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J. Biochem, 2004, Vol. 135, No. 5 589-596
© 2004 The Japanese Biochemical Society

CELL

Effects of Partial Agonists and Mg2+ Ions on the Interaction of M2 Muscarinic Acetylcholine Receptor and G Protein G{alpha}i1 Subunit in the M2-G{alpha}i1 Fusion Protein

Qingli Zhang*,1, Michiko Okamura1, Zeng-Dong Guo§,1, Shunsuke Niwa,2,3 and Tatsuya Haga{ddagger},1,2,3

1 Department of Neurochemistry, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033; 2 Institute for Biomolecular Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588; and 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Japan

We have expressed a M2-G{alpha}i1 fusion protein in insect cells, in which the G protein {alpha}i1 subunit was fused with a mutant of the muscarinic receptor M2 subtype without glycosylation sites and the central part of the third intracellular loop. The M2-G{alpha}i1 fusion protein showed GTP-sensitive, high-affinity agonist binding. Displacement curves by GDP of [35S]GTP{gamma}S binding shifted to the right in the presence of muscarinic agonists. The extent of the shift was greater for full agonists (120–150 fold) than for partial agonists (25–35 fold), and virtually no shift was observed for antagonists. The affinity for GDP decreased with increasing MgCl2 concentration in the presence of an agonist but was not affected by MgCl2 in the presence of an antagonist. These results indicate that the apparent affinity for GDP of the M2-G{alpha}i1 fusion protein bound to a ligand represents the efficacy of the given ligand, and that Mg2+ is required for the agonist-bound M2 to interact with G{alpha}i1, reducing its affinity for GDP. We propose that the agonist-M2-G{alpha}i1 complex represents the transition state for the GDP-GTP exchange reaction catalyzed by agonist-bound receptors, and that the complex has different affinities for GDP depending on the species of the ligand bound to M2 receptors.

* Present address: Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

§ Present address: Department of Pharmacology, China Medical University, Shenyang, China.

Present address: Amano, Watanabe & Associates, 3-40-4 Shiba, Tokyo 105-0014.

{ddagger} To whom correspondence should be addressed. Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Tel: +81-3-5992-1033, Fax: +81-3-5992-1034, E-mail: tatsuya.haga{at}gakushuin.ac.jp


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