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J. Biol. Chem., Vol. 279, Issue 26, 27709-27718, June 25, 2004

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A Fluorescence Resonance Energy Transfer-based Sensor Indicates that Receptor Access to a G Protein Is Unrestricted in a Living Mammalian Cell^*

Inaki Azpiazu and N. Gautam¶

From the Departments of Anesthesiology and Genetics, Washington University School of Medicine, St. Louis, Missouri 63110

Fluorescence recovery after photobleaching of muscarinic receptors and G protein subunits tagged with cyan or yellow fluorescent protein showed that receptors and G proteins were mobile and not immobilized on the cell membrane. The cyan fluorescent protein-tagged G and yellow fluorescent protein-tagged G subunits were used to develop sensors that coupled selectively with the M2 and M3 muscarinic receptors. In living Chinese hamster ovary cells, imaging showed that sensors emitted a fluorescence resonance energy transfer signal that was abrogated on receptor activation. When sequentially activated with highly expressed muscarinic receptors and endogenous receptors expressed at low levels, sensor molecules were sensitive to the sequence of activation and the receptor numbers. The results distinguish between models proposing that receptor and G protein types interact freely with each other on the cell membrane or that they function as mutually exclusive multimolecular complexes by providing direct support for the former model in these cells.

Received for publication, April 2, 2004

^* This work was supported by National Institutes of Health Grant GM46963. 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 text and figures.

^¶ To whom correspondence should be addressed: Box 8054, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-8568; Fax: 314-362-8571; E-mail: gautam{at}morpheus.wustl.edu.

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