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J. Biol. Chem., Vol. 280, Issue 48, 40144-40151, December 2, 2005

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Inhibition of Serotonin 5-Hydroxytryptamine2C Receptor Function through Heterodimerization

RECEPTOR DIMERS BIND TWO MOLECULES OF LIGAND AND ONE G-PROTEIN^*

From the Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT_2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT_2C receptor was created and coexpressed with wild-type 5-HT_2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser¹³⁸ to Arg (S138R) produced a 5-HT_2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT_2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT_2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT_2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT_2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT_2C receptors inhibit wild-type 5-HT_2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.

Received for publication, July 8, 2005 , and in revised form, August 30, 2005.

^* This work was supported by National Institutes of Health Grants MH057019 (to K. H.-D.) and RR017926 (to J. E. M.). 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.

¹ To whom correspondence should be addressed: Center for Neuropharmacology and Neuroscience, MC-136, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Tel.: 518-262-6357; Fax: 518-262-5799; E-mail: daviskh{at}mail.amc.edu.

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