scholarly journals Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells

1987 ◽  
Vol 245 (2) ◽  
pp. 501-505 ◽  
Author(s):  
G Milligan
Keyword(s):  
Pertussis Toxin ◽  
Cellular Proliferation ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
High Affinity ◽  
Glioma C6 ◽  
Guanine Nucleotide Binding

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.

scholarly journals Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates

1988 ◽  
Vol 249 (3) ◽  
pp. 653-659 ◽  
Author(s):  
F R McKenzie ◽  
E C H Kelly ◽  
C G Unson ◽  
A M Spiegel ◽  
G Milligan
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Opioid Peptides ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
High Affinity ◽  
C Terminus ◽  
Inhibitory G Protein ◽  
Guanine Nucleotide Binding

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.

scholarly journals Differential effects of suramin on the coupling of receptors to individual species of pertussis-toxin-sensitive guanine-nucleotide-binding proteins

1988 ◽  
Vol 251 (1) ◽  
pp. 201-205 ◽  
Author(s):  
S J Butler ◽  
E C H Kelly ◽  
F R McKenzie ◽  
S B Guild ◽  
M J O Wakelam ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Hybrid Cell ◽  
Calf Serum ◽  
Opioid Peptide ◽  
Individual Species ◽  
Foetal Calf Serum ◽  
Maximal Velocity ◽  
Gtpase Activity ◽  
High Affinity

The anti-helminthic drug suramin inhibited the basal high-affinity GTPase activity of both C6 BU1 glioma and NG 108-15 neuroblastoma x glioma hybrid-cell membranes with an IC50 (concentration causing half-maximal inhibition) value close to 30 micrograms/ml. This effect was shown to occur via a non-competitive mechanism in which the binding affinity of the G-proteins for GTP was not altered, but the maximal velocity of the subsequent hydrolysis was reduced. In NG 108-15 membranes, both opioid peptides and foetal-calf serum stimulated high-affinity GTPase activity in a pertussis-toxin-sensitive manner. These effects have previously been shown to be mediated by different G-proteins [McKenzie, Kelly, Unson, Spiegel & Milligan (1988) Biochem. J. 249, 653-659]. Suramin completely prevented the opioid-peptide-stimulated increase in GTP hydrolysis, but did not prevent the opioid peptide from binding to its receptor. Suramin, however, did not block the foetal-calf-serum-stimulated GTPase response. This selective action of suramin provides further evidence for distinct roles for two separate pertussis-toxin-sensitive G-proteins in signal transduction in NG 108-15 membranes and provides the first evidence for a selective effect of a drug on the functions of different G-proteins.

scholarly journals GTP analogues promote release of the α subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells

1988 ◽  
Vol 254 (2) ◽  
pp. 391-396 ◽  
Author(s):  
G Milligan ◽  
I Mullaney ◽  
C G Unson ◽  
L Marshall ◽  
A M Spiegel ◽  
...  
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Alpha Subunit ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Gamma Subunits ◽  
Guanine Nucleotide Binding

The major pertussis-toxin-sensitive guanine nucleotide-binding protein of rat glioma C6 BU1 cells corresponded immunologically to Gi2. Antibodies which recognize the alpha subunit of this protein indicated that it has an apparent molecular mass of 40 kDa and a pI of 5.7. Incubation of membranes of these cells with guanosine 5′-[beta gamma-imido]triphosphate, or other analogues of GTP, caused release of this polypeptide from the membrane in a time-dependent manner. Analogues of GDP or of ATP did not mimic this effect. The GTP analogues similarly caused release of the alpha subunit of Gi2 from membranes of C6 cells in which this G-protein had been inactivated by pretreatment with pertussis toxin. The beta subunit was not released from the membrane under any of these conditions, indicating that the release process was a specific response to the dissociation of the G-protein after binding of the GTP analogue. Similar nucleotide profiles for release of the alpha subunits of forms of Gi were noted for membranes of both the neuroblastoma x glioma hybrid cell line NG108-15 and of human platelets. These data provide evidence that: (1) pertussis-toxin-sensitive G-proteins, in native membranes, do indeed dissociate into alpha and beta gamma subunits upon activation; (2) the alpha subunit of ‘Gi-like’ proteins need not always remain in intimate association with the plasma membrane; and (3) the alpha subunit of Gi2 can still dissociate from the beta/gamma subunits after pertussis-toxin-catalysed ADP-ribosylation.

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 Persistent activation of the α subunit of Gs promotes its removal from the plasma membrane

1989 ◽  
Vol 260 (3) ◽  
pp. 837-841 ◽  
Author(s):  
G Milligan ◽  
C G Unson
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Membrane Attachment ◽  
Guanine Nucleotide Binding ◽  
Gs Alpha

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5′-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document