The receptor-Galpha fusion protein as a tool for ligand screening: a model study using a nociceptin receptor-Galphai2 fusion protein.
As a model system to screen endogenous ligands for G(i)-coupled receptors, we have prepared and characterized a fusion protein of nociceptin receptor and alpha subunit of G(i2). We detected nociceptin binding to the fusion protein by measuring stimulation of [(35)S]GTPgammaS binding with an EC(50) of 2.0 nM and a gain of approximately five times. The stimulation by nociceptin of [(35)S]GTPgammaS 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 IC(50) values for GDP to displace 100 pM [(35)S]GTPgammaS were estimated to be 2 micro M, 0.4 micro M, and 0.05 micro M in the presence of full agonist (nociceptin), partial agonist (F/G-NC), and antagonist (NBZH), respectively. We also detected the activity to stimulate [(35)S]GTPgammaS 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 Galpha(i) is useful for screening of endogenous ligands.
Takeda
Shigeki
S
Department of Neurochemistry, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033. stakeda@bce.gunma-u.ac.jp
Okada
Tomoaki
T
Okamura
Michiko
M
Haga
Tatsuya
T
Isoyama-Tanaka
Junko
J
Kuwahara
Hiromiki
H
Minamino
Naoto
N
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D001921:Brain / Q000378:metabolism; D002845:Chromatography; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D018847:Opioid Peptides / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D011957:Receptors, Opioid / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D013552:Swine; D000094942:Nociceptin Receptor; D000097629:Nociceptin
D017931:DNA Primers; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008024:Ligands; D018847:Opioid Peptides; D011518:Proto-Oncogene Proteins; D011957:Receptors, Opioid; D011993:Recombinant Fusion Proteins; D000094942:Nociceptin Receptor; D000097629:Nociceptin