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G Milligan

Publications and source records attributed to G Milligan.

At least 163 records · Page 9Linked to original sources
PubMed1994

The gonadotrophin-releasing hormone receptor of alpha T3-1 pituitary cells regulates cellular levels of both of the phosphoinositidase C-linked G proteins, Gq alpha and G11 alpha, equally.

The alpha T3-1 cell line, an immortalized gonadotroph cell line, expresses high levels of the gonadotrophin-releasing hormone (GnRH) receptor. Sustained exposure of these cells to the GnRH receptor agonist des-Gly10-[D-Ala6]luteinizing hormone-releasing hormone ethylamide resulted in a substantial down-regulation of cellular levels of a combination of the alpha subunits of the phospholipase C-beta 1-linked G proteins Gq and G11, as assessed by immunoblotting with an antiserum able to identify these two proteins equally. This effect was dependent upon the concentration of agonist used (EC50 = 4 nM) and on the time of the treatment (t1/2 = 6 hr) when a maximally effective concentration of agonist (1 microM) was used. Comparison of agonist regulation of inositol phosphate generation and Gq alpha/G11 alpha down-regulation demonstrated that effects on inositol phosphate production were approximately 3-fold more potent. In contrast to Gq alpha/G11 alpha, membrane-associated levels of Gs alpha and G12 alpha, the G proteins that transduce stimulatory and inhibitory regulation, respectively, of adenylyl cyclase, were not altered by agonist treatment. Analysis of mRNA by reverse transcriptase/polymerase chain reaction indicated the coexpression by alpha T3-1 cells of mRNA corresponding to both Gq alpha and G11 alpha. Immunoblotting with antisera selective for either Gq alpha or G11 alpha confirmed their coexpression. Resolution of membranes from untreated and agonist-treated alpha T3-1 cells under sodium dodecyl sulfate-polyacrylamide gel electrophoresis conditions able to separate Gq alpha from G11 alpha indicated that G11 alpha was more prevalent than Gq alpha at steady state but that agonist treatment regulated cellular levels of both of these G proteins in a nonselective manner. Sustained activation of protein kinase C with phorbol myristate acetate was unable to mimic agonist regulation of cellular Gq alpha/G11 alpha levels, as was treatment of alpha T3-1 cells with the selective protein kinase C inhibitor chelerythrine. These data suggest that the GnRH receptor is able to interact functionally with both Gq alpha and G11 alpha in alpha T3-1 cells and that sustained exposure to a GnRH receptor agonist selectively regulates the cellular levels of the G proteins that interact with the receptor.

Animals↗
PubMed1993

Distribution and relative levels of expression of the phosphoinositidase-C-linked G-proteins Gq alpha and G11 alpha: absence of G11 alpha in human platelets and haemopoietically derived cell lines.

Membranes of a variety of clonal cell lines, including neuroblastoma x glioma hybrid NG108-15, glioma C6, Rat 1 and CHO fibroblasts and the pituitary-derived cell lines alpha T3 and GH3 were immunoblotted with an antiserum (CQ) raised against a synthetic peptide corresponding to the C-terminal decapeptide of the alpha subunits of the phosphoinositidase-C-linked G-proteins Gq and G11. In SDS-PAGE conditions able to resolve these two polypeptides, direct evidence was obtained for co-expression of these two G-proteins in all of the above cell lines. The ratio of these two G-proteins varied substantially (alpha 11/alpha q = 0.25-2.5) between the cell lines. In human platelets and in a range of haemopoietically derived human cell lines including U937 (monoblasts), Raji (Burkitts lymphoma) and Jurkat (mature T cell) expression of G11 alpha was not detected. This was not due to the inability of the antiserum to identify human G11 alpha as other human cell lines co-expressed both G-proteins. A third, unidentified CQ reactive polypeptide of similar mobility was resolved and present in all cell lines examined.

Animals↗
PubMed1993

Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by alpha 2-adrenergic receptors expressed in fibroblasts.

