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 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.

scholarly journals Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and β2-adrenoceptor-expressing NG108-15 cells

1995 ◽  
Vol 308 (1) ◽  
pp. 275-281 ◽  
Author(s):  
G D Kim ◽  
I C Carr ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
Nucleotide Binding ◽  
Dose Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
Dose Dependent ◽  
Stimulation Of

Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha-subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2-adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose-effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost-stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor.

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.

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