Showing papers by "Graeme Milligan published in 1994"

Muscarinic M-current inhibition via G alpha q/11 and alpha-adrenoceptor inhibition of Ca2+ current via G alpha o in rat sympathetic neurones.

Abstract: 1. Microinjection of selective antibodies into superior cervical ganglion (SCG) neurones has identified the G-protein alpha-subunits mediating muscarinic receptor inhibition of M-type K+ current (IK(M)) and alpha-adrenoceptor inhibition of Ca2+ current (ICa). 2. Antibodies specific for G alpha q/11, but not those for G alpha o, reduced M-current inhibition by the muscarinic agonist oxotremorine-M, whereas anti-G alpha o antibodies, but not anti-G alpha q/11 or anti-G alpha i1-3 antibodies, reduced calcium current inhibition by noradrenaline. 3. Immunoblots with specific anti-G-protein antibodies demonstrated the presence of both G alpha q and G alpha 11, while G alpha o1 (but virtually no G alpha o2) was present. 4. We conclude that M1 muscarinic receptor inhibition of IK(M) is transduced by G alpha q and/or G alpha 11, and that G alpha o transduces alpha-adrenoceptor inhibition of ICa.

N-terminal fatty acylation of the alpha-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation.

Abstract: The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

Regulation of basal adenylate cyclase activity in neuroblastoma x glioma hybrid, NG108-15, cells transfected to express the human beta 2 adrenoceptor: evidence for empty receptor stimulation of the adenylate cyclase cascade.

Abstract: Clones of neuroblastoma x glioma hybrid, NH108-15, cells expressing differing levels of the human beta 2 adrenoceptor were isolated. Two clones were examined in detail, beta N22 which expressed some 4000 fmol/mg of membrane protein and clone beta N17 which expressed approx. 300 fmol/mg of membrane protein of the receptor. In beta N22 cells ‘basal’ adenylate cyclase activity measured in the presence of Mg2+ was significantly greater than that in wild-type NG108-15 or beta N17 cells. Both isoprenaline and iloprost were able to stimulate adenylate cyclase activity in each of beta N22 and beta N17 membranes. However, the EC50 for isoprenaline stimulation of adenylate cyclase in membranes of beta N22 cells (6 nM) was significantly lower than that in membranes of beta N17 cells (80 nM), whereas the EC50 for iloprost stimulation of adenylate cyclase (approx. 25 nM) was the same in the two clones and in parental NG108-15 cells. The high basal adenylate cyclase activity of beta N22 cell membranes was not a reflection of higher levels of expression of the adenylate cyclase catalytic unit, as adenylate cyclase activity measured in the presence of Mn2+ was equivalent in membranes of each of wild-type NG108-15 cells and clones beta N22 and beta N17. Basal adenylate cyclase activity measured in the presence of Mg2+ in clone beta N22 was significantly reduced, however, by the beta-receptor antagonist propranolol, whereas this agent was without effect on basal adenylate cyclase activity in membranes of wild-type NG108-15 cells. These data indicate that the elevated basal adenylate cyclase cascade in NG108-15 cells expressing high levels of the beta 2 adrenoceptor represents empty receptor activation of the signalling cascade.

Agonist-induced transfer of the alpha subunits of the guanine-nucleotide-binding regulatory proteins Gq and G11 and of muscarinic m1 acetylcholine receptors from plasma membranes to a light-vesicular membrane fraction.

Abstract: A clone of a Chinese hamster ovary (CHO) cell line expressing high levels of the human muscarinic M1 acetylcholine (Hm1) receptor undergoes a substantial agonist-specific down-regulation of both Hm1 receptors and the α subunits of the guanine-nucleotide-binding (G)-proteins Gq and G11 which is accompanied by a desensitization of inositol-phospholipid-specific-phospholipase-C response [Mullaney, I., Dodd, M. W., Buckley, N. J. & Milligan, G. (1993) Biochem. J. 289, 125–131]. To examine early events in this process, the effect of agonist on subcellular distribution of Gqα and G11α and of Hm1 receptors was assessed after short-term and long-term treatment with carbachol. Short-term (30 min) incubation with carbachol (1 mM) induced a simultaneous transfer of a proportion of both Gqα and G11α and Hm1 receptors from plasma membranes to distinct light vesicular membranes. The total number of receptors and of Gqα and G11α in each cell remained unchanged under these conditions. A similar transfer was noted for the G-protein Gsα but not for intrinsic plasma membrane markers. The plasma membrane, as well as light vesicular membrane, pool of Gsα subunit was unaffected by further sustained incubation with carbachol (16 h), whereas Hm1 receptors and both Gqα and G11α proteins were down-regulated to 25% and 40%, respectively, when compared with untreated cells. Such observations support the idea that down-regulation of both the Hm1 receptor and its associated inositol-phospholipid-specific-phospholipase-C-linked G-proteins is produced by two sequential steps. The first is a transfer of signal-transducing polypeptides from the plasma membrane to a non-plasma membrane light vesicle fraction. The second step is represented by an agonist-specific down-regulation pathway. Both the Hm1 receptor and Gqα/G11α appear to follow similar sequestration and down-regulation patterns.

