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  • 1
    Electronic Resource
    Electronic Resource
    Agonists activate Gi1α or Gi2α fused to the human mu opioid receptor differently (2002)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 81 (2002), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: As preferential coupling of opioid receptor to various inhibitory Gα subunits is still under debate, we have investigated the selectivity of the human mu opioid receptor fused to a pertussis toxin insensitive C351I Gi1α or C352I Gi2α in stably transfected HEK 293 cells. Overall agonist binding affinities were increased for both fusion constructs when compared to the wild type receptor. [35S]GTPγS binding was performed on pertussis toxin treated cells to monitor coupling efficiency of the fusion constructs. Upon agonist addition hMOR-C351I Gi1a exhibited an activation profile similar to the non-fused receptor while hMOR-C352I Gi2α was poorly activated. Interestingly no correlation could be drawn between agonist binding affinity and efficacy. Upon agonist addition, forskolin-stimulated cAMP production, as measured using a reporter gene assay, was inhibited by signals transduced via the fused Gi1α and Gi2α mainly. In contrast both fusion constructs were able to initiate ERK-MAPK phosphorylation via coupling to endogenous G proteins only. In conclusion our data indicate that hMOR couples more efficiently to Gi1α than Gi2α and that the coupling efficacy is clearly agonist-dependent.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.2002.00946.x
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  • 2
    Electronic Resource
    Electronic Resource
    Differential capacities of the RGS1, RGS16 and RGS–GAIP regulators of G protein signaling to enhance α2A-adrenoreceptor agonist-stimulated GTPase activity of Go1α (2001)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 78 (2001), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Recombinant RGS1, RGS16 and RGS–GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of Go1α promoted by agonists at the α2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an α2A-adrenoreceptor–Go1α fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor–G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 〉 RGS1 〉 RGS–GAIP. Both RGS1 and RGS16 reduced the potency of the α2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with Go1α to alter the conformation of the α2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS–GAIP show a high degree of selectivity to regulate α2A-adrenoreceptor-activated Go1α rather than Gi1α, Gi2α or Gi3α and different capacities to inactivate this G protein.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.2001.00479.x
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  • 3
    Electronic Resource
    Electronic Resource
    Regional Distribution and Quantitative Measurement of the Phosphoinositidase C-Linked Guanine Nucleotide Binding Proteins G1α and Gqα in Rat Brain (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 61 (1993), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Levels of the guanine nucleotide binding proteins G11α and Gqα, which produce receptor regulation of phosphoinositidase C., were measured immunologically in 13 regions of rat central nervous system. This was achieved by immunoblotting membranes from these regions with antisera (CQ series) that identify these two polypeptides equally, following separation of the membranes using sodium dodecyl sulphate-polyacrylamide gel electrophoresis conditions that can resolve Gqα and G11α. In all regions examined, Gqα was more highly expressed than G11α. Ratios of levels of Gqα to G11α varied between the regions from 5:1 to 2:1. Quantitative measurements of the levels of Gqα and G11α in each region were obtained by comparison with known amounts of purified liver Gqα and G11α and with E. coli expressed recombinant Gqα. Areas that expressed Gqα highly included olfactory bulb (930 ng/ mg of membrane protein), frontal cortex (700 ng/mg of membrane protein), parietal occipital cortex (670 ng/mg of membrane protein), caudate putamen (1,003 ng/mg of membrane protein), hippocampus (1,045 ng/mg of membrane protein), hypothalamus (790 ng/mg of membrane protein), and cerebellum (950 ng/mg of membrane protein). More modest levels were observed in thalamus (450 ng/mg of membrane protein), pituitary (480 ng/mg of membrane protein), optic chiasma (330 ng/mg of membrane protein), and spinal cord (350 ng/mg of membrane protein). Gna was more evenly expressed with values ranging from about 170 ng/mg of membrane protein in spinal cord and optic chiasma to close to 300 ng/mg of membrane protein in regions expressing high levels of Gqα. A third polypeptide could be identified by the CQ antisera in all brain regions. The possibility that this polypeptide is the α subunit of G14 is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1993.tb03595.x
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  • 4
    Electronic Resource
    Electronic Resource
    Expression of the Human β2-Adrenoceptor in NCB20 Cells Results in Agonist Activation of Adenylyl Cyclase and Agonist-Mediated Selective Down-Regulation of Gsα (1995)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Murine neuroblastoma × embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human β2-adrenoceptor under the control of a β-actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the β-adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild-type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein α subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane-associated levels of Gsα in membranes of clone L9 cells and a 50% reduction in Gsα levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gsα in wild-type NCB20 cells, and such treatment had no effect on the levels of other G protein α subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half-maximal loss of Gsα in clone D1 was achieved within 1–2 h of addition of agonist. Dose-response curves to isoprenaline in clone D1 indicated that half-maximal down-regulation of Gsα was produced by ∼1 nM agonist. Measurement of Gs mRNA levels in both clones, however, using both reverse transcriptase-polymerase chain reaction and northern blotting revealed no significant change following treatment with isoprenaline.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1995.65020545.x
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  • 5
    Electronic Resource
    Electronic Resource
