G protein .beta..gamma. subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of .alpha. subunits by pertussis toxin but differential interactions with Gs.alpha.

Biochemistry ◽  
1989 ◽  
Vol 28 (2) ◽  
pp. 611-616 ◽  
Author(s):  
Patrick J. Casey ◽  
Michael P. Graziano ◽  
Alfred G. Gilman
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Bovine Brain ◽  
Adp Ribosylation ◽  
Catalytic Support ◽  
Alpha Subunits ◽  
Gamma Subunits

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.

scholarly journals G-proteins in skeletal muscle. Evidence for a 40 kDa pertussis-toxin substrate in purified transverse tubules

1988 ◽  
Vol 254 (2) ◽  
pp. 405-409 ◽  
Author(s):  
M Toutant ◽  
J Barhanin ◽  
J Bockaert ◽  
B Rouot
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
Adp Ribosylation ◽  
Alpha Subunits ◽  
Low Salt ◽  
T Tubules

In muscle, it has been established that guanosine 5′-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein

1992 ◽  
Vol 70 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Richard W. Lerner ◽  
Gary D. Lopaschuk ◽  
Peter M. Olley
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Cyclase Activity ◽  
Prostaglandin E ◽  
Adenylyl Cyclase Activity ◽  
Adp Ribosylation ◽  
Number Of Binding Sites ◽  
Guanine Nucleotide Binding

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 μM 5′-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 ± 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 ± 0.7 nM) to one site of intermediate affinity (KD = 0.52 ± 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein. They also demonstrate that the interaction of this Gi protein with the PGE2 receptor is important in the regulation of PGE2 binding to its receptor.Key words: prostaglandin E2, sarcolemma, heart, adenylyl cyclase, G protein.

scholarly journals Phospholipase C-β1 is regulated by a pertussis toxin-insensitive G-protein

1991 ◽  
Vol 280 (3) ◽  
pp. 753-760 ◽  
Author(s):  
T F J Martin ◽  
J E Lewis ◽  
J A Kowalchyk
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Gh3 Cells ◽  
Phospholipid Vesicles ◽  
Protein Tyrosine Phosphorylation ◽  
Gtp Binding ◽  
Gamma Subunits ◽  
Gh3 Cell

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.

scholarly journals Metabotropic glutamate receptor analogues inhibit p[NH]ppG-stimulated phospholipase C activity in bovine brain coated vesicles: involvement of a pertussis toxin-sensitive G-protein

1995 ◽  
Vol 307 (3) ◽  
pp. 851-857 ◽  
Author(s):  
M Martín ◽  
J M Sanz ◽  
M Ros ◽  
A Cubero
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Glutamate Receptor ◽  
Pertussis Toxin ◽  
Metabotropic Glutamate Receptors ◽  
Metabotropic Glutamate Receptor ◽  
Bovine Brain ◽  
Coated Vesicles ◽  
Inhibitory Effects ◽  
Metabotropic Glutamate

Guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG)-stimulated phospholipase C (PLC) activity in bovine brain coated vesicles is inhibited by glutamate agonists. In the present study we show that quisqualic acid (QA), (+/-)-trans-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD), glutamic acid and ibotenic acid inhibited p[NH]ppG-stimulated PLC by 44, 41, 36 and 25% respectively. Carbachol also produced an inhibition of p[NH]ppG-stimulated PLC by 45%. The inhibition caused by trans-ACPD and QA was dose-dependent. DL-2-Amino-3-phosphonopropionic acid and (RS)-alpha-methyl-4-carboxyphenylglycine, specific antagonists of metabotropic glutamate receptors (mGluRs), abolished these inhibitory effects. trans-ACPD inhibition of p[NH]ppG-stimulated PLC was also observed in the presence of ionotropic glutamate receptor antagonists. When carbachol and QA or trans-ACPD were combined, additive inhibitory effects were observed. Preincubation of bovine brain coated vesicles with pertussis toxin abolished the inhibitory effects of mGluR analogues and carbachol on p[NH]ppG-stimulated PLC activity. The presence of Gs alpha and pertussis toxin substrates, Gi alpha and Go alpha subunits as well as PLC beta 1 in bovine brain coated vesicles has been confirmed by immunoblot. These results support the coupling of mGluRs to a PLC in an inhibitory manner through a pertussis toxin-sensitive G-protein in bovine brain coated vesicles.

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