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J. Biochem, 2004, Vol. 135, No. 5 597-604
© 2004 The Japanese Biochemical Society

CELL

The Receptor-G{alpha} Fusion Protein as a Tool for Ligand Screening: a Model Study Using a Nociceptin Receptor-G{alpha}i2 Fusion Protein

Shigeki Takeda1,2,*,§, Tomoaki Okada3,¶, Michiko Okamura1, Tatsuya Haga1,2,3, Junko Isoyama-Tanaka4, Hiromiki Kuwahara4 and Naoto Minamino4

1 Department of Neurochemistry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033; 2 Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Tokyo 171-8588; 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST); and 4 National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565

As a model system to screen endogenous ligands for Gi-coupled receptors, we have prepared and characterized a fusion protein of nociceptin receptor and {alpha} subunit of Gi2. We detected nociceptin binding to the fusion protein by measuring stimulation of [35S]GTP{gamma}S binding with an EC50 of 2.0 nM and a gain of approximately five times. The stimulation by nociceptin of [35S]GTP{gamma}S 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]GTP{gamma}S 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]GTP{gamma}S binding to the fusion protein in the brain extract derived from 2–3 g 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 G{alpha}i is useful for screening of endogenous ligands.

* To whom correspondence should be addressed at: Department of Nano-Material Systems, Graduate School of Engineering, Gunma University. Tel/Fax: +81-277-30-1434, E-mail: stakeda{at}bce.gunma-u.ac.jp

§ Present address: Department of Nano-Material Systems, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515.

Present address: Division of Cell Biology, The Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma Nara 630-0101.


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