Aequorin-based functional assays for G-protein-coupled receptors, ion channels, and tyrosine kinase receptors.
Aequorin is a photoprotein originating from jellyfish, whose luminescent activity is dependent on the concentration of calcium ions. Due to the high sensitivity and low background linked to luminescent assays, as well as to its absence of toxicity and its large linear dynamic range, aequorin has been used as an intracellular calcium indicator since its discovery in the early 1960s. The first applications of aequorin involved its microinjection in cells. The cloning of its gene in 1985 opened the way to the stable expression of aequorin in cell lines or even entire organisms. Here we present the validation of aequorin as a functional assay for the screening of G-protein-coupled receptors, ion channels, and tyrosine kinase receptors, as well as for their pharmacological characterization in agonist and antagonist detection assays. We optimized our cell suspension-based assay and determined that the most sensitive assay was performed at room temperature, with mitochondrially expressed aequorin and using coelenterazine derivative h for reconstitution of aequorin. The robustness of the assay and the current availability of luminometers with integrated injectors allow aequorin to fit perfectly with high throughput functional assays requirements.
Dupriez
Vincent J
VJ
Euroscreen s.a., 47 Rue Adrienne Bolland, B-6041 Gosselies, Belgium. vdupriez@euroscreen.be
Maes
Karlien
K
Le Poul
Emmanuel
E
Burgeon
Emmanuel
E
Detheux
Michel
M
D000331:Aequorin / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011955:Receptors, Drug / Q000378:metabolism; D013997:Time Factors
D007473:Ion Channels; D007477:Ions; D011956:Receptors, Cell Surface; D011955:Receptors, Drug; D000331:Aequorin; D004815:Epidermal Growth Factor; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins