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J. Biol. Chem., Vol. 278, Issue 27, 24552-24562, July 4, 2003

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A Combinatorial G Protein-coupled Receptor Reconstitution System on Budded Baculovirus

EVIDENCE FOR G_i AND G_o COUPLING TO A HUMAN LEUKOTRIENE B₄ RECEPTOR^*

Kazuyuki Masuda  , Hiroshi Itoh ¶, Toshiko Sakihama , Chiyuki Akiyama ||, Kazuaki Takahashi ||, Rie Fukuda  ||, Takehiko Yokomizo **  ¶¶, Takao Shimizu ** , Tatsuhiko Kodama  and Takao Hamakubo  || ||||

From the Laboratory for Systems Biology and Medicine and the ^||Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, the Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., Yokohama 222-8567, the ^¶Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0101, and the ^**Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo and the Japan Science and Technology Corp. (CREST and ^¶¶PRESTO), Tokyo 113-0033, Japan

To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B₄ receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [³H]leukotriene B₄ binding activity (K_d = 3.67 nM). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the G_i isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(,-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing G_i112 (K_d = 0.17 nM). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and G_i1 without G₁₂ did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various G subunits were combinatorially expressed in BV with BLT1 and G₁₂. The BLT1 BV co-expressing G_oA12 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to G_i112. Co-expression of other G isoforms such as G_s, G₁₁, G₁₄, G₁₆, G₁₂, or G₁₃ did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that G_o as well as G_i couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.

Received for publication, March 19, 2003 , and in revised form, April 28, 2003.

^* This study was supported in part by The project for Technological Development of Biological Resources in Bioconsortia on Research and Development of New Industrial Science and Technology Frontiers, which was performed by Industrial Science, Ministry of Economy, Trade and Industry and entrusted by New Energy Development Organization and by the Research for the Future Program of the Japan Society for the Promotion of Science. This study was also supported in part by a Grant-in-aid for Science Research (B2) 13558084 and by Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|||| To whom correspondence should be addressed: Dept. of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8904, Japan. Tel.: 81-3-5452-5231; Fax: 81-3-5452-5232; E-mail: hamakubo{at}med.rcast.u-tokyo.ac.jp.

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