Abstract
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-HT2C) 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-HT2C 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-HT2C 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-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C 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-HT2C receptors inhibit wild-type 5-HT2C 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.
Highlights
G-protein-coupled receptors (GPCRs)2 represent one of the largest families of signaling proteins in the human genome and are targets for a wide variety of therapeutic agents
5-HT stimulated [3H]inositol phosphate (IP) production in human embryonic kidney 293 (HEK293) cells transfected with plasmid containing wild-type 5-HT2C receptor cDNA, but not in cells transfected with plasmid containing Ser138 to Arg (S138R) 5-HT2C receptor cDNA (Fig. 1)
These results demonstrate that mutation of Ser138 to Arg eliminates the ability of the 5-HT2C receptor to bind ligand and to stimulate phospholipase C
Summary
G-protein-coupled receptors (GPCRs) represent one of the largest families of signaling proteins in the human genome and are targets for a wide variety of therapeutic agents. Radioligand binding, inositol phosphate (IP) signaling, confocal imaging of receptor endocytosis, and fluorescence resonance energy transfer (FRET) were evaluated in HEK293 cells coexpressing wild-type and inactive mutant S138R 5-HT2C receptors to determine the effect of dimerization on receptor function and to determine the ligand/dimer/G-protein stoichiometry. The results of this study show that S138R receptors form heterodimers with wild-type 5-HT2C receptors in living cells and have an inhibitory effect on 5-HT2C receptor function. Ligand/Dimer/G-protein Stoichiometry negative effect of an inactive mutant GPCR on wild-type receptor function through heterodimerization, resulting in the expression of nonfunctional receptor complexes on the plasma membrane. Other studies using isolated membranes [21] and purified receptors [22] suggest that each GPCR dimer interacts with a single G-protein We tested this model in intact living cells following coexpression of wild-type and inactive 5-HT2C receptors. Our results suggest that each GPCR dimer binds two molecules of ligand, resulting in the activation of one G-protein, and indicate that dimerization is essential for 5-HT receptor function
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