Showing papers by "Graeme Milligan published in 1988"

Techniques used in the identification and analysis of function of pertussis toxin-sensitive guanine nucleotide binding proteins

Abstract: All receptors that interact with effector systems to modulate the intracellular levels of a second messenger appear to do so via the intermediacy of members of a family of guanine nucleotide binding proteins (Gproteins). Rodbell and coworkers, whilst studying the ability of the peptide hormone glucagon to stimulate adenylate cyclase activity in hepatocytes, were the first to demonstrate definitively a specific requirement for guanine nucleotides in hormonal function (Rodbell et al., 1971a). Since these pioneering studies, it has become increasingly clear that a considerable number of unique but highly homologous G-proteins are the sites of action for guanine nucleotides in these processes. The purpose of this review will be to discuss the techniques that are currently in widespread use to identify and assess the functions of individual members of a subfamily of these signal-transducing G-proteins which are substrates for ADP-ribosylation catalysed by pertussis toxin.

Purification of heterotrimeric GTP-binding proteins from brain: identification of a novel form of Go.

Abstract: Using high-resolution Mono-Q anion-exchange chromatography, we purified four distinct GTP-binding proteins from bovine brain. Each consists of alpha and associated beta/gamma subunits, and each is a substrate for pertussis toxin catalyzed ADP-ribosylation. We defined the relationship between the alpha subunits of the purified proteins and cloned cDNAs encoding putative alpha subunits (1) by performing immunoblots with peptide antisera with defined specificity and (2) by comparing the migration on two-dimensional gel electrophoresis of the purified proteins, and of the in vitro translated products of cDNAs encoding alpha subunits. Purified G proteins with alpha subunits of 39, 41, and 40 kDa (G39, G41, and G40 in order of abundance) correspond to the products of Go, Gi1, and Gi2 cDNAs. We purified a novel G protein with an alpha subunit slightly above 39 kDa (G39*). G39* is less abundant than G39, elutes earlier than G39 on Mono-Q chromatography, and has a more basic pI (6.0 vs 5.6) than G39. G39 and G39*, however, are indistinguishable on immunoblots with a large number of specific antisera. The data suggest that G39* may represent a novel form of Go, differing in posttranslational modification rather than primary sequence.

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

Abstract: 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.

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.

Abstract: 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.

Differential regulation of amounts of the guanine-nucleotide-binding proteins Gi and Go in neuroblastoma x glioma hybrid cells in response to dibutyryl cyclic AMP.

Abstract: Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like processes. Pertussis-toxin-catalysed ADP-ribosylation indicated that amounts of guanine-nucleotide-binding proteins (G-proteins), which are substrates for this toxin, were approximately doubled in membranes from the 'differentiated' cells in comparison with the control cells. Immunoblotting of membranes derived from either untreated or dibutyryl cyclic AMP-treated cells with anti-peptide antisera specific for the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi and Go demonstrated that amounts of these G-proteins were reciprocally modulated during the differentiation process. In comparison with the untreated cells, the amount of Gi in the 'differentiated' cells was decreased, whereas the amount of Go was substantially increased. Stimulation of high-affinity GTPase activity in response to opioid peptides, which in this cell line interact with an opioid receptor of the delta subclass, was much decreased, and inhibition of adenylate cyclase activity was almost entirely attenuated in the 'differentiated'-cell membranes in comparison with membranes of untreated cells. Opioid receptor number was also decreased in membranes of the dibutyryl cyclic AMP-treated cells in comparison with the control cells. These data demonstrate that relatively small changes in the observed pattern of pertussis-toxin-catalysed ADP-ribosylation of membranes can mask more dramatic alterations in amounts of the individual pertussis-toxin-sensitive G-proteins, and further demonstrate the importance of methodologies able to discriminate between the different gene products.

Selective deficiency of guanine nucleotide-binding protein go in two dopamine-resistant pituitary tumors

Abstract: G proteins were quantitated by immunoblot in normal rat anterior pituitary, an estrone-induced pituitary adenoma, and in two transplantable pituitary tumors resistant to dopamine, 7315a and MtTW15. Antisera specific for the aQ and 6 subunits or the ai subunit of G proteins were tested with all preparations. While the a-,- and 6 subunits were found to be present in variable concentrations in all preparations, the a Q subunit was very 1 ow or undetectable in the transplantable tumors. These findings suggest that tumor resistance to the dopamine inhibitory actions on prolactin release and on tumor growth may be due to the deficiency of a Go protein.

Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells.

