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Biomedical subjects

G Milligan

Publications and source records attributed to G Milligan.

At least 91 records · Page 5Linked to original sources
PubMed1997

Interaction of the G-protein G11alpha with receptors and phosphoinositidase C: the contribution of G-protein palmitoylation and membrane association.

Wild-type and palmitoylation defective mutants of the murine G protein G11alpha were transfected into HEK293 cells. Wild-type G11alpha was membrane associated, Cys9Ser Cys10Ser G11alpha was present in the soluble fraction whilst both Cys9Ser G11alpha and Cys10Ser G11alpha were distributed between the fractions. Expression of the rat TRH receptor resulted in agonist stimulation of inositol phosphate accumulation. The degree of stimulation produced by TRH following co-transfection of the palmitoylation-resistant forms of G11alpha compared to the wild-type protein correlated with the amount of membrane-associated G protein.

Animals↗
PubMed1997

Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants.

The alpha2A-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 alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha 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 alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-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 alpha-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 Gi1alpha, Gi2alpha and Gi3alpha 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 Gi1alpha, Gi2alpha and Gi3alpha 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 Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys --> Gly mutants of Gi1alpha, Gi2alpha and Gi3alpha required 10-15-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 alpha2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

Adenylate Cyclase Toxin↗
PubMed1997

Inverse agonism and the regulation of receptor number.

Inverse agonists are ligands that preferentially stabilize inactive conformations of G protein-coupled receptors. In a range of systems, sustained treatment with inverse agonists can produce substantially greater upregulation of receptor levels than antagonists. The use of constitutively active mutant receptors can exaggerate this effect but may also allow agonists and antagonists to mimic the effect by preventing denaturation of the mutant receptor polypeptide. In this review Graeme Milligan and Richard Bond consider the basis for these effects and their therapeutic implications.

Adrenergic beta-2 Receptor Agonists↗
PubMed1997

Agonist-mediated tyrosine phosphorylation of isoforms of the shc adapter protein by the delta opioid receptor.

Maximally effective concentrations of the opioid agonist D-ala2-D-leu5-enkephalin resulted in some 2-3-fold enhancement of tyrosine phosphorylation of the p52 Shc adapter protein in a clone of Rat-1 fibroblasts transfected to express stably the murine delta opioid receptor. More limited modifications of the tyrosine phosphorylation status of the p46 and p66 forms of Shc were observed in parallel. Epidermal growth factor caused some 10-12-fold enhancement of tyrosine phosphorylation of p52 Shc and marked increases in the p46 and p66 forms. The effect of D-ala2-D-leu5-enkephalin was prevented by pretreatment of the cells with pertussis toxin and was not observed in non-transfected parental fibroblasts whereas the effect of epidermal growth factor was still manifest in both these situations. Half-maximal effects of D-ala2-D-leu5-enkephalin on p52 Shc tyrosine phosphorylation were produced with sub-nanomolar concentrations, in agreement with previous results on the tyrosine phosphorylation of p44MAPK (Burt et al., 1996). p52 Shc became tyrosine phosphorylated more rapidly than p44MAPK in response to D-ala2-D-leu5-enkephalin and its enhanced tyrosine phosphorylation was maintained for at least 10 min.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Tailoring cAMP-signalling responses through isoform multiplicity.

Multiple forms of cAMP phosphodiesterases (PDE), adenylate cyclase and protein kinase A (PKA) allow cells to tailor the responsiveness of the cAMP-signalling system and to allow for its dynamic adjustment. Multiple forms of these enzymes confer spatial and temporal characteristics on cAMP signalling so as to affect compartmentalised responses within a single cell type. The ability to breach the PKA activation threshold can depend upon either or both the activation of adenylate cyclase and inhibition of specific PDE isoforms.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Differential G-protein expression during B- and T-cell development.

