Biochemical and Biophysical Characterization of Serotonin 5-HT2CReceptor Homodimers on the Plasma Membrane of Living Cells

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Abstract
While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT2C receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT2C receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT2C receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT2C receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT2C receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.