scholarly journals The presence of free G protein beta/gamma subunits in human neutrophils results in suppression of adenylate cyclase activity.

1987 ◽  
Vol 262 (2) ◽  
pp. 589-594
Author(s):  
G M Bokoch
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Human Neutrophils ◽  
Gamma Subunits

scholarly journals Calmodulin binding distinguishes between βγ subunits of activated G proteins and transducin

1992 ◽  
Vol 283 (3) ◽  
pp. 683-690 ◽  
Author(s):  
L A Mangels ◽  
R R Neubig ◽  
H E Hamm ◽  
M E Gnegy
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Bovine Brain ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Dependent Manner ◽  
Protein Activation ◽  
Calmodulin Binding ◽  
Gamma Subunits

The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.

scholarly journals Inhibition of adenylate cyclase activity by galanin in rat insulinoma cells is mediated by the G-protein Gi3

1994 ◽  
Vol 303 (2) ◽  
pp. 369-375 ◽  
Author(s):  
P de Mazancourt ◽  
P K Goldsmith ◽  
L S Weinstein
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Pancreatic Beta Cells ◽  
Protein S ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Rinm5f Cells ◽  
Insulinoma Cells ◽  
Galanin Receptors ◽  
Rat Insulinoma

Galanin inhibits adenylate cyclase activity and insulin secretion and modulates ion channels in pancreatic beta-cells through pertussis-toxin-sensitive G-protein(s). Antibodies directed against the C-terminal region of specific G-protein alpha-subunits were used to determine which G-protein(s) couple galanin receptors to inhibition of adenylate cyclase in the rat insulinoma cell line RINm5F. Preincubation of membranes with EC antibody (anti-alpha i3) decreased the inhibition of forskolin-stimulated adenylate cyclase activity by galanin (100 nM) by 45% compared with control IgG (P < 0.05) whereas preincubation with AS (anti-alpha i1, alpha i2) or GO (anti-alpha o) antibodies had no significant effect. To confirm these results, RINm5F cells were exposed intermittently over a 4-day period to phosphorothioate oligodeoxynucleotides that were either sense or antisense to alpha i1, alpha i2, alpha i3 or alpha o. Oligodeoxynucleotides antisense to alpha i2, alpha i3 and alpha o specifically decreased the levels of the targeted alpha-subunit in membranes. alpha i1 was undetectable in these cells. Inhibition of adenylate cyclase activity by galanin was largely abolished in membranes from cells exposed to the oligodeoxynucleotide antisense to alpha i3, whereas all other oligodeoxynucleotides had no significant effect on this pathway. Indirect immunofluorescence and immunoblotting of specific membrane fractions with EC antibody show significant localization of alpha i3 to intracellular membrane compartments. These results suggest that Gi3 is the G protein that couples galanin receptors to inhibition of adenylate cyclase activity in RINm5F cells.

Positive coupling of beta-like adrenergic receptors with adenylate cyclase in the cnidarian Renilla koellikeri

1993 ◽  
Vol 182 (1) ◽  
pp. 131-146 ◽  
Author(s):  
E. W. Awad ◽  
M. Anctil
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Radioligand Binding ◽  
Guanine Nucleotides ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Binding Studies ◽  
Beta Adrenergic ◽  
Renilla Koellikeri ◽  
Stimulation Of

Coupling of the previously characterized beta1- and beta2-like adrenoceptors in the sea pansy Renilla koellikeri with adenylate cyclase was examined in membrane preparations from this cnidarian. Adenylate cyclase activity was stimulated by several guanine nucleotides, such as GTP, Gpp(NH)p and GTPgammaS. Fluoride ions and cholera toxin greatly enhanced the enzyme activity, whereas forskolin had no effect on basal or isoproterenol-induced stimulation of the enzyme. The stimulation of adenylate cyclase activity by several beta-adrenergic agonists in different parts of the animal reflected a positive coupling with the beta2- and beta1-like adrenoceptors in autozooid and peduncle tissues, respectively. In addition, isoproterenol-induced stimulation of adenylate cyclase activity was dependent on guanine nucleotides, suggesting coupling mediated by a G protein. The pharmacological profile of various antagonists on isoproterenol-sensitive adenylate cyclase in autozooid and peduncle tissues matched that of previous radioligand binding studies. Isoproterenol-induced stimulation of adenylate cyclase activity in rachidial tissues was partially inhibited by trifluoperazine of (+/−)CGP12177 and was completely blocked in the presence of both antagonists. This suggests that coupling of the enzyme occurs with beta1- and beta2-like adrenoceptors, both being present in the rachis. Serotonin and dopamine were also found to stimulate adenylate cyclase activity. Their stimulatory effect was additive to isoproterenol-induced activation, suggesting the presence of dopaminergic and serotonergic receptors in the tissues of the sea pansy. Along with the data presented previously on beta-adrenergic binding, this study suggests that elements of receptor-dependent G protein signal transduction originated early in invertebrate evolution.