The molecular mechanisms underlying B- and T-cell development are, as yet, poorly understood. However, as G proteins regulate a diverse range of biological responses including growth, proliferation and differentiation, we have investigated differential expression of G proteins during B- and T-cell development with the aim of identifying key signals involved in lymphocyte maturation. Differential expression of beta 1/2 and alpha-subunits of the Gs-, i- and q-families was found throughout lymphoid development. Most strikingly, G alpha i1 and G alpha i1 were very weakly, or not expressed in pre-, immature and mature B cells, thymocytes or mature T cells, but strongly induced in mature B-lymphoblastoid cell lines, some of which have been used as models of germinal centre B cells, suggesting that expression of these G proteins may correlate with the later stages of B-cell development. In contrast, G alpha 16 expression was highest in T cells and pre-B cells and progressively declined with B-cell maturation. These findings suggest that G proteins, and the signals they regulate, such as ion channels and/or adenylate cyclase (G alpha s/i) and phospholipase C (G beta gamma and G alpha 11/16) are differentially regulated in lymphoid cells in a maturation-and lineage-dependent manner.

Animals↗
PubMed1997

Regulation of spontaneous activity of the delta-opioid receptor: studies of inverse agonism in intact cells.

Adenylyl cyclase activity was measured following labelling of the cellular ATP pool with [3H]adenine in intact Rat-1 fibroblasts that had been stably transfected to express the murine delta-opioid receptor (clone D2). Basal [3H]cyclic AMP accumulation was low and was increased substantially by the addition of the diterpene forskolin. The synthetic enkephalin D-Ala2,D-Leu5 enkephalin (DADLE) produced strong inhibition of forskolin-amplified [3H]cyclic AMP production, whereas the delta-opioid ligand ICI174864 augmented forskolin-amplified adenylyl cyclase activity. Naloxone was unable to mimic the effects of ICI174864, and coincubation of the cells with these two ligands attenuated the effect of ICI174864. The EC50 (9.4 +/- 0.6 x 10(-8) M) for ICI174864 augmentation of forskolin-stimulated adenylyl cyclase was equal to its estimated Ki. Pertussis toxin pretreatment of clone D2 cells prevented both this effect of ICI174864 and the inhibition produced by DADLE. Use of a Cytosensor microphysiometer demonstrated that treatment of clone D2 cells with DADLE increased and that with ICI174864 decreased the basal rate of cellular proton extrusion. By using these two distinct experimental strategies, ICI174864 was shown to function in a manner anticipated for an inverse agonist, demonstrating that such effects can be observed in intact cells and are not restricted to assays performed on membrane preparations.

Adenine↗
PubMed1997

How do G-proteins stay at the plasma membrane?

G-protein alpha-subunits are modified by combinations of fatty acyl groups. G-protein gamma-subunits are isoprenylated. These modifications play central roles in membrane association of the G-proteins. Attachment of long-chain fatty acyl chains to G-protein alpha-subunits via a thioester linkage allows the possibility of dynamic regulation of membrane attachment and function.

Acylation↗
PubMed1997

Inverse agonism at adrenergic and opioid receptors: studies with wild type and constitutively active mutant receptors.

Ligands which display inverse agonism at G protein-coupled receptors do so by decreasing the intrinsic ability of a receptor to active the cellular G protein population in the absence of an agonist ligand. Expression of the murine delta opioid receptor in Rat-1 fibroblasts resulted in the inverse agonist ICI174864 being able to cause inhibition of basal high affinity GTPase activity and of the binding of [35S]GTP gamma S in membranes of a clone (D2) of these cells which expresses high levels of the receptor. These effects were blocked by co-addition of the neutral antagonist TIPP[psi], demonstrating a requirement for the delta opioid receptor, and by pertussis toxin pretreatment of the cells, showing them to be produced via a Gi-like G protein. The inverse agonist properties of ICI174864 could also be demonstrated in whole cells. Stimulation of forskolin-amplified adenylyl cyclase activity was produced by ICI174864 following [3H]adenine prelabelling of the cells. Constitutively activated mutants of receptors should provide a convenient means to detect inverse agonists. Incubation of cells either transiently or stably transfected with a constitutively activated mutant of the human beta 2-adrenoceptor with the beta 2-inverse agonists betaxolol or sotalol, which are both able to inhibit CAM beta 2-adrenoceptor-mediated basal adenylyl cyclase activity, resulted in a strong upregulation of levels of the receptor. In the stable cells lines this effect was prevented by co-incubation with neutral antagonists but could not be reproduced by an adenylyl cyclase P-site ligand which also inhibited basal adenylyl cyclase levels.

