Abstract
The alpha1-adrenergic agonist phenylephrine stimulated phospholipase D (PLD) activity in Rat 1 fibroblasts transfected to express either the wild-type hamster alpha1B-adrenoceptor or a constitutively active mutant (CAM) form of this receptor. The EC50 for agonist stimulation of PLD activity was substantially lower at the CAM receptor than at the wild-type receptor as previously noted for phenylephrine stimulation of phosphoinositidase C activity. Sustained treatment of cells expressing the CAM alpha1B-adrenoceptor with phentolamine resulted in a marked up-regulation in levels of this receptor with half-maximal effects produced within 24 h and with an EC50 of approx. 40 nM. Such an up-regulation could be produced with a range of other ligands generally viewed as alpha1-adrenoceptor antagonists but equivalent treatment of cells expressing the wild-type alpha1B-adrenoceptor was unable to mimic these effects. After sustained treatment of the CAM alpha1B-adrenoceptor expressing cells with phentolamine, basal PLD activity was increased and phenylephrine was now able to stimulate PLD activity to greater levels than in vehicle-treated CAM alpha1B-adrenoceptor-expressing cells. The EC50 for phenylephrine stimulation of PLD activity was not altered, however, by phentolamine pretreatment and the associated up-regulation of the receptor. After phentolamine-induced up-regulation of basal PLD activity, a range of alpha1-antagonists were shown to possess the characteristics of inverse agonists of the CAM alpha1B-adrenoceptor as they were able to substantially decrease the elevated basal PLD activity.
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Selected References
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- Allen L. F., Lefkowitz R. J., Caron M. G., Cotecchia S. G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11354–11358. doi: 10.1073/pnas.88.24.11354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Collins S., Caron M. G., Lefkowitz R. J. From ligand binding to gene expression: new insights into the regulation of G-protein-coupled receptors. Trends Biochem Sci. 1992 Jan;17(1):37–39. doi: 10.1016/0968-0004(92)90425-9. [DOI] [PubMed] [Google Scholar]
- Cook S. J., Wakelam M. J. Hydrolysis of phosphatidylcholine by phospholipase D is a common response to mitogens which stimulate inositol lipid hydrolysis in Swiss 3T3 fibroblasts. Biochim Biophys Acta. 1991 Apr 17;1092(2):265–272. doi: 10.1016/0167-4889(91)90166-u. [DOI] [PubMed] [Google Scholar]
- Costa T., Herz A. Antagonists with negative intrinsic activity at delta opioid receptors coupled to GTP-binding proteins. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7321–7325. doi: 10.1073/pnas.86.19.7321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cotecchia S., Lattion A. L., Diviani D., Cavalli A. Signalling and regulation of the alpha 1B-adrenergic receptor. Biochem Soc Trans. 1995 Feb;23(1):121–125. doi: 10.1042/bst0230121. [DOI] [PubMed] [Google Scholar]
- Dohlman H. G., Thorner J., Caron M. G., Lefkowitz R. J. Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem. 1991;60:653–688. doi: 10.1146/annurev.bi.60.070191.003253. [DOI] [PubMed] [Google Scholar]
- Lee T. W., Wise A., Cotecchia S., Milligan G. 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. Biochem J. 1996 Nov 15;320(Pt 1):79–86. doi: 10.1042/bj3200079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lefkowitz R. J., Cotecchia S., Samama P., Costa T. Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci. 1993 Aug;14(8):303–307. doi: 10.1016/0165-6147(93)90048-O. [DOI] [PubMed] [Google Scholar]
