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Biomedical subjects

A R Burt

Publications and source records attributed to A R Burt.

9 recordsLinked to original sources
PubMed1998

Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1alpha: effect on alpha2A-adrenoceptor-Gi1alpha fusion protein activation.

Fusion proteins were constructed between the porcine alpha2A-adrenoceptor and either wild-type (Cys351) or a pertussis toxin-resistant (Gly351) form of the G protein Gi1alpha. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The alpha2A-adrenoceptor-wild type Gi1alpha fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly351 Gi1alpha. Treatment of the fusion proteins as agonist-regulated enzymes allowed measurement of Vmax and turnover number for adrenaline-stimulation of the GTPase activity of each fusion construct. The turnover number of the alpha2A-adrenoceptor-Cys351 Gly Gi1alpha fusion protein was only 44'S, of that for the alpha2A-adrenoceptor-wild type Gi1alpha fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist-occupied receptor to activate the mutant.

Animals↗
PubMed1998

Agonist occupation of an alpha2A-adrenoreceptor-Gi1alpha fusion protein results in activation of both receptor-linked and endogenous Gi proteins. Comparisons of their contributions to GTPase activity and signal transduction and analysis of receptor-G protein activation stoichiometry.

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the alpha subunit of the G protein Gi1 and the porcine alpha2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder from endogenous Gialpha. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist. Although the Km for GTP of the fusion protein-linked Gi was lower than for the endogenous G protein, Vmax measurements demonstrated that adrenaline activated some 5 mol of endogenous Gi/mol of fusion protein-linked Gi. The isolated alpha2A-adrenoreceptor could activate Gs; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous Gialpha was greater than for the fusion protein-constrained C351G Gi1alpha. alpha2A-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated alpha2A-adrenoreceptor-C351G Gi1alpha fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment. These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1997

Mitogenic signalling by delta opioid receptors expressed in rat-1 fibroblasts involves activation of the p70s6k/p85s6k S6 kinase.

The regulation of mitogenic signalling pathways by G-protein-coupled receptors has been studied in Rat-1 fibroblasts stably transfected with the murine delta opioid receptor. We showed recently that stimulation of this receptor led to the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinase [Burt, Carr, Mullaney, Anderson and Milligan (1996) Biochem. J. 320, 227-235]. The present study has examined the role of the ribosomal S6 kinase p70(s6k) in mitogenic signalling by the delta opioid receptor. Treatment of Rat-1 fibroblasts expressing this receptor with the synthetic enkephalin [d-Ala,d-Leu]-enkephalin (DADLE) led to a dose-dependent increase in p70(s6k) enzyme activity. Activation of p70(s6k) was dependent on the level of delta opioid receptor expressed and was sustained above basal levels for several hours. Immunoblotting revealed that p70(s6k) was subject to increased phosphorylation, the extent of which coincided temporally with enzyme activation. Activation of p70(s6k) by DADLE, but not by platelet-derived growth factor, was blocked by pretreatment of cells with pertussis toxin. Activation of p70(s6k) was also partly blocked by wortmannin, indicating that phosphoinositide 3-OH kinase is required for full activation of p70(s6k) by opioid receptor agonists. Activation of the delta opioid receptor in transfected cells led to increased DNA synthesis. This increase was prevented by rapamycin, which also completely blocked activation of p70(s6k) by DADLE. In addition, prevention of the activation of p42 and p44 MAP kinases also blocked the induction of DNA synthesis by DADLE. These results suggest that the activation of both MAP kinases and p70(s6k) might be crucial to the induction of mitogenic responses by Gi-linked receptors such as the delta opioid receptor.

Androstadienes↗
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↗
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

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↗
PubMed1991

Correlations of cardiac function and SCL-90R in heart transplantation candidates.

Measurements of cardiac function and Symptom Checklist-90R (SCL-90R) data were retrospectively analyzed in two independent groups of cardiac transplant candidates in an effort to identify organic correlates of depressive syndromes. In the first study, no significant inverse correlations were found between depression measures and cardiac index as had been predicted. However, elevated right atrial pressure was associated with increased scores of the SCL-90R depression subscale (DEP) and global symptom index (GSI). A significant positive correlation was also found between the phobia subscale and cardiac index. In a second sample, again, cardiac index did not correlate inversely with DEP or GSI. The phobia-cardiac index correlation was replicated but the right atrial pressure correlations were not. Combining both groups, there were significant correlations between six SCL-90R subscales (including DEP and GSI) and cardiac index. All were positive correlations, refuting the initial hypothesis and suggesting patients with the best cardiac function reported the worst psychological distress and that patients with the worst cardiac function reported the least psychological distress. Possible explanations for these findings are discussed.

Cardiac Output↗
PubMed1987

Adjunctive hysterectomy following radiation therapy for bulky carcinoma of the uterine cervix: prognostic implications of tumor persistence.

Twenty-five patients underwent adjunctive extrafascial hysterectomy 14-60 days following completion of external and intracavitary irradiation for bulky carcinoma of the uterine cervix. Review of the operative histopathology and correlation with subsequent patient outcomes suggests that morphologically persistent cancer is prognostically ominous and that cancer near the surgical margin is viable in the majority of instances.

Adenocarcinoma↗