Abstract
In this paper we propose a powerful procedure to measure functional activation of the mouse δ-opioid receptor transiently expressed in mammalian cells. Receptor stimulation was assessed using a population of electroporated COS cells, transfected at a 50% efficiency. Under those conditions, agonist-promoted activation of the receptor was measured by [35S]GTPγS binding. Both BW373U86, an alkaloid compound, and DADLE, a peptide agonist, elicited increase of specific [35S]GTPγS binding representing 300% of basal level. Maximal activation was compared to that obtained for the cloned receptor stably expressed in CHO cells. Agonist efficacy was similar in both expressions systems, demonstrating the high sensitivity of the proposed method applied to transient expression. Finally dose-response curves were found highly reproducible across transfection experiments, opening the possibility for a direct comparison of distinct recombinant receptor preparations. This method represents a powerfull tool for the study of opioid signal transduction at the receptor level. It may also be extended to investigate signalling properties of other Gi/Go coupled receptors.
References
Simon, E. J., Hiller, J. M., and Edelman, I. 1973. Stereospecific binding of the potent narcotic analgesic [3H]etorphine to rat brain homogenate. Proc. Natl. Acad. Sci. USA 70:1947–1949.
Terenius, L. 1973. Stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex. Acta. Pharmacol. Toxicol. 32:317–319.
Pert, C. B., and Snyder, S. H. 1973. Oplate receptor: demonstration in nervous tissue. Science 179:1011–1014.
Goldstein, A. and Naidu, A. 1989. Multiple opioid receptors: ligand selectivity profiles and binding signatures. Mol. Pharmacol. 36:265–272.
Law, P. Y. and Loh, H. H. 1992. Second messengers in the transduction of multiple opioid receptors' signals. Pages 39–68, in Drugs of abuse and neurobiology (Watson, R. R., ed.), CRC Press, Boca Raton, FL.
North, R. A. 1993. Opioid action on membrane ion channels. Pages 773–793, in Herz, A. (ed), Handbook of Experimental Pharmacology, Opioids I. Springer-Verlag, Berlin, Vol. 104/I.
Childers, S. R. 1993. Opioid receptor-coupled second messenger systems. Pages 189–208, in Herz, A. (ed) Handbook of Experimental Pharmacology, Opioids I. Springer-Verlag, Berlin, Vol. 104/I.
Cox, B. M. 1993. Opioid receptor-G protein interactions: acute and chronic effects of opioids. Pages 145–180, in: Herz A. (ed). Handbook of Experimental Pharmacology, Opioids I, Springer-Verlag, Berlin, Vol. 104/I.
Evans, C. J., Keith, D. E., Morrison, H., Magendzo, K., and Edwards, R. H. 1992. Cloning of a δ-opioid receptor by functional expression. Science 258:1952–1955.
Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C., and Hirth, C. G. 1992. The δ-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc. Natl. Acad. Sci. U.S.A. 89:12048–12052.
Kieffer, B. L. 1995. Recent advances in molecular recognition and signal transduction of active peptides: receptors for opioid peptides. Cell. Mol. Neurobiol. 15:615–635.
Satoh, M. and Minami, M. 1995. Molecular pharmacology of the opioid receptors. Pharmacol. Ther. 68:343–364.
Tallent, M., Dichter, M. A., Bell, G. I., and Reisine, T. 1994. The cloned kappa opioid receptor couples to an N-type calcium current in undifferentiated PC-12 cells. Neurosci. 63:1033–1040.
Piros, E. T., Prather, P. L., Loh, H. H., Law, P. Y., Evans, C. J., and Hales, T. G. 1995. Ca2+ channel and adenylyl cyclase modulation by cloned μ-opioid receptors in GH3 cells. Mol. Pharmacol. 47:1041–1049.
Prather, P. L., McGinn, T. M., Erickson, L. J., Evans, C. J., Loh, H. H., and Law, P. Y. 1994. Ability of δ-opioid receptors to interact with multiple G-proteins is dependent of receptor density. J. Biol. Chem. 269:21293–21302.
Chabrakarti, S., Prather, P. L., Yu, L., Law, P. Y., and Loh, H. H. 1995. Expression of the μ-opioid receptor in CHO cells: ability of μ-opioid ligands to promote α-azidoanilido [32P]GTP labeling of multiple G protein α subunits. J. Neurochem. 64:2534–2543.
Prather, P. L., McGinn, T. M., Claude, P. A., Liu-Chen, L. Y., Loh, H. H., and Law, P. Y. 1995. Properties of a κ-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual Gα subunit and is similar to that of other opioid receptor. Mol. Brain. Res. 29:336–346.