    Identification of Two Distinct Isoforms of the Guanine Nucleotide Binding Protein Go in Neuroblastoma × Glioma Hybrid Cells: Independent Regulation During Cyclic AMP-Induced Differentiation (1990)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 55 (1990), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Three distinct antipeptide antisera generated against synthetic peptides that represent parts of the primary sequence of the α-subunit of the (pertussis toxin-sensitive) guanine nucleotide binding protein Go were used in two-dimensional immunoblots of membranes of neuroblastoma × glioma (NG108–15) cells. Each antiserum identified two distinct polypeptides of some 39 kDa. These had apparent isoelectric points of 5.5 and 5.8. Differentiation of NG108–15 cells in response separately to dibutyryl cyclic AMP (cAMP), 8-bromo cAMP, forskolin, and prostaglandin E1 produced elevated levels of Goα, as has previously been noted in onedimensional immunoblots. Two-dimensional analysis demonstrated that the cAMP-induced increases in levels of Goα were only of the more acidic isoform. The two isoforms were both substrates for pertussis toxin-catalysed ADP-ribosylation and did not appear to represent differentially phosphorylated forms of the same polypeptide. Separation of the two forms of Goα could be achieved in one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis when 4 M deionized urea was included in the resolving gel. The more slowly migrating band was the acidic form and corresponded exactly in mobility with the major form of Go from both rat and mouse brain. There was no equivalent in brain of the more rapidly migrating form of Go from the cells. In agreement with the data from two-dimensional gels, only the more slowly migrating form was expressed in considerably higher amounts following cAMP-induced differentiation of NG108–15 cells. Of these two forms of “Go,” the acidic species is equivalent to Go from brain, but the basic form is not identical with Go, which has been purified from bovine brain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1990.tb05773.x
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  • 6
    Electronic Resource
    Electronic Resource
    Robustness of G Proteins in Alzheimer's Disease: An Immunoblot Study (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 57 (1991), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Many of the neurotransmitter systems that are altered in senile dementia of the Alzheimer type are known to mediate their effects via G proteins, yet the integrity of guanine nucleotide-binding proteins (G proteins) in Alzheimer's diseased brains has received minimal investigation. The aim of this study was to establish whether the level of Gα subunits of five G proteins was altered in Alzheimer's disease. We used immunoblotting (Western blotting) to compare the amounts of Gi1, Gi2, GsH (heavy molecular weight), GSL (light molecular weight), and Go in the frontal cortex and hippocampus, two regions severely affected by the disease, and the cerebellum, which is less severely affected. The number of senile plaques was also quantified. We report that there was no significant difference in the level of these Gα subunits between Alzheimer's diseased and age-matched postmortem brains. These results suggest that alterations in the amount of G protein α subunits are not a feature of Alzheimer's disease.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1991.tb02092.x
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  • 7
    Electronic Resource
    Electronic Resource
    Regulation of Spontaneous Activity of the δ-Opioid Receptor: Studies of Inverse Agonism in Intact Cells (1997)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 69 (1997), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [3H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine δ-opioid receptor (clone D2). Basal [3H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin d-Ala2,d-Leu5 enkephalin (DADLE) produced strong inhibition of forskolin-amplified [3H]cyclic AMP production, whereas the δ-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC50 (9.4 ± 0.6 × 10−8M) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated Ki. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1997.69052115.x
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  • 8
    Electronic Resource
    Electronic Resource
    The human δ opioid receptor activates Gi1α more efficiently than Go1α (2001)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 76 (2001), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: To assess the relative capacity of the human δ opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the α-subunits of either Gi1 or Go1, containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in-frame with the C-terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [3H]naltrindole with high affinity. d-ala2,d-leu5 Enkephalin effectively inhibited forskolin-stimulated adenylyl cyclase activity in intact cells in a concentration-dependent, but pertussis toxin-insensitive, manner. The high-affinity GTPase activity of both constructs was also stimulated by d-ala2,d-leu5 enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to d-ala2,d-leu5 enkephalin was more than three times greater for Gi1α than for Go1α. As the effect of agonist in both cases was to increase Vmax without increasing the observed Km for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of Gi1α compared with Go1α. An equivalent fusion protein between the human µ opioid receptor-1 and Gi1α produced a similar d-ala2,d-leu5 enkephalin-induced GTP turnover number as the δ opioid receptor−Gi1α fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of Gi1α.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.2001.00196.x
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  • 9
    Electronic Resource
    Electronic Resource
    Insulin activates glycerol-3-phosphate acyltransferase (de novo phosphatidic acid synthesis) through a phospholipid-derived mediator. Apparent involvement of Gi.alpha. and activation of a phospholipase C (1990)
    s.l. : American Chemical Society
    Biochemistry 29 (1990), S. 8735-8740 
    Details
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1021/bi00489a033
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  • 10
    Electronic Resource
    Electronic Resource
    Immunochemical and electrophoretic characterization of the major pertussis toxin substrate of the RAW264 macrophage cell line (1988)
    s.l. : American Chemical Society
    Biochemistry 27 (1988), S. 2040-2046 
    Details
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1021/bi00406a034
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