The alpha 2-adrenergic receptors are linked to inhibition of adenylylcyclase and, under certain circumstances, to stimulation of phospholipid hydrolysis via pertussis toxin-sensitive G proteins. Here we show that alpha 2-adrenergic receptors can couple to an alternative signaling pathway. When expressed in Rat-1 cells, stimulation of the alpha 2A receptor, which couples to Gi2 and Gi3, causes rapid, transient activation of the protooncogene product p21ras as measured by an increase in the amount of bound GTP. Furthermore, alpha 2A receptor stimulation causes rapid phosphorylation of the p42 mitogen-activated protein (MAP) kinase. Pertussis toxin completely inhibits both p21ras activation and MAP kinase phosphorylation, but both responses appear to be independent of adenylylcyclase inhibition or phospholipase stimulation. Thus, alpha 2-adrenergic receptors can couple to the p21ras-MAP kinase pathway via Gi, which may explain the mitogenic potential of alpha 2 agonists in certain cell types; together with previous results, these findings further suggest that activation of this pivotal signaling pathway may be a common event in the action of Gi-coupled receptors.

Animals↗
PubMed1993

Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer's disease.

The aim of this study was to assess the effect of Alzheimer's disease has on the functional integrity of several signal transduction proteins. The relative levels of the G-protein alpha subunits Gs alpha-L, Gs alpha-S, Gi alpha-2 and G(o) alpha were measured by western blotting and found to be unchanged in membranes prepared from Alzheimer-diseased frontal cortex or hippocampus compared to control brains. However the activity of the G-protein associated enzyme, high affinity GTPase, was found to be reduced in the frontal cortex (reduced by 25%) and by a similar magnitude in the hippocampus (reduced by 27%) of Alzheimer subjects. The same membrane preparations were also assayed for the activity of adenylate cyclase. Basal enzyme activity was not significantly altered in Alzheimer diseased hippocampus, but was markedly reduced (by 45%) in the frontal cortex. The ability of fluoride and aluminium ions to stimulate adenylate cyclase was not significantly changed in either brain region. This suggests that G-proteins, especially Gs, are still able to interact with this enzyme. These results indicate that although the presence of Alzheimer's disease does not significantly alter G-protein levels, changes have taken place in the overall activity of these proteins. However this alteration does not affect their ability to stimulate adenylate cyclase activity.

Adenylyl Cyclases↗
PubMed1993

Enhanced degradation of the phosphoinositidase C-linked guanine-nucleotide-binding protein Gq alpha/G11 alpha following activation of the human M1 muscarinic acetylcholine receptor expressed in CHO cells.

Treatment of CHO cells stably expressing the human M1 muscarinic acetylcholine (HM1) receptor with the cholinergic agonist carbachol results in a reduction in cellular levels of Gq alpha/G11 alpha. Half-maximal effects are produced by 3 h, and a new steady state of some 50% of the resting levels of Gq alpha/G11 alpha is subsequently established [Mullaney, Dodd, Buckley and Milligan, (1993) Biochem. J. 289, 125-131]. To analyse the mechanism of this effect, we examined the rate of turnover of Gq alpha/G11 alpha in these HM1-expressing cells in the presence and absence of carbachol (1 mM). In untreated cells the measured removal of 35S-labelled Gq alpha/G11 alpha was adequately described by a monoexponential curve with a half-time (t0.5) of 18.0 +/- 2.2 h. When the cells were treated with carbachol a more complex pattern of Gq alpha/G11 alpha degradation was observed. Upon addition of the agonist, the rate of degradation initially increased markedly (t0.5 = 2.9 +/- 0.2 h). The maintained presence of the agonist was unable, however, to sustain the enhanced rate of degradation. Beyond 8 h of treatment with carbachol, degradation of Gq alpha/G11 alpha returned to a rate close to that observed in untreated cells (t0.5 = 18.5 +/- 1.3 h). Parallel experiments indicated that the effect of carbachol was specific for Gq alpha/G11 alpha, as the t0.5 of Gi2 alpha (approx. 30 h) was not affected by the agonist. Analysis of Gq alpha/G11 alpha mRNA levels by reverse transcriptase/PCR indicated that there was no difference in cells maintained in the absence and presence of carbachol. Such data demonstrate that agonist-induced establishment of a new steady-state level of Gq alpha/G11 alpha results from an initial receptor-mediated enhancement of protein turnover followed by a desensitization of the receptor response.

