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J. Biol. Chem., Vol. 283, Issue 8, 4665-4673, February 22, 2008

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Direct Detection of the Interaction between Recombinant Soluble Extracellular Regions in the Heterodimeric Metabotropic -Aminobutyric Acid Receptor^*

Rei Nomura, Yoshikazu Suzuki¹, Akira Kakizuka, and Hisato Jingami^¶2

From the Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, the Laboratory of Functional Biology, Kyoto University Graduate School of Biostudies & Solution Oriented Research for Science and Technology (JST), Sakyo-Ku, Kyoto 606-8501, and ^¶The Graduate Courses for Integrated Research Training, Kyoto University Faculty of Medicine, Sakyo-Ku, Kyoto 606-8501, Japan

The -aminobutyric acid, type B (GABA_B) receptor is a heterodimeric receptor consisting of two complementary subunits, GABA_B1 receptor (GBR1) and GABA_B2 receptor (GBR2). GBR1 is responsible for GABA binding, whereas GBR2 is considered to perform a critical role in signal transduction toward downstream targets. Therefore, precise communication between GBR1 and GBR2 is thought to be essential for the proper signal transduction process. However, biochemical data describing the interaction of the two subunits, especially for the extracellular regions, are not sufficient. Thus we began by developing a protein expression system of the soluble extracellular regions. One of the soluble recombinant GBR1 proteins exhibited a ligand binding ability, which is similar to that of the full-length GBR1, and thus the ligand-binding domain was determined. Direct interaction between GBR1 and GBR2 extracellular soluble fragments was confirmed by co-expression followed by affinity column chromatography and a sucrose density gradient sedimentation. In addition, we also found homo-oligomeric states of these soluble extracellular regions. The interaction between the two soluble extracellular regions caused the enhancement of the agonist affinity for GBR1 as previously reported in a cell-based assay. These results not only open the way to future structural studies but also highlight the role of the interaction between the extracellular regions, which controls agonist affinity to the heterodimeric receptor.

Received for publication, June 25, 2007 , and in revised form, December 28, 2007.

^* This work was supported by a research grant endorsed by the New Energy and Industrial Technology Development Organization. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Present address: Otsuka Pharmaceutical Co., Ltd., 224-18, Hiraishi-Ebisuno, Kawauchi-cho, Tokushima 771-0195, Japan.

² To whom correspondence should be addressed: Tel.: 81-75-753-9493; Fax: 81-75-753-9495; E-mail: jingami{at}mfour.med.kyoto-u.ac.jp.

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