Conclusions

Studies using recombinant cell systems have provided evidence consistent with the hypothesis that 5-HT2C receptors are expressed on the plasma membrane as homodimers (Herrick-Davis et al. 2004, 2005, 2006, 2007; Mancia et al. 2008). Homodimerization occurs regardless of whether the receptors are in an inactive or active conformation, and the homodimers do not dissociate upon agonist or inverse agonist binding (Herrick-Davis et al. 2007). 5-HT2C receptor homodi-merization begins during receptor biosynthesis within the ER and is a naturally occurring step in 5-HT2C receptor maturation and processing (Herrick-Davis et al. 2006). Homodimerization may be a prerequisite for normal receptor trafficking and expression on the plasma membrane, as it may be necessary for passing ER quality-control checkpoints that determine functionality. It is also possible that dimerization in the ER may be a prerequisite for trafficking to the plasma membrane as dimers may represent the basic metabotropic signaling unit. The results or our experiments with the 5-HT2C receptor suggest a ligand/ dimer/G-protein stoichiometry of 2:1:1, consistent with a model in which one receptor dimer binds two molecules of ligand and one G protein (Herrick-Davis et al. 2005).

A review of the literature concerning the GPCR dimer interface indicates that TMD I and TMD IV-V are among the most frequently cited regions for class A GPCR dimer/oligomer formation. Consistent with these reports, cysteine cross-linking experiments involving the 5-HT2C receptor reported the formation of dis-ulfide bonds between residues in TMD I and between residues located in TMD IV and V (Mancia et al. 2008). Previous studies have suggested that GPCR may use dimer interfaces at both TMD I and TMD IV-V to form oligomeric complexes (Milligan 2004; Klco et al. 2003; Fotiadis et al. 2006). However, higher-order oligomers of 5-HT2C receptors were not observed following cysteine cross-linking (Mancia et al. 2008). Based on these results and on experiments using receptor-Galpha fusion proteins, it was concluded that 5-HT2C receptor dimers are quasisymmetrical at the TMD IV-V interface and asymmetrical with respect to G-protein coupling (Mancia et al. 2008). The cysteine cross-linking study revealed different receptor conformations at the putative TMD IV-V dimer interface for the agonist and antagonist bound states of the 5-HT2C receptor (Mancia et al. 2008). These results suggest that if 5-HT2C receptors do in fact use a TMD IV-V interface for homodimerization, then it is likely that conformational changes in one pro-tomer of the dimer could be translated across the dimer interface to the adjacent protomer.

Studies demonstrating that native GPCR form dimers/oligomers in vivo are lacking. Studies have reported dimeric/oligomeric D2-dopamine receptors solubilized from brain membranes (Zawarynski et al. 1998), and coimmunoprecipi-tation of native mu- and delta-opiod receptors solubilized from spinal cord (Waldhoer et al. 2005). For 5-HT2C receptors, we have detected the presence of immunoreactive bands the predicted size of receptor homodimers on Western blots of solubilized membrane proteins prepared from freshly isolated choroid plexus tissue. To date, the most compelling evidence for native GPCR dimer/oligomer formation in intact tissue comes from atomic force microscopy studies of rhodopsin receptors in native disk membranes of mouse retina (Liang et al. 2003). While this study showed images of mouse retinal rhodopsin receptors organized in rows of dimmers, the structural organization of native GPCR in neuronal membranes may likely differ from that of rhodopsin receptors in the retina.

Future studies should aim toward studying dimer/oligomer formation of native receptors in live primary neuronal cultures. This will require the development of new tools and techniques specifically designed for labeling and monitoring native receptors in their natural cellular environment. For example, monoclonal antibodies (directed toward extracellular domains) that recognize the native receptor conformation would be valuable tools. If such antibodies were available, then fluorescent-labeled Fab fragments could be generated and used to directly label native receptors in live primary cultures for analysis by fluorescence correlation spectroscopy (FCS). FCS measures fluctuations in the fluorescence intensity of fluorescent molecules diffusing through a very small confocal volume, providing near single molecule detection sensitivity (Briddon and Hill 2008). FCS has been used to monitor ligand-receptor interactions and to identify receptor dimers/oligomers in recombinant cell systems transfected with cDNAs encoding fluorescent fusion proteins. The next step would be to perform FCS experiments using fluorescent Fab fragments to label native receptors. Photon-counting histogram analysis of the FCS data could be used to provide information about the number of fluorescent molecules traveling together within a protein complex, such as dimers or tetramers/oligomers (Chen and Muller 2007). Novel techniques and approaches, as described above, will be essential for elucidating the true dimeric/oligomeric structure of native GPCR in their natural physiological environment.

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