Dascal, N., Schreibmayer, W., Lim, N. F., Wang, W., Chavkin, C., Di, M. L., Labarca, C., Kieffer, B. L., Gaveriaux, R. C., Trollinger, D., Lester, H. A. and Davidson, N. 1993. Atrial G protein-activated K+ channel: expression cloning and molecular properties. Proc. Natl. Acad. Sci. U.S.A. 90:10235–10239.
Lai, H. W. L., Minami, M., Satoh, M., and Wong, Y. H. 1995. Gz coupling to the rat κ-opioid receptor. FEBS. 360:97–99.
Henry, D. J., Grandy, D. K., Lester, H. A., Davidson, N. and Chavkin, C. 1995. κ-opioid receptors couple to inwardly rectifying potassium channels when coexpressed byXenopus oocytes. Mol. Pharmacol. 47:551–557.
Law, P. Y., McGinn, T. M., Wick, M. J., Erickson, L. J., Evans, C. J., and Loh, H. H. 1994. Analysis of delta-opioid receptor activities stably expressed in CHO cell lines: function of receptor density? J. Pharmacol. Exp. Ther. 271:1686–1694.
Tsu, R. C., Chan, J. S. C., and Wong, Y. H. 1995. Regulation of multiple effectors by the cloned δ-opioid receptor: stimulation of phospholipase C and Type II adenylyl cyclase. J. Neurochem. 64: 2700–2707.
Kovoor, A., Henry, D., and Chavkin, C. 1995. Agonist-induced desensitization of the μ-opioid receptor-coupled potassium channel (GIRK1). J. Biol. Chem. 270:589–595.
Ma, G. H., Miller, R. J., Kuznetsov, A., and Philipson, L. H. 1995. κ-opioid receptor activates an inwardly rectifying K+ channel by a G protein-linked mechanism: coexpression inXenopus oocytes. Mol. Pharmacol. 47:1035–1040.
Traynor, J. R., and Nahorski, S. R. 1995. Modulation by μ-opioid agonists of guanosine-5′-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47:848–854.
Sim, L. J., Selley, D. E., and Childers, S. R. 1995. In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5′-[γ-[35S]thio]-triphosphate binding. Neurobiology 92:7242–7246.
Thomas, D. R., Faruq, S. A., Balcarek, J. M., and Brown, A. M. 1995. Pharmacological characterization of [35S]-GTPγS binding to Chinese hamster ovary cell membranes stably expressing cloned human 5-HT1D receptor subtypes. J. Receptor Signal Transduction Res. 15:199–211.
Burford, N. T., Tobin, A. B., and Nahorski, S. R. 1995. Coupling of muscarinic m1, m2 and m3 acetylcholine receptors, expressed in Chinese hamster ovary cells, to pertussis toxin-sensitive guanine nucleotide-binding proteins. Eur. J. Pharmacol. Mol. Sect. 19: 343–351.
Simonin, F., Befort, K., Gaveriaux-Ruff, C., Matthes, H., Nappey, V., Lannes, B., Micheletti, G., and Kieffer, B. 1994. The human δ-opioid receptor: genomic organization, cDNA cloning, functional expression and distribution in human brain. Mol. Pharmacol. 46:1015–1021.
Befort, K., Tabbara, L., Bausch, S., Chavkin, C., Evans, C., and Kieffer, B. 1996. The conserved aspartate residue in the third putative tranmembrane domain of the δ-opioid receptor is not the anionic counterpart for cationic opiate binding but is a constituent of the receptor binding site. Mol. Pharm. 49:216–223.
Malatynska, E., Wang, Y., Knapp, R. J., Santoro, G., Li, X., Waite, S., Roeske, W. R., and Yamamura, H. I. 1995. Human δ-opioid receptor: a stable cell line for functional studies of opioids. Neuroreport. 6:613–616.
Chakrabarti, S., Law, P. Y., and Loh, H. H. 1995. Neuroblastoma Neuro2A cells stably expressing a cloned μ-opioid receptor: a specific cellular model to study acute and chronic effects of morphine. Mol. Brain Res. 30:269–278.
Author information
Authors and Affiliations
Corresponding author
Additional information
Special issue dedicated to Dr. Eric J. Simon.
Rights and permissions
About this article
Cite this article
Befort, K., Tabbara, L. & Kieffer, B.L. [35S]GTPγS binding: A tool to evaluate functional activity of a cloned opioid receptor transiently expressed in COS cells. Neurochem Res 21, 1301–1307 (1996). https://doi.org/10.1007/BF02532371
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF02532371