The palmitoylation status of the G-protein G(o)1 alpha regulates its activity of interaction with the plasma membrane.

Abstract: Plasmids containing cDNAs encoding either the wild-type guanine-nucleotide-binding protein G(o)1 alpha or the palmitoylation-negative cysteine-3-to-serine (C3S) mutant of G(o)1 alpha were transfected into Rat 1 cells, and clones stably expressing immunoreactivity corresponding to these polypeptides were isolated. Clones C5B (expressing wild-type G(o)1 alpha) and D3 (expressing the mutant form) were selected for detailed study. Immunoprecipitation of whole cell lysates of each clone labelled with either [3H]palmitate or [3H]myristate demonstrated incorporation of [3H]myristate into both wild-type and the C3S mutant of G(o)1 alpha, but that incorporation of hydroxylamine-sensitive [3H]palmitate was restricted to the wild type. When membrane and cytoplasmic fractions were prepared from cells of either the C5B or D3 clones, although immunodetection of wild-type G(o)1 alpha was observed only in the membrane fraction, the C3S mutant was present in both membrane and cytoplasmic fractions. Furthermore, a significant proportion of the C3S G(o)1 alpha immunoreactivity was also detected in the cytoplasmic fraction if immunoprecipitation of recently synthesized G(o)1 alpha was performed from fractions derived from cells pulse-labelled with [35S]Trans label. Pretreatment of cells of both clones C5B and D3 with pertussis toxin led to complete ADP-ribosylation of the cellular population of G(o)1 alpha in both cell types, irrespective of whether the polypeptide was subsequently found in the membrane or cytoplasmic fraction following cellular disruption. By contrast, separation of membrane and cytoplasmic fractions before pertussis-toxin-catalysed [32P]ADP-ribosylation allowed modification only of the membrane-associated G(o)1 alpha (whether wild-type or the C3S mutant). This labelling was decreased substantially by incubation of the membranes with guanosine 5'-[beta gamma-imido]triphosphate. No cytoplasmic G-protein beta subunit was detected immunologically, and the non-membrane-associated C3S G(o)1 alpha from D3 cells migrated as an apparently monomeric 40 kDa protein on a Superose 12 gel-filtration column. Membrane-associated wild-type and C3S G(o)1 alpha appeared to interact with guanine nucleotides with similar affinity, as no alteration in the dose-response curves for guanine-nucleotide-induced maintenance of a stable 37 kDa tryptic fragment was noted for the two forms of G(o)1 alpha. Chemical depalmitoylation of membranes of clone C5B with neutral 1 M hydroxylamine caused a release of some 25-30% of each of G(o)1 alpha, Gi2 alpha and Gq alpha/G11 alpha from the membranes. Equivalent treatment of D3 cells caused an equivalent release of Gi2 alpha and Gq alpha/G11 alpha, but was unable to cause any appreciable release of the CS3 form of G(o)1 alpha, which was membrane-bound.

Agonist regulation of cellular Gs alpha-subunit levels in neuroblastoma x glioma hybrid NG108-15 cells transfected to express different levels of the human beta 2 adrenoceptor.

Abstract: Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, Gs. Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of Gs alpha. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human beta 2-adrenoceptor cDNA under the control of the beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of beta N22 cells to the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-protein alpha subunits, including Gs alpha. In beta N22 cells the IP prostanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down-regulation of cellular Gs alpha levels. Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.

Quantitative stoichiometry of the proteins of the stimulatory arm of the adenylyl cyclase cascade in neuroblastoma x glioma hybrid, NG108‐15 cells

Abstract: To understand the details of regulation of guanine-nucleotide-binding-protein-linked transmembrane cellular-signalling cascades, it is important to know the absolute levels of each polypeptide component and the stoichiometry of their interactions Amounts of the IP prostanoid receptor, the stimulatory G protein of the adenylyl cyclase cascade (Gsα) and the functional complex of Gsα with adenylyl cyclase, which acts as the cyclic AMP generator, were measured in membranes of neuroblastoma x glioma hybrid, NG108-15, cells As measured by the specific binding of [3H]prostaglandin E1, the IP prostanoid receptor was present in some 100000 copies/cell Gsα assessed by quantitative immunoblotting with recombinantly expressed protein, was present in considerably higher levels (1250000 copies/cell) However, the maximal formation of a complex of Gsα and adenylyl cyclase represented only some 17500 copies/cell The previously established 8:1 stoichiometry of concurrent downregulation of Gsα and the IP prostanoid receptor in these cells [Adie, E J, Mullaney, I, McKenzie, F R & Milligan, G (1992) Biochem J 285, 529–536] indicates that full-agonist occupation of the receptor should be able to activate some 65% of the expressed Gs Despite the potential 70-fold excess of Gsα over the Gsα/adenylyl cyclase complex, IP prostanoid-receptor-agonist-mediated reduction of Gsα levels by some 35% resulted in a 25% reduction in the maximal formation of the Gsα/adenylyl cyclase complex Such results demonstrate that adenylyl cyclase is quantitatively the least highly expressed component of this signalling cascade and suggests that much of the cellular Gsα may not have access to adenylyl cyclase

Beta 3-adrenoceptor agonist-induced down-regulation of Gs alpha and functional desensitization in a Chinese hamster ovary cell line expressing a beta 3-adrenoceptor refractory to down-regulation.

