Regulation of Oligomeric Organization of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Observed by Spatial Intensity Distribution Analysis

• Published in: Journal of Biological Chemistry Vol. 290; no. 20; pp. 12844 - 12857
• Main Authors: Ward, Richard J., Pediani, John D., Godin, Antoine G., Milligan, Graeme
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.05.2015; Elsevier BV; American Society for Biochemistry and Molecular Biology
• Subjects: epidermal growth factor receptor (EGFR); G protein-coupled receptor (GPCR); Green Fluorescent Proteins; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Humans; Multiprotein Complexes; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Protein Multimerization; Protein Multimerization - drug effects; Protein Multimerization - physiology; Protein Structure and Folding; Protein Structure, Quaternary; Receptor, Serotonin, 5-HT2C; Receptor, Serotonin, 5-HT2C - genetics; Receptor, Serotonin, 5-HT2C - metabolism; Recombinant Fusion Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; serotonin; Serotonin 5-HT2 Receptor Antagonists; Serotonin 5-HT2 Receptor Antagonists - pharmacology; seven-helix receptor; single-molecule biophysics; G protein-coupled receptor (GPCR); single-molecule biophysics; serotonin; seven-helix receptor; epidermal growth factor receptor (EGFR)
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M115.644724

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor. The quaternary organization of G protein-coupled receptors remains a subject of considerable debate. The serotonin 5-HT2C receptor is a mixture of monomers, dimers, and higher-order oligomers. These become predominantly monomers upon antagonist treatment. SpIDA indicates that the 5-HT2C receptor exists as mixtures of forms that are regulated in an antagonist-dependent manner. SpIDA delivers new insights into receptor quaternary structure.