Stimulatory guanine nucleotide-binding protein and adenylate cyclase activities in Bio 14.6 cardiomyopathic hamsters at the hypertrophic stage

1992 ◽  
Vol 110 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Takayoshi Ikegaya ◽  
Akira Kobayashi ◽  
Rong Bang Hong ◽  
Hisamichi Masuda ◽  
Masanori Kaneko ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Cardiomyopathic Hamsters ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Protein

scholarly journals Inhibition of subunit dissociation and release of the stimulatory G-protein, Gs, by βγ-subunits and somatostatin in S49 lymphoma cell membranes

1991 ◽  
Vol 280 (2) ◽  
pp. 303-307 ◽  
Author(s):  
L A Ransnäs ◽  
D Leiber ◽  
P A Insel
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Cell Membranes ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Subunit Dissociation ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Protein

We examined the interaction between the stimulatory guanine-nucleotide-binding protein, Gs, and the inhibitory guanine-nucleotide-binding protein, Gi, in cell membranes of S49 lymphoma cells. In these cells, beta-adrenergic receptors stimulate the activity of adenylate cyclase via Gs, whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, Gi. Using an antibody that selectively recognizes alpha s, the monomeric, but not the heterotrimeric, alpha-subunit of Gs, we quantified the extent of dissociation of Gs in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 microM-isoprenaline, 100 microM free Mg2+ and 100 microM-GTP produced substantial subunit dissociation of Gs, which was reversible by addition of purified beta gamma-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated Gs (kinhib. for somatostatin = 51 +/- 12 nM) and in the activity of adenylate cyclase (kinhib. = 121 +/- 20 nM). By contrast, after addition of a 10-fold molar excess of beta gamma-dimer relative to alpha s, there was a 2-3 min lag, after which the beta gamma-dimer re-associated Gs. Isoprenaline-induced dissociation of Gs was accompanied by a release of alpha s from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of alpha s by the beta-agonist and reversal by somatostatin. Membrane release of Gs by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conclude that in native cell membranes somatostatin-induced activation of Gi dissociates Gi and interferes with the Gs activation cycle by providing beta gamma-dimer, which acts to prevent or reverse formation of monomeric alpha s. Because alpha s can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated alpha s is likely to be an important factor in modulating the activity of adenylate cyclase.

scholarly journals Evidence for two GTPases activated by thrombin in membranes of human platelets

1986 ◽  
Vol 237 (3) ◽  
pp. 669-674 ◽  
Author(s):  
R Grandt ◽  
K Aktories ◽  
K H Jakobs
Keyword(s):  
Adenylate Cyclase ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Hydrolysis ◽  
Platelet Membranes ◽  
Guanine Nucleotide Binding ◽  
Stimulation Of ◽  
Nucleotide Binding Protein

Thrombin inhibits adenylate cyclase and stimulates GTP hydrolysis by high-affinity GTPase(s) in membranes of human platelets at almost identical concentrations. Both of these thrombin actions are similar to those observed with agonist-activated alpha 2-adrenoceptors coupling to the inhibitory guanine nucleotide-binding protein N1. However, stimulation of GTP hydrolysis caused by adrenaline (alpha 2-adrenoceptor agonist) and by thrombin at maximally effective concentrations was partially additive, whereas with regard to adenylate cyclase inhibition no additive response was observed. Furthermore, treatment of platelet membranes with pertussis toxin, which inactivates Ni and largely abolishes thrombin- and adrenaline-induced adenylate cyclase inhibition and adrenaline-induced GTPase stimulation, decreased the thrombin-induced stimulation of GTP hydrolysis by only about 30%. Additionally, the thiol reagent N-ethylmalemide (NEM) at rather low concentrations abolished thrombin- and adrenaline-induced stimulation of GTP hydrolysis was decreased by only 30-40% by treatment of platelet membranes with even high concentrations of NEM. Treatment with cholera toxin, which inhibits GTPase activity of the Ns (stimulatory guanine nucleotide-binding) protein, has no effect on thrombin-stimulated GTP hydrolysis. The data suggest that thrombin interaction with its receptor sites in platelet membranes leads to stimulation of two GTP-hydrolysing enzymes. One of these enzymes is apparently Ni and is also activated by agonist-activated alpha 2-adrenoceptors and is inactivated by pertussis toxin and NEM treatment. The other GTP-hydrolysing enzyme activated by thrombin may represent a guanine nucleotide-binding protein apparently involved in the coupling of thrombin receptors to the phosphoinositide phosphodiesterase.

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