Abstract: Chinese hamster ovary (CHO) cells transfected to express human beta 2- or beta 3-adrenoceptors (beta 2-CHO and beta 3-CHO cells) were exposed to the beta-adrenoceptor agonist isoprenaline at various concentrations and for differing times. Sustained exposure of the beta 2-CHO but not beta 3-CHO cells to isoprenaline resulted in a time- and concentration-dependent down-regulation of the receptor as measured by a reduction in specific binding of [125I]cyanopindolol. Such maintained exposure of cells expressing either receptor to the agonist produced a marked down-regulation of immunologically detectable levels of the alpha subunit of the stimulatory guanine-nucleotide-binding protein Gs. This effect was specific for Gs because levels of both G12 alpha and Gq alpha/G11 alpha were unaltered by isoprenaline treatment of both beta 2-CHO and beta 3-CHO cells. The effect of isoprenaline on Gs alpha down-regulation was some 30-fold more potent in the beta 2-CHO than in the beta 3-CHO cells. Time courses of isoprenaline-induced down-regulation of Gs alpha were not different, however, in the two cell lines. Isoprenaline treatment of the beta 3-CHO cells produced a desensitization of agonist-mediated regulation of adenylyl cyclase, manifested by a 4-fold reduction in the potency and a 30% reduction in maximal effect of the agonist, whereas desensitization of the beta 2-CHO cells was considerably greater (25-fold reduction in potency and 70% reduction in maximal effect). These results demonstrate that agonist-induced down-regulation of the G-protein which interacts with a receptor can be produced by both beta 2- and beta 3-adrenoceptors. Despite apparent concurrence of down-regulation of receptors and G-proteins in other systems [e.g. Adie, Mullaney, McKenzie and Milligan (1992) Biochem. J. 285, 529-536], agonist-induced receptor down-regulation does not appear to be a prerequisite for down-regulation of the G-protein. Furthermore, the results suggest that agonist-induced down-regulation of a G-protein may be sufficient, in the absence of receptor regulation, to induce some agonist desensitization of effector function.

The turkey erythrocyte beta-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein alpha subunits.

Abstract: By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response.

Specificity and functional applications of antipeptide antisera which identify G-protein alpha subunits.

Abstract: Publisher Summary Several attempts to define the specificity of interactions among receptors, G proteins, and effectors made considerable use of exotoxins produced by the bacteria Vibrio cholerae and Bordetella pertussis. These toxins respectively modulated stimulatory and inhibitory regulation of cyclic adenosine monophosphate (AMP) production by causing mono-adenosine diphosphate (ADP)-ribosylation of the G proteins which allow communication among receptors and adenylyl cyclase. Both toxins interfered with the functional interactions among receptors and G proteins. In the case of cholera toxin-catalyzed ADP-ribosylation of Gs, the G protein was converted to an essentially irreversibly activated form which no longer required agonist occupation of a receptor to produce regulation of adenylyl cyclase, whereas in the case of pertussis toxin-catalyzed ADP-ribosylation of the “Gi-like” G proteins, the covalent modification prevented productive coupling among receptors and these G proteins. However, with the realization that there could be multiple G-protein gene products which acted as substrates for these toxins expressed in a single cell or tissue, it became clear that more selective tools would be required to discriminate among receptor functions transmitted to effector moieties by the individual G proteins.

Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of Gi by lysophosphatidic acid (LPA) in wild-type and α2C10 adrenoceptor-transfected Rat 1 fibroblasts

Abstract: Lysophosphatidic acid (LPA) stimulated high-affinity GTPase activity in membranes of Rat 1 fibroblasts. This effect was dose-dependent, with maximal effects at 10 microM LPA, and was attenuated by pertussis toxin but not by cholera toxin pretreatment of the cells, indicating that the effect was likely to be produced by a Gi-like G-protein. LPA stimulation of high-affinity GTPase was also observed in a clone of Rat 1 fibroblasts that had been transfected to express the human alpha 2C10 adrenoceptor. The alpha 2 adrenoceptor agonist UK14304 also stimulated high-affinity GTPase activity in membranes of these cells, but not in parental Rat 1 cells. LPA was also able to promote cholera toxin-catalysed [32P]ADP-ribosylation of Gi. This effect of LPA was also prevented by pretreatment of the cells with pertussis toxin but not cholera toxin. LPA-stimulated cholera toxin-catalysed [32P]ADP-ribosylation of Gi in membranes of the alpha 2C10 adrenoceptor-expressing clone was additive with that produced by UK14304. Dose-response curves for LPA in the two assays of G-protein activation were coincident. The results presented herein demonstrate conclusively that the pertussis toxin-sensitive effects of LPA in Rat 1 fibroblasts and a clone of these cells expressing the alpha 2C10 adrenoceptor are produced directly by the activation of Gi.