Muscarine Receptors Regulating Electrically Evoked Release of Acetylcholine in Hippocampus Are Linked to Pertussis Toxin-Sensitive G Proteins but Not to Adenylate Cyclase

Rat islets express a pertussis toxin sensitive G-protein involved in receptor-mediated inhibition of insulin secretion. This has been assumed previously to represent “Gi” which couples inhibitory receptors to adenylate cyclase. Incubation of islet G-proteins with32P-NAD and pertussis toxin resulted in the labelling of a band of molecular weight 40,000. This band was very broad and did not allow resolution of individual components. Incubation of the radiolabelled proteins with an anti-Go antiserum resulted in specific immunoprecipitation of a32P-labelled band. These results demonstrate that the complement of pertussis toxin sensitive G-proteins in rat islets includes Go.

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Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2Dopamine Receptors in the A1Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

Journal of Neurochemistry ◽

10.1111/j.1471-4159.1990.tb04949.x ◽

1990 ◽

Vol 55 (5) ◽

pp. 1631-1638 ◽

Cited By ~ 7

Author(s):

Toshihiko Murayama ◽

Yuriko Itahashi ◽

Yasuyuki Nomura

Keyword(s):

Cerebral Cortex ◽

Adenylate Cyclase ◽

G Proteins ◽

Pertussis Toxin ◽

Rat Cerebral Cortex ◽

Adenylate Cyclase System

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Dual Regulation of Phospolipase C Activity by G Proteins

Physiology ◽

10.1152/physiologyonline.1993.8.2.61 ◽

1993 ◽

Vol 8 (2) ◽

pp. 61-63

Author(s):

H Deckmyn ◽

C Van Geet ◽

J Vermylen

Keyword(s):

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Inhibitory Pathway ◽

Close Parallelism

Some subtypes of phosphatidylinositide-specific phospholipase C (PLC) are activated via pertussis toxin-sensitive or -insensitive G proteins. However, a G protein-dependent PLC inhibitory pathway also may exist. The resultant picture is of dual regulation of PLC, showing a close parallelism with the dual regulation of adenylate cyclase.

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G-proteins of fat-cells Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate

Biochemical Journal ◽

10.1042/bj2400035 ◽

1986 ◽

Vol 240 (1) ◽

pp. 35-40 ◽

Cited By ~ 39

Author(s):

P J Rapiejko ◽

J K Northup ◽

T Evans ◽

J E Brown ◽

C C Malbon

Keyword(s):

Adenylate Cyclase ◽

G Proteins ◽

Pertussis Toxin ◽

Hormonal Regulation ◽

Alpha Subunit ◽

Fat Cells ◽

Fat Cell ◽

Alpha Subunits ◽

Inositol 1,4,5 Trisphosphate ◽

Rat Fat Cells

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.

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Both Pertussis Toxin-Sensitive and Insensitive G-Proteins Link Melatonin Receptor to Inhibition of Adenylate Cyclase in the Ovine Pars Tuberalis

Journal of Neuroendocrinology ◽

10.1111/j.1365-2826.1990.tb00639.x ◽

1990 ◽

Vol 2 (6) ◽

pp. 773-776 ◽

Cited By ~ 69

Author(s):

P. J. Morgan ◽

G. Davidson ◽

W. Lawson ◽

P. Barrett

Keyword(s):

Adenylate Cyclase ◽

G Proteins ◽

Pertussis Toxin ◽

Pars Tuberalis ◽

Melatonin Receptor

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Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses

Biochemical Journal ◽

10.1042/bj2520297 ◽

1988 ◽

Vol 252 (1) ◽

pp. 297-300 ◽

Cited By ~ 17

Author(s):

H S Banga ◽

R K Walker ◽

L K Winberry ◽

S E Rittenhouse

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Marked Decrease ◽

Human Platelets ◽

Adp Ribosylation

Thrombin stimulates phospholipase C and inhibits adenylate cyclase in human platelets. We have studied the effect of purified S1 monomer, the ADP-ribosylating subunit of pertussis toxin, on these receptor-coupled G-protein-dependent activities. ADP-ribosylation of a 41 kDa protein is associated with a marked decrease in the ability of thrombin to inhibit cyclic AMP formation, but has little effect on phospholipase C. Therefore adenylate cyclase and phospholipase C appear to be modulated by different G-proteins.

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Activation of G proteins by (Rp) and (Sp) diastereomers of guanosine 5′-[β-thio]triphosphate in hamster fibroblasts. Differential stereospecificity of Gi, Gs and Gp

Biochemical Journal ◽

10.1042/bj2840327 ◽

1992 ◽

Vol 284 (2) ◽

pp. 327-332 ◽

Cited By ~ 1

Author(s):

S Paris ◽

F Eckstein

Keyword(s):

Adenylate Cyclase ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Biochemical Characterization ◽