scholarly journals A mnemonical or negative-co-operativity model for the activation of adenylate cyclase by a common G-protein-coupled calcitonin-gene-related neuropeptide (CGRP)/amylin receptor

1993 ◽  
Vol 293 (1) ◽  
pp. 229-236 ◽  
Author(s):  
M Bushfield ◽  
A Savage ◽  
N J Morris ◽  
M D Houslay
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
Kinetic Mechanism ◽  
Dose Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Hill Coefficient ◽  
Single Receptor ◽  
Calcitonin Gene ◽  
Dose Dependent Fashion

Both amylin and calcitonin-gene-related neuropeptide (CGRP) activated adenylate cyclase activity in hepatocyte membranes around 5-fold in a dose-dependent fashion, with EC50 values of 120 +/- 14 and 0.3 +/- 0.14 nM respectively. Whereas amylin exhibited normal activation kinetics (Hill coefficient, h approximately 1), CGRP showed kinetics indicative of either multiple sites/receptor species having different affinities for this ligand or a single receptor species exhibiting apparent negative co-operativity (h approximately 0.21). The CGRP antagonist CGRP-(8-37)-peptide inhibited adenylate cyclase stimulated by EC50 concentrations of either amylin or CGRP. Inhibition by CGRP-(8-37) was selective in that markedly lower concentrations were required to block the action of amylin (IC50 = 3 +/- 1 nM) compared with that of CGRP itself (IC50 = 120 +/- 11 nM). Dose-effect data for inhibition of CGRP action by CGRP-(8-37) showed normal saturation kinetics (h approximately 1), whereas CGRP-(8-37) inhibited amylin-stimulated adenylate cyclase activity in a fashion which was indicative of either multiple sites or apparent negative co-operativity (h approximately 0.24). Observed changes in the kinetics of inhibition by CGRP-(8-37) of CGRP, but not amylin-stimulated adenylate cyclase, at concentrations of agonists below their EC50 values militated against a model of two distinct populations of non-interacting receptors each able to bind both amylin and CGRP. A kinetic model is proposed whereby a single receptor, capable of being activated by both CGRP and amylin, obeys either a mnemonical kinetic mechanism or one of negative co-operativity with respect to CGRP but not to amylin. The relative merits of these two models are discussed together with a proposal suggesting that the activation of adenylate cyclase by various G-protein-linked receptors may be described by a mnemonical model mechanism.

scholarly journals Enhanced expression of inhibitory guanine nucleotide regulatory protein in spontaneously hypertensive rats. Relationship to adenylate cyclase inhibition

1992 ◽  
Vol 288 (1) ◽  
pp. 79-85 ◽  
Author(s):  
M B Anand-Srivastava
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Spontaneously Hypertensive Rats ◽  
Regulatory Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Heart Sarcolemma

We have previously shown that the stimulatory effects of guanine nucleotides, N-ethylcarboxamide-adenosine and other agonists on adenylate cyclase activity were diminished in aorta and heart sarcolemma of spontaneously hypertensive rats (SHR) [Anand-Srivastava (1988) Biochem. Pharmacol. 37, 3017-3022]. In the present studies, we have examined whether the decreased response of these agonists is due to the defective GTP-binding proteins (G-proteins) which couple the receptors to adenylate cyclase, and have therefore measured the levels of G-proteins in aorta and heart from SHR and their respective Wistar-Kyoto (WKY) controls by using pertussis toxin (PT)- and cholera toxin (CT)-catalysed ADP-ribosylations and immunoblotting techniques using specific antibodies against G-proteins. The labelling with [32P]NAD+ and PT identified a 40/41 kDa protein in heart and aorta from WKY and SHR and was significantly increased in the hearts (approximately 100%) and aorta (approximately 30-40%), from SHR as compared with WKY. Immunoblotting revealed an increase in the levels of the G-protein alpha-subunits Gi alpha-2 and Gi alpha-3 in heart and Gi alpha-2 in aorta, whereas no change in Go alpha was observed in heart from SHR and WKY. On the other hand, no differences were observed in CT labelling or immunoblotting of stimulatory G-protein (Gs) in heart and aorta from WKY and SHR. In addition, CT stimulated the adenylate cyclase activity in heart sarcolemma from WKY and SHR to a similar extent. These results were correlated with adenylate cyclase inhibition and stimulation by various hormones. Angiotensin II (AII), atrial natriuretic factor (ANF) and oxotremorine-mediated inhibition was found to be greater in SHR as compared with WKY, whereas the stimulatory effects of adrenaline, isoprenaline, dopamine and forskolin were diminished in SHR aorta as compared to WKY. These results indicate that regulatory protein G(i) is more expressed in SHR, which may be associated with the decreased responsiveness of stimulatory hormones and increased sensitivity of inhibitory hormones to stimulate/inhibit adenylate cyclase activity. It may thus be suggested that the enhanced G(i) activity may be one of the mechanisms responsible for the diminished vascular tone and impaired myocardial functions in hypertension.

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