Adrenergic beta-Antagonists↗
PubMed1996

Up-regulation of a constitutively active form of the beta2-adrenoceptor by sustained treatment with inverse agonists but not antagonists.

In neuroblastoma X glioma hybrid, NG1O8-15, cells transfected to stably express a constitutively active mutant (CAM) form of the human beta2-adrenoceptor, the beta-adrenoceptor ligands sotalol and betaxolol functioned as inverse agonists as they reduced basal adenylyl cyclase activity whereas the antagonists dihydroalprenolol and propranolol did not. Maintained presence of the CAMbeta2-adrenoceptor inverse agonists but not the antagonists in the culture medium of the cells resulted in a substantial, concentration-dependent, up-regulation of the CAMbeta2-adrenoceptor. Up-regulation of the CAMbeta2-adrenoceptor by the inverse agonists was prevented by co-incubation of the cells with either propranolol or dihydroalprenolol. Neither maintained elevation of cAMP levels nor the inhibition of adenylyl cyclase activity altered the ability of the inverse agonist ligands to cause receptor up-regulation.

Adrenergic beta-2 Receptor Antagonists↗
PubMed1996

Characterization of heterotrimeric G-proteins in adult Acanthocheilonema viteae.

Heterotrimeric G-proteins have been found in eukaryotic cells, from yeast to humans, but have received little attention, to date, with respect to parasitic organisms. We now present the first report of the characterization of heterotrimeric G-proteins expressed in a filarial nematode, Acanthocheilonema viteae. Using a combination of (i) affinity labelling with [alpha-32P]GTP; (ii) ADP-ribosylation with cholera toxin and pertussis toxin; (iii) Western blotting with a panel of anti-G-protein antibodies; and (iv) reverse transcriptase-PCR with degenerate G-protein oligonucleotide primers followed by hybridization analysis using oligonucleotides specific for individual G-protein subunits, we demonstrate that adult A. viteae expresses homologues of the beta 1- and/or beta 2-like subunits and alpha-subunits of the Gs, G1, Gq and G12 subfamilies found in mammals. The role which these G-proteins may play in the biology of the organism is discussed.

Adenosine Diphosphate Ribose↗
PubMed1996

Overexpression of G(s)alpha in NG108-15, neuroblastomaXglioma cells: effects on receptor regulation of the stimulatory adenylyl cyclase cascade.

NeuroblastomaXglioma hybrid, NG108-15 cells were stably transfected with an epitope tagged variant of G(s)alpha (HA-G(s)alpha). The introduced HA-G(s)alpha was able to interact with the IP prostanoid receptor and was able to stimulate adenylyl cyclase activity as measured by an enhanced capacity of membrane extracts to reconstitute NaF-dependent adenylyl cyclase activity to membranes of S49 lymphoma cyc- cells. Despite this, neither the maximal stimulation nor the potency of agonist ligands at the IP prostanoid, A2 adenosine or secretin receptors was altered substantially compared to the parental cells although the basal adenylyl cyclase activity was increased. These data indicate that cellular levels of G(s)alpha do not limit signal transduction capacity in NG108-15 cells, whereas enhanced expression of adenylyl cyclase allows greater maximal cAMP generation following receptor activation (MacEwan, D.J., Kim, G.D. and Milligan, G. (1996) Biochem. J. 318, 1033-1039).

Adenosine↗
PubMed1996

A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1996

Agonist activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse delta opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation.

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse delta opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5'-[gamma-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42MAPK and p44MAPK could be achieved. EC50 values for DADLE stimulation of GTPase activity and for activation of p44MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC50 value for DADLE stimulation of p44MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8-1.0). DADLE activation of p44MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44MAPK in both clones in a pertussis toxinsensitive manner, but both the kinetics and the concentration-response curve for activation of p44MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a G1-dependent manner, and show that only a very small proportion of the cellular G1 population is required to be activated to result in full phosphorylation of the p42MAPK and p44MAPK MAP kinases.

Animals↗
PubMed1996

Agonist regulation of adenylate cyclase activity in neuroblastoma x glioma hybrid NG108-15 cells transfected to co-express adenylate cyclase type II and the beta 2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity.

Stable cell lines, derived from NG108-15 cells and transfected to express both the beta 2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the beta 2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5'-[beta, gamma-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced beta 2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration-effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones over-expressing adenylate cyclase type II. A maximally effective concentration of the beta-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5'-N-ethylcarbox-amidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.

