[35S]GTP gamma S binding: a tool to evaluate functional activity of a cloned opioid receptor transiently expressed in COS cells.
In this paper we propose a powerful procedure to measure functional activation of the mouse delta-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]GTP gamma S binding. Both BW373U86, an alkaloid compound, and DADLE, a peptide agonist, elicited increase of specific [35S]GTP gamma S 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 powerful 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.
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CNRS UPR 9050 Laboratoire des Récepteurs et Protéines Membranaires, ESBS, Illkirch, France.
Tabbara
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Kieffer
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D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D051379:Mice; D009270:Naloxone / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D017465:Receptors, Opioid, delta / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013462:Sulfur Radioisotopes; D014162:Transfection
D001549:Benzamides; D010879:Piperazines; D017465:Receptors, Opioid, delta; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C083877:BW 373U86; D009270:Naloxone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D016308:Enkephalin, Leucine-2-Alanine