Abstract
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.
Highlights
The quaternary organization of G protein-coupled receptors remains a subject of considerable debate
Determination of the Quantal Brightness of Membrane-localized A206K enhanced green fluorescent protein (eGFP)—Spatial intensity distribution analysis (SpIDA) can be used to assess the oligomeric size of complexes of a protein tagged with an appropriate fluorophore, such as monomeric A206K eGFP, at various locations in a cell by statistical analysis of suitable laser confocal scanning
We have previously expressed transiently in HEK293T cells either monomeric A206K eGFP or a tandem of this protein in which the two copies of the fluorescent polypeptide were linked by a short peptide sequence (Arg-Ile-Leu-GlnSer-Thr-Val-Pro-Arg-Ala-Arg-Asp-Pro-Pro-Val-Asp-Ile)
Summary
The quaternary organization of G protein-coupled receptors remains a subject of considerable debate. This is one of the most contentious areas in current biology with very different conclusions being reached These range from opinions that consider most of the receptor population to exist as monomer, with only random collisions suggesting quaternary structure [17,18,19,20,21], to others that indicate the vast majority, or even all, of the receptor exists as either dimers or higher-order oligomers [22,23,24,25]. Treatment with a number of antagonists of the 5-HT2C receptor (reversibly) reduced this complexity to favor monomeric and dimeric forms of the receptor These results demonstrate that this receptor can exist in multiple forms, that interaction with ligands can alter this ensemble substantially, and that methods that report averages of receptor organizational structure fail to identify the level of complexity present
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