scholarly journals Gi3 does not contribute to the inhibition of adenylate cyclase when stimulation of an α2-adrenergic receptor causes activation of both Gi2 and Gi3

1992 ◽  
Vol 284 (2) ◽  
pp. 565-568 ◽  
Author(s):  
S J McClue ◽  
E Selzer ◽  
M Freissmuth ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
G Proteins ◽  
Adrenergic Receptor ◽  
Genomic Dna ◽  
Gtpase Activity ◽  
Adrenergic Agonists ◽  
Dna Encoding ◽  
Receptor Stimulation ◽  
High Affinity ◽  
Stimulation Of

Agonist occupancy of the alpha 2-C10 adrenergic receptor in a stable clone (1C) of Rat 1 fibroblasts produced by transfection of cells with genomic DNA encoding this receptor causes the activation of both of the pertussis-toxin-sensitive G-proteins Gi2 and Gi3 [Milligan, Carr, Gould, Mullaney & Lavan (1991) J. Biol. Chem. 266, 6447-6455]. An IgG fraction from an antiserum (I3B) which identifies the C-terminal decapeptide of Gi3 alpha only was able to inhibit partially receptor stimulation of high-affinity GTPase activity. An equivalent fraction from an antiserum (AS7) able to identify the C-terminal decapeptide of Gi1 alpha + Gi2 alpha, but not Gi3 alpha, was also able to inhibit partially receptor stimulation of GTPase activity, and the effects of the two antisera were additive. By contrast, agonist-mediated inhibition of forskolin-amplified adenylate cyclase activity was abolished completely by the IgG fraction of antiserum AS7, but was not decreased by treatment with antiserum 13B. Based on the proportion of agonist-stimulated high-affinity GTPase which was prevented by each antiserum and on the measured membrane levels of Gi2 and Gi3, calculations indicated that essentially all of the cellular Gi3, but only 15% of the available Gi2, can be activated by the alpha 2-C10 adrenergic receptor in these cells. These results demonstrate that, although Gi3 is activated by alpha 2-adrenergic agonists in membranes of clone 1C cells, it does not contribute to the transduction of receptor-mediated inhibition of adenylate cyclase.

scholarly journals δ-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2

1990 ◽  
Vol 267 (2) ◽  
pp. 391-398 ◽  
Author(s):  
F R McKenzie ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Opioid Receptor ◽  
Protein S ◽  
Guanine Nucleotide ◽  
Gtpase Activity ◽  
Guanine Nucleotide Binding Protein ◽  
High Affinity ◽  
Guanine Nucleotide Binding

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.

scholarly journals Interactions of the α2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants

1997 ◽  
Vol 321 (3) ◽  
pp. 721-728 ◽  
Author(s):  
Alan WISE ◽  
Marie-Ange WATSON-KOKEN ◽  
Stephen REES ◽  
Melanie LEE ◽  
Graeme MILLIGAN
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Resistant Mutant ◽  
Cysteine Residue ◽  
Gtpase Activity ◽  
Wild Type ◽  
High Affinity ◽  
Agonist Stimulation ◽  
Stimulation Of

The α2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of ‘Gi-like’ pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine α2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1α, Gi2α and Gi3α or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The α2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5ƀ-[γ-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein α-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1α, Gi2α and Gi3α resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys → Gly mutations of Gi1α, Gi2α and Gi3α were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1a was correlated highly both with the level of expression of this G-protein and with the level of expression of the α2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys → Gly mutants of Gi1α, Gi2α and Gi3α required 10Ő15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the α2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

Downregulation of caveolin by chronic β-adrenergic receptor stimulation in mice

1997 ◽  
Vol 273 (6) ◽  
pp. C1957-C1962 ◽  
Author(s):  
Naoki Oka ◽  
Kuniya Asai ◽  
Raymond K. Kudej ◽  
John G. Edwards ◽  
Yoshiyuki Toya ◽  
...  
Keyword(s):  
G Proteins ◽  
Adrenergic Receptor ◽  
Skeletal Muscles ◽  
Ventricular Myocardium ◽  
Northern Blotting ◽  
Receptor Stimulation ◽  
Caveolin 1 ◽  
Gradient Fractionation ◽  
The Brain ◽  
Isoproterenol Infusion

Caveolae, flask-shaped invaginations of cell membranes, are believed to play pivotal roles in transmembrane transportation of molecules and cellular signaling. Caveolin, a structural component of caveolae, interacts directly with G proteins and regulates their function. We investigated the effect of chronic β-adrenergic receptor stimulation on the expression of caveolin subtypes in mouse hearts by immunoblotting and Northern blotting. Caveolin-1 and -3 were abundantly expressed in the heart and skeletal muscles, but not in the brain. Continuous (−)-isoproterenol, but not (+)-isoproterenol, infusion via osmotic minipump (30 μg ⋅ g−1 ⋅ day−1) for 13 days significantly downregulated both caveolin subtypes in the heart. The expression of caveolin-1 was reduced by 48 ± 6.1% and that of caveolin-3 by 28 ± 4.0% ( P < 0.01, n = 8 for each). The subcellular distribution of caveolin subtypes in ventricular myocardium was not altered as determined by sucrose gradient fractionation. In contrast, the expression of both caveolin subtypes in skeletal muscles was not significantly changed. Our data suggest that the expression of caveolin subtypes is regulated by β-adrenergic receptor stimulation in the heart.

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