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J. Biol. Chem., Vol. 283, Issue 34, 23150-23160, August 22, 2008

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Ric-8A Catalyzes Guanine Nucleotide Exchange on G_i1 Bound to the GPR/GoLoco Exchange Inhibitor AGS3^*

Celestine J. Thomas, Gregory G. Tall, Anirban Adhikari^¶, and Stephen R. Sprang¹

From the Center for Bimolecular Structure and Dynamics and the Division of Biological Science, University of Montana, Missoula, Montana 59812, the Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, and the ^¶Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including G subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various G·GDP subunits and forms a stable nucleotide-free Ric-8A:G complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes G subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for G·GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:G_il and that of Ric-8A with the AGS3-C:G_il·GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:G_i1·GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:G_i1·GDP. Subsequent dissociation of AGS3-C and GDP from G_i1 yields a stable nucleotide free Ric-8A·G_i1 complex that, in the presence of GTP, dissociates to yield Ric-8A and G_i1·GTP. AGS3-C does not induce dissociation of the Ric-8A·G_i1 complex, even when present at very high concentrations. The action of Ric-8A on AGS3:G_i1·GDP ensures unidirectional activation of G subunits that cannot be reversed by AGS3.

Received for publication, March 28, 2008 , and in revised form, May 26, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants R01-DK46371 (to S. R. S.) and GM34497 (to Alfred G. Gilman). 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 Figs. 1 and 2.

¹ To whom correspondence should be addressed. Tel.: 406-243-6028; E-mail: stephen.sprang{at}umontana.edu.

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