Animals↗
PubMed1993

The human muscarinic M1 acetylcholine receptor, when express in CHO cells, activates and downregulates both Gq alpha and G11 alpha equally and non-selectively.

CHO cells express both of the phosphoinositidase C-linked G-proteins Gq and G11. G11 alpha is some 2.5-fold more highly expressed than Gq alpha in membranes of these cells. Following transfection and stable expression of CHO cells with DNA encoding the human muscarinic M1 acetylcholine (HM1) receptor, chronic treatment of the cells with the cholinergic agonist carbachol resulted in down-regulation of membrane levels of both Gq alpha and G11 alpha. Dose-response curves to carbachol produced identical EC50 values for agonist-induced down-regulation of the two G-proteins and both were down-regulated with the same time course. These data indicate that the HM1 receptor interacts with the activates both Gq alpha and G11 alpha equivalently and non-selectively in a whole cell system in which the receptor has access to both G-proteins.

Animals↗
PubMed1993

Immunological analysis of glucose transporters expressed in different regions of the rat brain and central nervous system.

Glucose is the major energy source for brain tissue. It is now well established that glucose crosses the blood-brain barrier by facilitated diffusion mediated by the erythrocyte-type (GLUT 1) glucose transporter. Northern blot analysis has indicated that brain tissue expresses two different glucose transporters, the erythrocyte-type transporter and the brain-type transporter (GLUT 3). Here we use a panel of antibodies specific for four glucose transporters to examine the distribution of these transporters in various regions of rat brain and central nervous system. We show that GLUT 1 and GLUT 3 are expressed in virtually all regions of the brain examined, but that the relative levels of the proteins differ. We also demonstrate that GLUT 2 appears to be widely expressed in all brain regions, but at apparently low levels. In addition, GLUT 4 is expressed in the pituitary, the hypothalamus and the medulla. These results are discussed in the light of potential glucose-regulated processes in the brain.

Animals↗
PubMed1993

A novel N-terminal motif for palmitoylation of G-protein alpha subunits.

We have examined the post-translational processing of G alpha subunits expressed endogenously in rat PC12 and NG108-15 rat/mouse hybrid cells, and after transfection of cDNA expression constructs into COS cells. Thioester-linked palmitoylation of alpha o, alpha s, alpha q/alpha 11 and alpha 12 has been detected by metabolic labelling with [3H]palmitate and immunoprecipitation. Palmitoylation of alpha o occurs post-translationally in cells treated with protein-synthesis inhibitors, suggesting possible dynamic acylation. Palmitoylation of the C-terminal CAAX motif has been excluded. Site-directed mutagenesis of alpha o has been used to implicate the site of modification as a cysteine residue next to the N-terminal myristoylated glycine, in a novel protein-lipid modification motif Met-Gly-Cys. The non-palmitoylated alpha o mutant is still myristoylated but shows reduced membrane binding, suggesting that reversible palmitoylation may regulate G alpha localization and function.

Acylation↗
PubMed1993

Agonist activation of transfected human M1 muscarinic acetylcholine receptors in CHO cells results in down-regulation of both the receptor and the alpha subunit of the G-protein Gq.

