The classic concept that GPCRs function as monomers has been challenged by the emerging evidence of GPCR dimerization and oligomerization. Rhodopsin (Rh) is the only GPCR whose native oligomeric arrangement was revealed by atomic force microscopy demonstrating that Rh exists as a dimer. However, the role of Rh dimerization in retinal physiology is currently unknown. In this study, we identified econazole and sulconazole, two small molecules that disrupt Rh dimer contacts, by implementing a cell-based high-throughput screening assay. Racemic mixtures of identified lead compounds were separated and tested for their stereospecific binding to Rh using UV-visible spectroscopy and intrinsic fluorescence of tryptophan (Trp) 265 after illumination. By following the changes in UV-visible spectra and Trp265 fluorescence in vitro, we found that binding of R-econazole modulates the formation of Meta III and quenches the intrinsic fluorescence of Trp265. In addition, electrophysiological ex vivo recording revealed that R-econazole slows photoresponse kinetics, whereas S-econazole decreased the sensitivity of rods without effecting the kinetics. Thus, this study contributes new methodology to identify compounds that disrupt the dimerization of GPCRs in general and validates the first active compounds that disrupt the Rh dimer specifically.-Getter, T., Gulati, S., Zimmerman, R., Chen, Y., Vinberg, F., Palczewski, K. Stereospecific modulation of dimeric rhodopsin.
Keywords: G-protein-coupled receptors; GPCR dimerization; chromophore; high-throughput screening; isomerization.