Recombinant aequorin as a reporter for receptor-mediated changes of intracellular Ca2+ -levels in Drosophila S2 cells.
The bioluminescent Ca(2+)-sensitive reporter protein, aequorin, was employed to develop an insect cell-based functional assay system for monitoring receptor-mediated changes of intracellular Ca(2)(+)-concentrations. Drosophila Schneider 2 (S2) cells were genetically engineered to stably express both apoaequorin and the insect tachykinin-related peptide receptor, STKR. Lom-TK III, an STKR agonist, was shown to elicit concentration-dependent bioluminescent responses in these S2-STKR-Aeq cells. The EC(50) value for the calcium effect detected by means of aequorin appeared to be nearly identical to the one that was measured by means of Fura-2, a fluorescent Ca(2)(+)-indicator. In addition, this aequorin-based method was also utilised to study receptor antagonists. Experimental analysis of the effects exerted by spantide I, II and III, three potent substance P antagonists, on Lom-TK III-stimulated S2-STKR-Aeq cells showed that these compounds antagonise STKR-mediated responses in a concentration-dependent manner. The rank order of inhibitory potencies was spantide III > spantide II > spantide I.
Torfs
Herbert
H
Laboratory for Developmental Physiology and Molecular Biology, Zoological Institute K.U. Leuven, Naamsestraat 59, 3000 Leuven, Belgium.
Poels
Jeroen
J
Detheux
Michel
M
Dupriez
Vincent
V
Van Loy
Tom
T
Vercammen
Linda
L
Vassart
Gilbert
G
Parmentier
Marc
M
Vanden Broeck
Jozef
J
D000331:Aequorin / Q000502:physiology*; D000818:Animals; D001059:Apoproteins / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004331:Drosophila melanogaster / Q000166:cytology / Q000378:metabolism; D019476:Insect Proteins / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008163:Luminescent Measurements; D009994:Osmolar Concentration; D018010:Receptors, Invertebrate Peptide / Q000819:agonists / Q000037:antagonists & inhibitors; D018039:Receptors, Tachykinin / Q000819:agonists / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000378:metabolism; D013373:Substance P / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D015320:Tachykinins / Q000494:pharmacology
D001059:Apoproteins; D019476:Insect Proteins; D018010:Receptors, Invertebrate Peptide; D018039:Receptors, Tachykinin; D011994:Recombinant Proteins; C107470:STKR receptor, Stomoxys calcitrans; D015320:Tachykinins; C060014:apoaequorin; C408989:spantide III; C065446:spantide II; C068632:locustatachykinin III protein, insect; D013373:Substance P; D000331:Aequorin; C042867:spantide; D002118:Calcium