A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor

Anal Biochem. 1997 Oct 1;252(1):115-26. doi: 10.1006/abio.1997.2308.


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.

Publication types

  • Comparative Study

MeSH terms

  • Adenosine-5'-(N-ethylcarboxamide) / metabolism
  • Adenosine-5'-(N-ethylcarboxamide) / pharmacology
  • Adenylate Cyclase Toxin
  • Aequorin / genetics
  • Aequorin / metabolism
  • Animals
  • Apoproteins / genetics
  • Apoproteins / metabolism
  • Biochemistry / methods*
  • CHO Cells / metabolism
  • Calcium / metabolism
  • Cricetinae
  • Cyclic AMP / metabolism
  • Cytoplasm / genetics
  • Cytoplasm / metabolism
  • GTP-Binding Proteins / metabolism*
  • Humans
  • Isoenzymes / metabolism
  • Luminescent Measurements*
  • Mitochondria / genetics
  • Mitochondria / metabolism
  • Pertussis Toxin
  • Phospholipase C beta
  • Purinergic P1 Receptor Agonists*
  • Receptor, Adenosine A2A
  • Receptors, Cell Surface / metabolism*
  • Receptors, Purinergic P1 / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Type C Phospholipases / metabolism
  • Virulence Factors, Bordetella / pharmacology


  • Adenylate Cyclase Toxin
  • Apoproteins
  • Isoenzymes
  • Purinergic P1 Receptor Agonists
  • Receptor, Adenosine A2A
  • Receptors, Cell Surface
  • Receptors, Purinergic P1
  • Recombinant Proteins
  • Virulence Factors, Bordetella
  • apoaequorin
  • Adenosine-5'-(N-ethylcarboxamide)
  • Aequorin
  • Cyclic AMP
  • Pertussis Toxin
  • Type C Phospholipases
  • Phospholipase C beta
  • GTP-Binding Proteins
  • Calcium