title: A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor.
abstract: Transient expression of apoaequorin in Chinese hamster ovary (CHO) cells and reconstitution with the co-factor coelenterazine resulted in a large, concentration-dependent agonist-mediated luminescent response following cotransfection with the endothelin ETA, angiotensin ATII, thyrotropin-releasing hormone (TRH), and neurokinin NK1 receptors, all of which interact pre-dominantly with the G alpha q-like phosphoinositidase-linked G-proteins. A substantially greater luminescence was obtained with mitochondrially targeted apoaequorin compared to cytoplasmically expressed apoaequorin. To generate a system amenable for the study of agonist activity at virtually any G-protein-coupled receptor the alpha subunit of the receptor promiscuous G-protein G alpha 16 was either transiently or stably expressed in CHO cells together with apoaequorin. In cells expressing G alpha 16, but not in its absence, agonists at a series of receptors which normally interact with either G alpha s or G alpha i were now able to cause a luminescent response from mitochondrially targeted apoaequorin. In the case of the A1 adenosine receptor, this response was clearly a result of activation of G alpha 16 and not a consequence of the release of the G alpha i-associated beta/gamma complex, as the luminescent response was unaffected by pertussis toxin treatment of the cells, whereas agonist-mediated inhibition of adenylyl cyclase activity was attenuated. These studies describe the use of coexpressed apoaequorin as a reporter for G-protein-coupled receptor-mediated calcium signaling. Furthermore, coexpression of G alpha 16 and apoaequorin provides a basis for a generic mammalian cell microplate assay for the assessment of agonist action at virtually any G-protein-coupled receptor, including orphan receptors for which the physiological signal transduction mechanism may be unknown.
authors.affiliation: Receptor Systems Unit, Glaxo Wellcome Research and Development, Stevenage, Herts, United Kingdom.
authors.forename: J
authors.initials: J
authors.lastname: Stables
authors.forename: A
authors.initials: A
authors.lastname: Green
authors.forename: F
authors.initials: F
authors.lastname: Marshall
authors.forename: N
authors.initials: N
authors.lastname: Fraser
authors.forename: E
authors.initials: E
authors.lastname: Knight
authors.forename: M
authors.initials: M
authors.lastname: Sautel
authors.forename: G
authors.initials: G
authors.lastname: Milligan
authors.forename: M
authors.initials: M
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Rees
mesh_terms: D019830:Adenosine-5'-(N-ethylcarboxamide) / Q000378:metabolism / Q000494:pharmacology; D037361:Adenylate Cyclase Toxin; D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000235:genetics / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D016466:CHO Cells / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008163:Luminescent Measurements; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D037342:Pertussis Toxin; D054799:Phospholipase C beta; D058906:Purinergic P1 Receptor Agonists; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D001059:Apoproteins; D007527:Isoenzymes; D058906:Purinergic P1 Receptor Agonists; D043705:Receptor, Adenosine A2A; D011956:Receptors, Cell Surface; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C060014:apoaequorin; D019830:Adenosine-5'-(N-ethylcarboxamide); D000331:Aequorin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins; D002118:Calcium