CHO cells stably transfected with cDNA encoding the human M1 muscarinic acetylcholine (HM1) receptor were treated with the cholinergic agonist carbachol at various concentrations for differing times. Levels of the HM1 receptor and of a range of G-proteins were subsequently measured. Carbachol treatment of the transfected cells caused a substantial down-regulation of cellular levels of the alpha subunit of Gq (Gq alpha), but did not significantly alter cellular levels of the alpha subunits of Gs or Gi2. A small decrease in levels of G-protein beta-subunit was also produced. Parallel assessment of agonist-induced down-regulation of the HM1 receptor demonstrated that it was lost in concert with the G-protein. Similar concentrations of carbachol (5 microM) were required to produce half-maximal stimulation of inositol phosphate generation and loss of each of the HM1 receptor and Gq alpha, and half-maximal losses of both receptor and Gq alpha were produced by 3 h of treatment with 1 mM-carbachol. By contrast, treatment of the non-transfected parental CHO cells, which do not express detectable levels of the receptor, with carbachol had no effect on cellular Gq alpha levels. Concurrent treatment of the HM1-expressing CHO cells with carbachol and cycloheximide indicated that suppression of protein synthesis de novo did not mimic the effect of carbachol, and hence even complete inhibition of transcription of the Gq alpha gene and/or translation of pre-existing Gq alpha mRNA could not account for the agonist-induced effect. We have previously noted that cellular levels of both Gs alpha [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093] and the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi1, Gi2 and Gi3 [Green, Johnson and Milligan (1990) J. Biol. Chem. 265, 5206-5210] can be regulated in certain cell systems by agonist activation of receptors expected to interact with these G-proteins. These results demonstrate that the same is true of Gq alpha and suggest that agonist-induced co-ordinate loss of receptors and associated G-proteins may be a more common feature than has been appreciated to date.

Animals↗
PubMed1993

Mechanisms of multifunctional signalling by G protein-linked receptors.

A number of G protein-linked receptors have recently been shown to regulate multiple effector pathways both when expressed endogenously and, more frequently, following transfection into heterologous systems. The mechanisms responsible for such signal bifurcation are beginning to be unravelled. In this Viewpoint article, Graeme Milligan analyses the information on these mechanisms and considers whether they are dependent upon either levels of expression of the receptor or the cell type.

Adenylyl Cyclases↗
PubMed1993

Agonist regulation of cellular G protein levels and distribution: mechanisms and functional implications.

Exposure of cells to agonists of receptors linked to G proteins can result in downregulation of cellular levels or redistribution of G proteins from membranes to the cytosol. Agonist-induced reductions in G protein levels have been observed for members of each of the Gs, Gi and Gq families of G proteins, are likely to be dependent upon the level of receptor expression, and are generally restricted to the G protein(s) with which the receptor interacts. The mechanisms responsible, reviewed here by Graeme Milligan, vary with cell type and include both second messenger-dependent and -independent enhanced protein degradation. Agonist-induced reduction in cellular G protein levels can provide one mechanism for the development of sustained heterologous desensitization.

Animals↗
PubMed1993

An arginine residue is the site of receptor-stimulated, cholera toxin-catalysed ADP-ribosylation of pertussis toxin-sensitive G-proteins.

Cholera toxin-catalysed [32P]ADP-ribosylations were performed in the absence of guanine nucleotides on membranes of a clone (1C) of Rat 1 fibroblasts which express high levels of the alpha 2-C10 adrenergic receptor. As well as incorporation of radioactivity into 45,000 and 42,000 M(r) polypeptides which represent forms of Gs alpha, there was weak labelling of a 40,000 M(r) polypeptide(s). Addition of the alpha 2 adrenergic agonist UK14304 to such assays enhanced markedly the incorporation of radioactivity into the 40,000 M(r) polypeptide(s) but did not alter labelling of the forms of Gs. We have previously demonstrated that the 40,000 M(r) polypeptide(s) labelled in such a manner represents a combination of the alpha subunits of Gi2 and Gi3 [Milligan et al. (1991) J. biol. Chem. 266, 6447-6455]. Mercuric acetate treatment of membranes prelabelled with [32P]ADP-ribose by exposure to pertussis toxin and [32P]NAD removed totally the radiolabel from both Gi2 and Gi3. However, cholera toxin-catalysed [32P]ADP-ribosylation of either the alpha subunits of the Gi-subtypes or of forms of Gs was unaffected by such treatment. By contrast, prolonged, but not short-term, exposure to neutral hydroxylamine removed radiolabel incorporated by cholera toxin from the Gi-subtypes and from Gs but did not remove [32P]ADP-ribose incorporated by pertussis toxin from the Gi-subtypes. It is concluded that ADP-ribosylation of pertussis toxin-sensitive G-proteins by cholera toxin, which can be induced by exposure of membranes to agonists for receptors which interact with that G-protein, is at an arginine residue. It is suggested that this residue may be Arg 179 in Gi2 alpha and Arg 178 in Gi3 alpha.

