Simple luminometric assay to detect phosphoinositol-linked receptor expression in Xenopus oocytes.
A simple and sensitive method to measure the expression of phosphoinositol-linked receptors in Xenopus laevis oocytes is described. Oocytes are co-injected with the calcium photoprotein aequorin and RNA, encoding the receptor of interest. The binding of ligand to the expressed receptor increases intracellular calcium that induces the aequorin to luminesce. With an autosampler-equipped luminometer, this provides a fully automated assay of receptor expression of oocytes. This method was applied to cloning the bombesin/GRP receptor expressed in Swiss 3T3 fibroblasts. Oocytes expressing the cloned BR showed up to a 10,000-fold increase in light emission in response to bombesin. The sensitivity of this procedure allowed detection of positive luminometer signals in single oocytes injected with RNA transcribed from cDNA pools as large as 25,000 clones. These findings show the potential value of this procedure for rapid screening of expression libraries, structure/function analysis of receptors and analysis of receptor antagonists or agonists.
Giladi
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Division of Neuroscience, Oregon Regional Primate Research Center, Beaverton 97006.
Spindel
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D000331:Aequorin / Q000378:metabolism; D000818:Animals; D001839:Bombesin / Q000037:antagonists & inhibitors; D002118:Calcium / Q000032:analysis; D049454:Firefly Luciferin / Q000378:metabolism; D015870:Gene Expression; D007267:Injections; D007295:Inositol Phosphates; D008163:Luminescent Measurements; D009865:Oocytes / Q000502:physiology; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic; D014982:Xenopus laevis
D007295:Inositol Phosphates; D018004:Receptors, Bombesin; D017981:Receptors, Neurotransmitter; D000331:Aequorin; D049454:Firefly Luciferin; D001839:Bombesin; D002118:Calcium