G protein-coupled receptors (GPCRs) make up the largest family of cell surface protein receptors and are involved in a number of diverse biological processes. The association of GPCRs, whether they be monomeric, dimeric, or oligomeric, is hypothesized to alter their signaling. Attaining crystallographic evidence of the dimeric or oligomeric associations of Class A GPCRs, specifically (non)visual opsins, remains a difficulty, as does establishing the stability of these associations. The purpose of this research was to quantify the association of (non)visual opsins, in situ, in the plasma membrane of live cells. We used a time-resolved fluorescence approach to accomplish this purpose. Pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) offered a way in which the dynamic interactions of (non)visual opsins could be quantified.
Throughout this dissertation, three projects will be presented. The first project focused on the dimeric association of rhodopsin, the light sensitive protein involved in scotopic vision. By transfecting low concentrations of rhodopsin into mammalian cells, we found a modest affinity for dimerization. The second project focused on the proteins involved in trichromatic photopic vision, cone opsins. Two of the three human cone opsins, OPN1LW (red) and OPN1MW (green) share a 95% sequence homology. Despite having such a homology, red and green cone opsin showed different affinities for dimerization. Red cone opsin was observed to have the highest affinity for dimeric association among the GPCRs studied. Green cone opsin was shown to primarily exist as a monomer. Mutagenesis was performed on both red and green cone opsin in an attempt to decrease red cone opsin dimerization affinity and increase green cone opsin dimerization affinity. The third project focused on melanopsin, a non-visual human opsin. Melanopsin is expressed in the ganglion cell layer (GCL) of the retina and plays a role in both circadian rhythm and the pupillary light response. The experiments in Chapter 5 demonstrate that melanopsin has a low dimerization affinity. The affinity is higher than our monomeric controls, but lower than that of both rhodopsin and red cone opsin. Establishing the native association of these visual and non-visual opsins in the retina is a key step in determining how the spatial organization of these proteins regulates their biological function. Experiments in chapters 3, 4, and 5 begin to connect dimerization to function, but more work is needed to quantify these relationships. This work also creates a paradigm in which GPCR dimerization can be quantified and contextualized, which is critical for developing new pharmaceutical treatments for this important class of proteins.