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Fig. 7.8 Live-cell confocal imaging and FRET from HEK 293 cells expressing 5-HT2C and 5-HT . (a) 5-HT2C/YFP expressed on the plasma membrane of transfected HEK 293 cells. (b) Intracellular 5-HT2Ctr/YFP 24 h posttransfection. Note that the pattern of 5-HT2Ctr fluorescence is consistent with the pattern of fluorescence obtained with the ER/YFP marker (as shown in Fig. 7.5a)

Fig. 7.9 Whole cell radioligand binding to intact HEK 293 cells following cotransfection with

[3H]mesulergine for 30 min at 37 °C (±1 uM mianserin to define specific binding) and washed with PBS to remove unbound radioligand. Three percent TCA in PBS was added to the cells (20 min at 37°C) to release plasma membrane bound radioligand. The TCA solution was removed from the monolayer of cells, transferred to a scintillation vial containing Ecoscint Cocktail, and counted in a Beckman scintillation counter. Data represent the mean ± SEM from five transfec-tions. *p < 0.01

5-HT + pcDNA3 vector (control) or 5-HT + 5-HT . Intact cells were incubated with 2.5 nM

Another possible way that dimerization could regulate the functional properties of 5-HT2C receptors is through heterodimers of different 5-HT2C receptor isoforms with different functional characteristics. RNA editing occurs in the second intracel-lular loop of the 5-HT2C receptor, changing amino acids 156, 158, and 160 from INI to VGV in the fully edited isoform and results in the expression of 14 different

5-HT2C receptor isoforms in the human brain (Burns et al. 1997; Niswender et al. 1999; Fitzgerald et al. 1999) with varying levels of constitutive activity (HerrickDavis et al. 1999). The unedited INI isoform has high basal activity, while the fully edited VGV isoform has little to no basal activity (Herrick-Davis et al. 1999). Furthermore, the VGV isoform differs in its binding affinity for several drugs including 5-HT and LDS (Burns et al. 1997; Fitzgerald et al. 1999). To determine if heterodimerization among 5-HT2C receptor isoforms is possible, a series of BRET measurements were made following coexpression of different receptor isoforms in HEK 293 cells (Fig. 7.10). Since it would not be possible to test all possible combinations of isoforms, BRET was measured between the most divergent and most similar isoforms. As expected from the results of our FRET experiments, the same BRET ratio was obtained for INI and VGV homodimers. Similar BRET ratios were produced following coexpression of INI and VGV, INI and VSV, and VSV with VGV (Fig. 7.10). These results suggest that there is no difference in the ability of the different isoforms to associate with one another.

Studies to address the functional significance of putative 5-HT2C isoform het-erodimers have not been performed. Based on previous studies characterizing the ligand binding and constitutive signaling capabilities (functional properties) of the different isoforms, the two isoforms with the most divergent functional properties are the unedited (INI) and fully edited (VGV) isoforms (Burns et al. 1997; Niswender et al. 1999; Fitzgerald et al. 1999; Herrick-Davis et al. 1999). It is tempting to speculate that an INI-VGV heterodimer may have different functional

Fig. 7.10 5-HT2C receptor isoform BRET. HEK 293 cells were transfected with cDNAs encoding different 5-HT2C isoforms as indicated. Emission spectra were collected immediately following the addition of coelenterazine f. BRET ratios were calculated from the emission spectra as previously described (Herrick-Davis et al. 2004; Angers et al. 2000) (Data represent the mean ± SEM from three experiments)

Fig. 7.10 5-HT2C receptor isoform BRET. HEK 293 cells were transfected with cDNAs encoding different 5-HT2C isoforms as indicated. Emission spectra were collected immediately following the addition of coelenterazine f. BRET ratios were calculated from the emission spectra as previously described (Herrick-Davis et al. 2004; Angers et al. 2000) (Data represent the mean ± SEM from three experiments)

properties than either homodimeric species. However, it is not clear whether a single cell would have a significant portion of both unedited and fully edited iso-forms to allow the formation of INI-VGV heterodimers. Studies from the original paper identifying 5-HT2C RNA editing reported the isoform distribution for several different brain regions (Burns et al. 1997). While multiple isoforms were identified in each brain region, it is noteworthy that the isoforms within an individual brain region tended to be similar in terms of their degree of RNA editing. For example, the majority of the isoforms identified in the choroid plexus were ones that were minimally edited and had the highest affinity for 5-HT and highest levels of constitutive activity, following expression in HEK 293 cells (Herrick-Davis et al. 1999). This observation suggests that it may be more likely to have heterodi-mers among isoforms with similar degrees of RNA editing. If this is the case, then heterodimers between VSV and VGV would be predicted to have the most dramatic change in functional characteristics since the VGV isoform has distinct differences in its ligand binding properties from the other isoforms, including VSV (Niswender et al. 1999; Fitzgerald et al. 1999). Of course, this remains speculative until it is demonstrated that native VSV and VGV isoforms exist as heterodimers in vivo.

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