A Bioluminescent Assay for Agonist Activity at Potentially Any G-Protein-Coupled Receptor

doi:10.1006/abio.1997.2308

Analytical Biochemistry

Volume 252, Issue 1, 1 October 1997, Pages 115-126

https://doi.org/10.1006/abio.1997.2308 Get rights and content

Abstract

Transient expression of apoaequorin in Chinese hamster ovary (CHO) cells and reconstitution with the cofactor coelenterazine resulted in a large, concentration-dependent agonist-mediated luminescent response following cotransfection with the endothelin ET_A, angiotensin AT_II, thyrotropin-releasing hormone (TRH), and neurokinin NK₁receptors, all of which interact predominantly with the Gα_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 Gα₁₆was either transiently or stably expressed in CHO cells together with apoaequorin. In cells expressing Gα₁₆, but not in its absence, agonists at a series of receptors which normally interact with either Gα_sor Gα_iwere now able to cause a luminescent response from mitochondrially targeted apoaequorin. In the case of the A₁adenosine receptor, this response was clearly a result of activation of Gα₁₆and not a consequence of the release of the Gα_i-associated β/γ 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 Gα₁₆and 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.

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Citation Excerpt :
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Regular ArticleA Bioluminescent Assay for Agonist Activity at Potentially Any G-Protein-Coupled Receptor

Abstract

Anal. Biochem.

Anal. Biochem.

Cell Calcium

J. Biol. Chem.

J. Biol. Chem.

Trends. Pharmacol. Sci.

Biochem. Biophys. Res. Commun.

Neuropeptides

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

Neuron

Science

Trends Biochem. Sci.

Endocrine Rev.

Cell

Regular Article
A Bioluminescent Assay for Agonist Activity at Potentially Any G-Protein-Coupled Receptor