Adenosine Diphosphate Ribose↗
PubMed1993

G-proteins in experimental hypertension: a study of spontaneously hypertensive rat myocardial and renal cortical plasma membranes.

OBJECTIVE: Spontaneously hypertensive rat (SHR) myocardium is known to exhibit reduced beta-adrenergic agonist-stimulated adenylyl cyclase activity. As G-proteins play a pivotal role in transducing information from the receptor to adenylyl cyclase, a study was undertaken to assess whether changes in G-protein expression and functioning could explain this. DESIGN: Studies were performed in plasma membrane homogenates from vascular tissue (myocardium) from 11-week-old SHR (weighing 255 g) and control rats from both Wistar-Kyoto (WKY) and Wistar strains. Similar studies were performed in non-vascular tissue (renal cortical plasma membranes) to assess whether any observed changes are part of a more widely distributed membrane defect. METHODS: G-protein function was inferred from studies of adenylyl cyclase activity and levels of G-protein subunits (Gs alpha, Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha and beta) were assessed by immunoblotting. RESULTS: Differences in adenylyl cyclase activity were seen in SHR compared with WKY rat myocardium: in WKY rats adenylyl cyclase activity was greater than in SHR under forskolin-stimulated conditions and in the presence of fluoride and several ligands which couple to the catalytic unit of adenylyl cyclase via Gs, including the receptor-linked species prostaglandin E1, glucagon and isoproterenol. However, with the exception of forskolin-stimulated activity, which in SHR was greater than in Wistar rats, SHR myocardial adenylyl cyclase activities were similar to those in Wistar rat membranes. Immunoblotting studies showed similar levels of G-protein subunits in all three strains. Studies of renal cortical plasma membranes failed to identify any differences in adenylyl cyclase activity or in G-protein subunit levels. CONCLUSIONS: SHR, WKY rats and Wistar rats exhibit differences in myocardial adenylyl cyclase activity which are not seen in renal cortical plasma membranes. These are not related to hypertension or to differences in G-protein levels, and probably reflect strain differences in Gs function.

Adenylyl Cyclases↗
PubMed1993

G-proteins in essential hypertension: a study of human platelet plasma membranes.

AIM OF STUDY: Guanine nucleotide regulatory (G) proteins act as key signal transducers for many hormones, growth factors and neurotransmitters, and have been shown to have an important influence on platelet function. As abnormal G-protein levels and activity have been reported in platelets from human non-insulin-dependent diabetics (NIDDM) we studied G-protein function in essential hypertension, a condition which is also associated with insulin resistance and in which abnormal platelet function has been reported. METHODS: G-protein function was deduced from studies of adenylyl cyclase activity in platelet membrane preparations from 14 untreated essential hypertensives and 14 controls matched as far as possible for age and sex. Levels of G-protein subunits (Gs alpha, Gi alpha 2 and beta-subunits) were assessed by immunoblotting, using platelets from 15 subjects with untreated essential hypertension and 15 controls. RESULTS: No changes in levels of G-proteins (Gs alpha, Gi alpha 2 and beta-subunits) were seen. However, in contrast to the observations in NIDDM, the studies of adenylyl cyclase function identified greater prostaglandin E1-stimulated activity in hypertensive platelet membranes than in controls (88.8 verus 72% stimulation, P = 0.018). This may have a physiological basis in protecting cells against a Ca2+ overload. CONCLUSION: These data are in opposition to the theory that a common defect in G-proteins can explain the association between hypertension and NIDDM.

Adenylyl Cyclases↗