Biomedical subjects

Graeme Milligan

Publications and source records attributed to Graeme Milligan.

76 records · Page 5Linked to original sources

PubMed2002

Generation and analysis of constitutively active and physically destabilized mutants of the human beta(1)-adrenoceptor.

Constitutive activity of wild-type and mutant forms of human beta(1)- and beta(2)-adrenoceptors was measured by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assays using fusion proteins between these receptors and G(s)alpha. Constitutive activity of the beta(1)-adrenoceptor is enhanced by mutation of Leu(322). The ability of ligands to suppress receptor instability and produce up-regulation is often associated with constitutively active mutants. Leu(322)Lysbeta(1)-adrenoceptor, but not wild type, was up-regulated by exposure to the beta(1)-adrenoceptor selective blocker betaxolol. More extensive sequence alterations of the beta(1)-adrenoceptor were generated to mimic the initially described constitutively active mutant (CAM) of the beta(2)-adrenoceptor that is up-regulated strongly by betaxolol. Substitution of amino acids 316 to 324 of the beta(1)-adrenoceptor with the equivalent alpha(1b)-adrenoceptor sequence did not result in up-regulation by betaxolol. However, these forms of both beta(1)- and beta(2)-adrenoceptors displayed substantial and equivalent constitutive activity. The addition of the Leu(322)Lys mutation into the alpha(1b)-adrenoceptor substituted beta(1)-adrenoceptor to produce the CAMKbeta(1)-adrenoceptor allowed substantially greater levels of up-regulation by betaxolol without enhancement of constitutive [(35)S]GTPgammaS binding. Arg(156)Alabeta(1)-adrenoceptor was up-regulated strongly by betaxolol but displayed lower constitutive activity than did other mutants. Binding of [(35)S]GTPgammaS binding to all the fusion proteins was increased substantially by isoprenaline. Despite the ability of betaxolol to cause up-regulation of many mutants, only for the CAMbeta(2)-adrenoceptor-G(s)alpha and CAMKbeta(1)-adrenoceptor-G(s)alpha fusion proteins was the basal binding of [(35)S]GTPgammaS decreased by betaxolol. Clear resolution between receptor constitutive activity and ligand suppression of receptor instability can be obtained for mutant beta-adrenoceptors, and potential inverse agonists do not function equally at phenotypically apparently equivalent CAM receptors.

Adrenergic beta-Antagonists↗

PubMed2002

The use of receptor G-protein fusion proteins for the study of ligand activity.

Fusion proteins in which the N-terminus of a G protein alpha subunit is attached in frame to the C-terminal tail of a G-protein-coupled receptor have become widely used as experimental systems to explore the quantitative details of ligand stimulation of specific receptor G-protein combinations. In part, this reflects that they function as agonist-activated GTPases that behave with simple Michaelan kinetics. They have also been used to explore the effects of mutation in both receptor and G protein on information transfer, ligand regulation of posttranslational acylation, and the mechanism and potential selectivity of regulators of G-protein signaling.

Animals↗

PubMedDate not supplied

Expression of Gsalpha, connexin-43, connexin-26, and EP1, 3, and 4 receptors in myometrium of prelabor singleton versus multiple gestations and the effects of mechanical stretch and steroids on Gsalpha.

OBJECTIVE: We tested the hypothesis that multiple pregnancies would be associated with altered expression of the following three groups of proteins that are key regulators of myometrial function: (i) Gsalpha, (ii) connexins-43 and 26, and (iii) prostanoid EP1, EP3, and EP4 receptors. METHODS: An in vitro model was used to determine the effects of mechanical stretch on myometrial cell Gsalpha expression. Then the effects of the steroid hormones beta-estradiol and progesterone were tested on Gsalpha expression in vitro. All in vitro studies were performed using myometrium from nonlaboring women. RESULTS: There were no differences in the expression of Gsalpha, prostaglandin E2 receptors, or gap junction proteins in myometrium of singleton versus multiple pregnancies. Mechanical stretch did not alter Gsalpha expression in vitro, and Gsalpha expression was unaffected by steroid hormones. CONCLUSION: These results suggest that the methods whereby stretch can promote myometrial contraction are complex or require additional factors than those tested here. Alternatively, cases of multiple gestation that do not result in preterm labor perhaps compensate for the increased stretch by preventing aberrant expression of the proteins investigated in this study.

Biomechanical Phenomena↗

PubMedDate not supplied

Ligand rescue of constitutively active mutant receptors.

Constitutively active mutants (CAMs) of G protein-coupled receptors are found naturally in disease states and they can be generated by point mutation. As these mutants are able to activate G proteins in the absence of a ligand, they are useful tools in the study of conformational changes leading to receptor activation and in the drug discovery process. Early studies on CAMs noted that they are often expressed at lower levels than their wild-type forms. In this review we discuss the mechanisms responsible for this reduced expression and also provide an explanation for the observation that challenging cells with receptor ligands can increase the CAM expression level. The application of these observations to the development of a high-throughput reporter assay suitable for ligand identification is also discussed.

Animals↗