Inositol Phosphates ◽

Chinese Hamster ◽

Lung Fibroblasts ◽

Guanine Nucleotide Binding ◽

The One

The effects of guanosine 5′-[beta-thio]triphosphate (GTP beta[S]) on G proteins have been examined in Chinese hamster lung fibroblasts (CCL39 line) permeabilized with alpha-toxin from Staphylococcus aureus. Although much less effective than guanosine 5′-[gamma-thio]triphosphate (GTP gamma[S]), both (Rp) and (Sp) diastereomers of GTP beta[S] were found to activate three G protein-mediated pathways: inhibition of forskolin-stimulated adenylate cyclase (mediated by Gi), potentiation of receptor-mediated activation of adenylate cyclase (mediated by Gs), and activation of phosphoinositide breakdown (mediated by Gp). Activation of Gi and Gs occurred above 3 microM-GTP beta[S], but activation of Gp only occurred above 100 microM-GTP beta[S]. Moreover, the order of effectiveness of the two diastereomers was not the same for the three G protein-mediated processes. Whereas both Gi and Gs were more effectively activated (about 5-fold) by (Sp)-GTP beta[S] than by (Rp)-GTP beta[S], Gp showed a marked preference for the (Rp) isomer. Indeed, (Rp)-GTP beta[S] induced the formation of inositol phosphates with a shorter latency and was a better competitor of GDP for binding to Gp than the (Sp) isomer. These results point to different guanine nucleotide-binding properties for Gi and Gs on the one hand and Gp on the other. At least two distinct Gp proteins, differing by their sensitivity to pertussis toxin, are present in CCL39 cells. Since pretreatment of cells with pertussis toxin completely suppressed the effects of (Rp)-GTP beta[S] on Gi, while only slightly attenuating its effects on Gp, we believe that it is the pertussis toxin-insensitive Gp which prefers the (Rp) isomer. Therefore (Rp)-GTP beta[S] may be a valuable tool for the selective activation and the biochemical characterization of this pertussis toxin-insensitive Gp.

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THE ROLE OF GTP-BINDING PROTEINS EXHIBITING GTPase ACTIVITY IN PLATELET ACTIVATION

10.1055/s-0038-1644773 ◽

1987 ◽

Author(s):

K H Jakobs ◽

P Gierschik ◽

R Grandt

Keyword(s):

Signal Transduction ◽

Adenylate Cyclase ◽

Phospholipase C ◽

G Protein ◽

G Proteins ◽

Pertussis Toxin ◽

Inositol Phosphate ◽

Adenylate Cyclase Inhibition ◽

Gi Protein ◽

Stimulation Of

Activation of platelets by agonists acting via cell surface-located receptors apparently involves as an early event in transmembrane signalling an interaction of the agonist-occupied receptor with a guanine nucleotide-binding regulatory protein (G-protein). The activated G-protein, then, transduces the information to the effector molecule, being responsible for the changes in intracellular second messengers. At least two changes in intracellular signal molecules are often found to be associated with platelet activation by agonists, i.e., increases in inositol trisphosphate and diacylglycerol levels caused by activation of a polyphosphoinositide-specific phospholipase C and decrease in cyclic AMP concentration caused by inhibition of adenylate cyclase.Both actions of platelet-activating agents apparently involve G-proteins as transducing elements. Generally, the function of a G-protein in signal transduction can be measured either by its ability to regulate the activity of the effector molecule (phospholipase C or adenylate cyclase) or the binding affinity of an agonist to its specific receptor or by the abitlity of the G-protein to bind and hydrolyze GTP or one of its analogs in response to agonist-activated receptors. Some platelet-activating agonists (e.g. thrombin) can cause both adenylate cyclase inhibition and phospholipase C activation, whereas others induce either inhibition of adenylate cyclase (e.g. α2-adrenoceptor agonists) or activation of phospholipase C (e.g. stable endoperoxide analogs) . It is not yet known whether the simultaneous activation of two signal transduction systems is due to activation of two separate G-proteins by one receptor, to two distinct receptors activating each a distinct G-protein or to activation of two effector molecules by one G-protein.For some of the G-proteins, rather specific compounds are available causing inactivation of their function. In comparison to Gs, the stimulatory G-protein of the adenylate cyclase system, the adenylate cyclase inhibitory Gi-protein is rather specifically inactivated by ADP-ribosylation of its a-subunit by pertussis toxin, “unfortunately” not acting in intact platelets, and by SH-group reactive agents such as N-ethylmaleimide and diamide, apparently also affecting the Giα-subunit. Both of these treatments completely block α2-adrenoceptor-induced GTPase stimulation and adenylate cyclase inhibition and also thrombin-induced inhibition of adenylate cyclase. In order to know whether the G-protein coupling receptors to phospholipase C is similar to or different from the Gi-protein, high affinity GTPase stimulation by agents known to activate phospholipase C was evaluated in platelet membranes. The data obtained indicated that GTPase stimulation by agents causing both adenylate cyclase inhibition and phospholipase C activation is reduced, but only partially, by the above mentioned Gi-inactivating agents, while stimulation of GTPase by agents stimulating only phospholipase C is not affected by these treatments. These data suggested that the G-protein regulating phospholipase C activity in platelet membranes is different from the Gi-protein and may also not be a substrate for pertussis toxin. Measuring thrombin stimulation of inositol phosphate and diacylglycerol formation in saponin-permeabilized platelets, apparently contradictory data were reported after pertussis toxin treatment, being without effect or causing even an increase in thrombin stimulation of inositol phosphate formation (Lapetina: BBA 884, 219, 1986) or being inhibitory to thrombin stimulation of diacylglycerol formation (Brass et al.: JBC 261, 16838, 1986). These data indicate that the nature of the phospholipase C-related G-protein(s) is not yet defined and that their elucidation requires more specific tools as well as purification and reconstitution experiments. Preliminary data suggest that some antibiotics may serve as useful tools to characterize the phospho-lipase-related G-proteins. The possible role of G-protein phosphorylation by intracellular signal molecule-activated protein kinases in attenuation of signal transduction in platelets will be discussed.

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