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J. Biol. Chem., Vol. 279, Issue 29, 30279-30286, July 16, 2004

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Visualization of G Protein Dimers Using Bimolecular Fluorescence Complementation Demonstrates Roles for Both and in Subcellular Targeting^*

Thomas R. Hynes, Linnan Tang, Stacy M. Mervine, Jonathan L. Sabo, Evan A. Yost, Peter N. Devreotes, and Catherine H. Berlot¶

From the The Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2623 and The Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

To investigate the role of subcellular localization in regulating the specificity of G protein signaling, we have applied the strategy of bimolecular fluorescence complementation (BiFC) to visualize dimers in vivo. We fused an amino-terminal yellow fluorescent protein fragment to and a carboxyl-terminal yellow fluorescent protein fragment to . When expressed together, these two proteins produced a fluorescent signal in human embryonic kidney 293 cells that was not obtained with either subunit alone. Fluorescence was dependent on assembly in that it was not obtained using ₂ and ₁, which do not form a functional dimer. In addition to assembly, BiFC complexes were functional as demonstrated by more specific plasma membrane labeling than was obtained with individually tagged fluorescent and subunits and by their abilities to potentiate activation of adenylyl cyclase by _s in COS-7 cells. To investigate isoform-dependent targeting specificity, the localization patterns of dimers formed by pair-wise combinations of three different subunits with three different subunits were compared. BiFC complexes containing either ₁ or ₂ localized to the plasma membrane, whereas those containing ₅ accumulated in the cytosol or on intracellular membranes. These results indicate that the subunit can direct trafficking of the subunit. Taken together with previous observations, these results show that the G protein , , and subunits all play roles in targeting each other. This method of specifically visualizing dimers will have many applications in sorting out roles for particular complexes in a wide variety of cell types.

Received for publication, February 9, 2004 , and in revised form, May 3, 2004.

^* This work was supported by National Institutes of Health Grant GM50369. 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 Video 1.

^¶ To whom correspondence should be addressed: The Weis Center for Research, Geisinger Clinic, 100 North Academy Ave., Danville, PA 17822-2623. Tel.: 570-271-8661; Fax: 570-271-6701; E-mail: chberlot{at}geisinger.edu.

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