A_2AReceptors are Colocalized with and Activate G_olfin Rat Striatum

Adenosine A_2AReceptors are Colocalized with and Activate G_olfin Rat Striatum

GTP analogue hydrolysis by the Gs protein: implication for the role of catalytic glutamine in the GTPase reaction

Distribution of G-protein ? subunits and neurotransmitter activation of G?i and G?q in the brain of the lobsterHomarus americanus

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m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application

Abstract: A novel photoaffinity label, .n-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In

Cited by 12 publications

References 22 publications

Adenosine A2AReceptors are Colocalized with and Activate Golfin Rat Striatum

Adenosine A2AReceptors are Colocalized with and Activate Golfin Rat Striatum

GTP analogue hydrolysis by the Gs protein: implication for the role of catalytic glutamine in the GTPase reaction

Distribution of G-protein ? subunits and neurotransmitter activation of G?i and G?q in the brain of the lobsterHomarus americanus

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Adenosine A_2AReceptors are Colocalized with and Activate G_olfin Rat Striatum

Adenosine A_2AReceptors are Colocalized with and Activate G_olfin Rat Striatum