The Receptor-G.ALPHA. Fusion Protein as a Tool for Ligand Screening: a Model Study Using a Nociceptin Receptor-G.ALPHA.i2 Fusion Protein

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As a model system to screen endogenous ligands for Gi-coupled receptors, we have prepared and characterized a fusion protein of nociceptin receptor and a subunit of Gi2. We detected nociceptin binding to the fusion protein by measuring stimulation of [35S] GTPγS binding with an EC50 of 2.0 nM and a gain of approximately five times. The stimulation by nociceptin of [35S] GTPγS binding to the fusion protein was clearly observed in the presence of an appropriate concentration of GDP, because the affinity for GDP was decreased in the presence of agonist. Full and partial agonists differed in their effects on apparent the affinity of the fusion protein for GDP: the IC50 values for GDP to displace 100 pM [35S] GTPγS were estimated to be 2 μM, 0.4 μM, and 0.05 μM in the presence of full agonist (nociceptin), partial agonist (F/G-NC), and antagonist (NBZH), respectively. We also detected the activity to stimulate [35S] GTPγS binding to the fusion protein in the brain extract derived from 2-3g wet weight tissue without false-positive results. The active component was identified as endogenous nociceptin itself. These results indicate that the fusion protein of GPCR and Gαi is useful for screening of endogenous ligands.
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