Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein.
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.
Herrick-Davis
Katharine
K
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA. daviskh@mail.amc.edu
Grinde
Ellinor
E
Harrigan
Timothy J
TJ
Mazurkiewicz
Joseph E
JE
D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D019204:GTP-Binding Proteins / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D044405:Receptor, Serotonin, 5-HT2C / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D058830:Serotonin 5-HT2 Receptor Antagonists; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D018076:DNA, Complementary; D008024:Ligands; D008164:Luminescent Proteins; D044405:Receptor, Serotonin, 5-HT2C; D058830:Serotonin 5-HT2 Receptor Antagonists; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D012694:Serine; D001120:Arginine; D019204:GTP-Binding Proteins