scholarly journals The Inhibitory G Protein Gi Identified as Pertussis Toxin-Catalyzed ADP-Ribosylation

2012 ◽  
Vol 35 (12) ◽  
pp. 2103-2111 ◽  
Author(s):  
Toshiaki Katada
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Adp Ribosylation ◽  
Inhibitory G Protein

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 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.

scholarly journals Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates

1988 ◽  
Vol 249 (3) ◽  
pp. 653-659 ◽  
Author(s):  
F R McKenzie ◽  
E C H Kelly ◽  
C G Unson ◽  
A M Spiegel ◽  
G Milligan
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Opioid Peptides ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
High Affinity ◽  
C Terminus ◽  
Inhibitory G Protein ◽  
Guanine Nucleotide Binding

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.

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