Development of a high-throughput bioassay to screen melatonin receptor agonists using human melatonin receptor...

Melatonin receptors belong to the superfamily of G-protein-coupled receptors and appear to couple with Gi type of G protein, which has an inhibitory effect on the adenylate cyclase. Normally, melatonin dose not induce transient elevation of intracellular calcium concentration in CHO cells stably expressing melatonin receptors. Accordingly, the cells are unable to be used for fluorescent imaging plate reader (FLIPR), which is the device used to measure the cellular signal as a calcium elevation. To overcome this issue we tried to transfect chimeric G protein, Gqi5, into CHO cells expressing melatonin receptors. The Gqi5 is a chimeric Gq protein containing the five carboxyl-terminal amino acids from Gi, which interact with Gi-coupled receptor and possess the function of evaluating calcium concentration through the Gq pathway. The transfected cells result in a calcium elevation in a concentration-response manner. The specificity of this assay was similar to that of radioreceptor binding assay. Therefore, this FLIPR assay, using melatonin receptor and Gqi5 expressing CHO cells, is available for clinical bioassay of melatonin and for the screening of specific ligands of melatonin.