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J. Biol. Chem. 276 (2): 1585-1593

© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Selective Interaction of AGS3 with G-proteins and the Influence of AGS3 on the Activation State of G-proteins*

Michael L. BernardDagger , Yuri K. Peterson, Peter Chung, Jane Jourdan, and Stephen M. Lanier§

From the Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29403

AGS3 (activator of G-protein signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. As an initial approach to define the role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of G-protein. Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT1-MF2. Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with Galpha i3 from cell and tissue lysates, indicating that a subpopulation of AGS3 and Galpha i exist as a complex in the cell. The coimmunoprecipitation of AGS3 and Galpha i was dependent upon the conformation of Galpha i3 (GDP GTPgamma S (guanosine 5'-3-O-(thio)triphosphate)). The regions of AGS3 that bound Galpha i were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro463-Ser650), each of which were capable of binding Galpha i. AGS3-GPR domains selectively interacted with Galpha i in tissue and cell lysates and with purified Galpha i/Galpha t. Subsequent experiments with purified Galpha i2 and Galpha i3 indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one Galpha i subunit at the same time. The AGS3-GPR domains effectively competed with Gbeta gamma for binding to Galpha t(GDP) and blocked GTPgamma S binding to Galpha i1. AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.

* This work was supported in part by Grants NS24821 and MH5993 (to S. M. L.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Medical Scientist Training Program fellowship, supported by National Institutes of Health Grant T32-GM08716.

§ To whom correspondence should be addressed: Dept. of Pharmacology, Medical University of South Carolina, 173 Ashley Ave., Charleston, SC 29403. Tel.: 843-792-2574; Fax: 843-792-2475; E-mail: laniersm@musc.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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