Abstract
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. FASEB J. 33, 9526–9539 (2019). www.fasebj.org.
| Original language | English |
|---|---|
| Pages (from-to) | 9526-9539 |
| Number of pages | 14 |
| Journal | FASEB Journal |
| Volume | 33 |
| Issue number | 8 |
| DOIs | |
| State | Published - 1 Aug 2019 |
| Externally published | Yes |
Bibliographical note
Publisher Copyright:© FASEB
Funding
The authors thank Drs. Philip Kiser [Physiology & Biophysics Department, University of California–Irvine, Irvine, CA, USA (UCI)], Timothy Kern (Ophthalmology Department, UCI), and Angele Nalbandian (Ophthalmology Department, UCI) for helpful comments on this manuscript. The authors wish to thank members of the Small Molecule Drug Development Core facilities at Case Western Reserve University for guidance through the development of our high-throughput screening (HTS) assay. This research was supported in part by grants from the U.S. National Institutes of Health (NIH) National Eye Institute (EY R 40 4864 and R24EY027283 to K.P., EY024992 to Y.C., and EY026651 to F.V.), an unrestricted Research to Prevent Blindness (RPB) grant to the Department of Ophthalmology at UCI; the Canadian Institute for Advanced Research (CIFAR); and the Alcon Research Institute (ARI). K.P. is the Leopold Chair of Ophthalmology. The authors declare no conflicts of interest.
| Funders | Funder number |
|---|---|
| University of California–Irvine, Irvine, CA, USA | |
| National Institutes of Health | |
| National Eye Institute | EY R 40 4864, EY024992, EY026651, R24EY027283 |
| Research to Prevent Blindness | |
| Canadian Institute for Advanced Research | |
| Alcon Research Institute | |
| Urban Child Institute |
Keywords
- G-protein-coupled receptors
- GPCR dimerization
- chromophore
- high-throughput screening
- isomerization