- MacEwan D. J., Kim G. D., Milligan G. 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. Biochem J. 1996 Sep 15;318(Pt 3):1033–1039. doi: 10.1042/bj3181033. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MacEwan D. J., Kim G. D., Milligan G. Analysis of the role of receptor number in defining the intrinsic activity and potency of partial agonists in neuroblastoma x glioma hybrid NG108-15 cells transfected to express differing levels of the human beta 2-adrenoceptor. Mol Pharmacol. 1995 Aug;48(2):316–325. [PubMed] [Google Scholar]
- MacEwan D. J., Milligan G. Inverse agonist-induced up-regulation of the human beta2-adrenoceptor in transfected neuroblastoma X glioma hybrid cells. Mol Pharmacol. 1996 Dec;50(6):1479–1486. [PubMed] [Google Scholar]
- MacEwan D. J., Milligan G. Up-regulation of a constitutively active form of the beta2-adrenoceptor by sustained treatment with inverse agonists but not antagonists. FEBS Lett. 1996 Dec 9;399(1-2):108–112. doi: 10.1016/s0014-5793(96)01300-2. [DOI] [PubMed] [Google Scholar]
- MacNulty E. E., McClue S. J., Carr I. C., Jess T., Wakelam M. J., Milligan G. Alpha 2-C10 adrenergic receptors expressed in rat 1 fibroblasts can regulate both adenylylcyclase and phospholipase D-mediated hydrolysis of phosphatidylcholine by interacting with pertussis toxin-sensitive guanine nucleotide-binding proteins. J Biol Chem. 1992 Feb 5;267(4):2149–2156. [PubMed] [Google Scholar]
- Milligan G., Bond R. A., Lee M. Inverse agonism: pharmacological curiosity or potential therapeutic strategy? Trends Pharmacol Sci. 1995 Jan;16(1):10–13. doi: 10.1016/s0165-6147(00)88963-4. [DOI] [PubMed] [Google Scholar]
- Mullaney I., Carr I. C., Milligan G. Analysis of inverse agonism at the delta opioid receptor after expression in Rat 1 fibroblasts. Biochem J. 1996 Apr 1;315(Pt 1):227–234. doi: 10.1042/bj3150227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parma J., Duprez L., Van Sande J., Cochaux P., Gervy C., Mockel J., Dumont J., Vassart G. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature. 1993 Oct 14;365(6447):649–651. doi: 10.1038/365649a0. [DOI] [PubMed] [Google Scholar]
- Perez D. M., Hwa J., Gaivin R., Mathur M., Brown F., Graham R. M. Constitutive activation of a single effector pathway: evidence for multiple activation states of a G protein-coupled receptor. Mol Pharmacol. 1996 Jan;49(1):112–122. [PubMed] [Google Scholar]
- Plevin R., Palmer S., Gardner S. D., Wakelam M. J. Regulation of bombesin-stimulated inositol 1,4,5-trisphosphate generation in Swiss 3T3 fibroblasts by a guanine-nucleotide-binding protein. Biochem J. 1990 Jun 15;268(3):605–610. doi: 10.1042/bj2680605. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Samama P., Cotecchia S., Costa T., Lefkowitz R. J. A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem. 1993 Mar 5;268(7):4625–4636. [PubMed] [Google Scholar]
- Scheer A., Fanelli F., Costa T., De Benedetti P. G., Cotecchia S. Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. EMBO J. 1996 Jul 15;15(14):3566–3578. [PMC free article] [PubMed] [Google Scholar]
- Schipani E., Kruse K., Jüppner H. A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science. 1995 Apr 7;268(5207):98–100. doi: 10.1126/science.7701349. [DOI] [PubMed] [Google Scholar]
- Shenker A., Laue L., Kosugi S., Merendino J. J., Jr, Minegishi T., Cutler G. B., Jr A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature. 1993 Oct 14;365(6447):652–654. doi: 10.1038/365652a0. [DOI] [PubMed] [Google Scholar]
- Smit M. J., Leurs R., Alewijnse A. E., Blauw J., Van Nieuw Amerongen G. P., Van De Vrede Y., Roovers E., Timmerman H. Inverse agonism of histamine H2 antagonist accounts for upregulation of spontaneously active histamine H2 receptors. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6802–6807. doi: 10.1073/pnas.93.13.6802. [DOI] [PMC free article] [PubMed] [Google Scholar]