Abstract: NG108-15 neuroblastoma x glioma hybrid cells express a major 45 kDa substrate for cholera toxin and a 40 kDa substrate(s) for pertussis toxin when ADP-ribosylation is performed in the presence of GTP. In the absence of exogenous GTP, however, cholera toxin was shown to catalyse incorporation of radioactivity into a 40 kDa protein as well as into the 45 kDa polypeptide. In membranes of cells which had been pretreated in vivo with pertussis toxin, the 40 kDa band was no longer a substrate for either pertussis or cholera toxin in vitro, whereas in membranes from cholera-toxin-pretreated cells the 40 kDa band was still a substrate for fresh cholera toxin in vitro and for pertussis toxin. In this cell line, opioid peptides have been shown to inhibit adenylate cyclase exclusively by interacting with Gi (inhibitory G-protein) and with no other pertussis-toxin-sensitive G-protein. Opioid agonists, but not antagonists, promoted the cholera-toxin-catalysed ADP-ribosylation of the 40 kDa polypeptide, hence demonstrating that this cholera-toxin substrate was indeed the alpha-subunit of Gi. These results demonstrate that Gi can be a substrate for either cholera or pertussis toxin under appropriate conditions.

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

Abstract: 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.

Immunochemical and electrophoretic characterization of the major pertussis toxin substrate of the RAW264 macrophage cell line.

Abstract: The pertussis toxin substrate from RAW264 macrophage cell membranes was characterized by two-dimensional gel electrophoresis and by immunoblots using antibodies directed against different guanine nucleotide binding proteins. RAW264 membranes were found to contain one major pertussis toxin substrate, which was recognized by both antibodies AS/6 and LE/3. The AS/6 antibody was made against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of transducin, and the LE/3 antibody was made against the peptide corresponding to amino acids 160-169 of a guanine nucleotide binding protein (Gi-2-alpha) cloned from a mouse macrophage cell line. The RAW264 pertussis toxin substrate was not recognized by either antibody CW/6 or antibody RV/3, which recognize the 41-kilodalton alpha-subunit of brain Gi (Gi-1-alpha) and Go-alpha, respectively. Pertussis toxin substrates from bovine brain were resolved into four major alpha-subunits by two-dimensional gel electrophoresis, and the LE/3 antibody recognized only one of the four proteins. The brain LE/3 reactive protein also reacted with the AS/6 antibody, migrated with a 40K molecular weight, and had an isoelectric point slightly more basis than the RAW264 pertussis toxin substrate. Therefore, the major pertussis toxin substrate in RAW264 cells appears to be Gi-2, and bovine brain contains a relatively minor amount of a closely related guanine nucleotide binding protein.

Treatment of streptozotocin-diabetic rats with metformin restores the ability of insulin to inhibit adenylate cyclase activity and demonstrates that insulin does not exert this action through the inhibitory guanine nucleotide regulatory protein Gi.

Abstract: Insulin caused the inhibition of glucagon-stimulated adenylate cyclase activity in liver plasma membranes, but failed to inhibit this activity in liver membranes from rats made diabetic by treatment with either alloxan or streptozotocin. Treatment of streptozotocin-diabetic rats with insulin, to normalize their blood glucose concentrations, restored this action of insulin. Rats treated with the biguanide drug metformin exhibited a decreased content of the inhibitory guanine nucleotide regulatory protein Gi in liver plasma membranes assessed both structurally, by using a specific polyclonal antibody (AS7), and functionally. Treatment of normal rats with metformin did not alter insulin's ability to inhibit adenylate cyclase in liver plasma membranes; however, metformin treatment of streptozotocin-diabetic rats completely restored this inhibitory action of insulin. Liver plasma membranes from streptozotocin-diabetic animals which either had or had not been treated with metformin had contents of Gi which were less than 10% of those seen in control animals. We conclude that: (i) insulin does not inhibit adenylate cyclase activity through the inhibitory guanine nucleotide regulatory protein Gi; (ii) streptozotocin- and alloxan-induced diabetes elicit a selective insulin-resistant state; and (iii) metformin can exert a post-receptor effect, at the level of the liver plasma membrane, which restores the ability of insulin to inhibit adenylate cyclase.

The use of anti-peptide antisera to probe interactions between receptors and guanine nucleotide binding proteins.

Abstract: FERGUS R. McKENZIE,* IAN MULLANEY,* CECILIA G. UNSON,? ALLEN M. SPIEGEU and GRAEME MILLIGAN*§ Molecitlur i-’harmucoloby Group, Depurtmetits of *Biochemistry arid $ ~’hurmucohby, University of Glusgow, Glusgow GI2 8QQ, Scotlund, U.K.; tDepartment of Biochemistry, The Rockefeller University, New York, NY 10021, U.S.A. and $Metabolic Diseuses Brunch, National institute of Ijiuhetes, Digestive and Kidney Diseuses, Nutionul institiites of Health, Bethesdu, M D 20892, U.S.A.