Adenylyl Cyclases↗
PubMed1996

Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors.

In order to study the regulation of the ribosomal protein S6 kinase, p70s6k, by G protein-coupled receptors, Rat-1 fibroblasts were stably transfected with two versions of the alpha2 adrenergic receptor. Stimulation of clone 1C cells, which express 3.5 pmol/mg of protein of the human alpha2C10 receptor, with the alpha2 agonist UK 14304 led to a transient increase in p70s6k activity. UK 14304 also activated p70s6k in a clone expressing the porcine alpha2A receptor (400 fmol/mg of protein). Lysophosphatidic acid (LPA), acting through endogenous G protein-coupled receptors, also activated p70s6k in alpha2 receptor-transfected and in nontransfected cells. Activation of p70s6k by both UK 14304 and LPA was accompanied by increased phosphorylation of the protein. Rapamycin completely blocked the activation of p70s6k by both agents. Activation of p70s6k by UK 14304 and by LPA, but not by platelet-derived growth factor (PDGF), was blocked by preincubation of cells with pertussis toxin. Wortmannin, a selective inhibitor of phosphoinositide (PI) 3-OH kinase, prevented activation of p70s6k by UK 14304, LPA, and PDGF. These data indicate that p70s6k is regulatable by Gi-coupled receptor agonists in a pertussis toxin-sensitive fashion in Rat-1 fibroblasts and that activation of p70s6k by such agents appears to involve an isoform of PI 3-kinase.

Adrenergic alpha-Agonists↗
PubMed1996

Analysis of inverse agonism at the delta opioid receptor after expression in Rat 1 fibroblasts.

A cDNA encoding the mouse delta opioid receptor was expressed stably in a Rat 1 fibroblast cell line. Expression of this receptor was demonstrated both in ligand binding studies and by reverse transcriptase-PCR. In membranes of clone D2 cells the opioid peptide [D-Ala(2)]-leucine enkephalin (DADLE) produced a robust, concentration-dependent, stimulation of basal high-affinity GTPase activity; the prototypic opioid antagonist naloxone and the highly selective and potent delta opioid ligands H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-Tic[Ch2-NH]Phe-Phe-OH (TIPP[psi]) had little effect but N,N-diallyl-Tyr-Aib-Aib-Phe-Leu (ICI174864) caused a marked dose-dependent inhibition of this activity (Tic, 1,2,3,4-tetrahydroisoquinolin-2-yl-carbonyl]; Aib, alpha-aminobutyric acid). This effect of ICI174864 was reversed by TIPP[psi] and attenuated after treatment of the cells with pertussis toxin. No stimulation by DADLE or inhibition by ICI174864 was observed in Rat 1 fibroblasts that did not express the delta opioid receptor. Basal binding of [(35)S]guanosine 5'-O-(3-thio-triphosphate) to membranes of clone D2 cells was also stimulated by DADLE and inhibited by ICI174864; both of these effects were reversed by co-incubation with TIPP[psi]. When cholera toxin-catalysed [(32)P]ADP-ribosylation was performed on membranes of clone D2 cells in the absence of guanine nucleotides, a 40 kDa G1-family polypeptide was labelled in addition to both the long and short isoforms of Gsalpha. Labelling of the 40 kDa polypeptide was enhanced by addition of DADLE and fully attenuated by addition of ICI174864. In contrast, labelling of the isoforms of Gsalpha was unaffected by either opioid ligand. Again, both the positive effect of DADLE and the inhibitory effect of ICI174864 were prevented by co-incubation with TIPP[psi] which, in isolation, had little effect on cholera toxin-catalysed [(32)P]ADP-ribosylation of either Gs or Gi. These data demonstrate that the delta opioid receptor displays a spontaneous activity when expressed in this genetic background. Attenuation of this activity is produced by ICI174864, which by acting as an 'inverse agonist' in this system, functionally uncouples the expressed receptor from the cellular G-protein population. The complete attenuation of agonist-independent cholera toxin-catalysed [(32)P]ADP-ribosylation of Gi demonstrated that ICI174864 acts as an inverse agonist with high intrinsic activity at this receptor.

Amino Acid Sequence↗