Quercetin effect on the stability and regeneration of the G-protein-coupled receptor rhodopsin
Perez, J.; Garriga, P.; Herrera-Hernandez, M.; S. Lupala, C.; Dong, X.
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The XXIX symposium of the Protein Society
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Data de presentació
G-protein coupled receptors (GPCRs) are transmembrane heptahelical receptors that constitute a large and widespread family of signal transduction proteins. A number of extracellular ligands, ranging from small molecules to GPCR-binding proteins, have been proposed as good candidates for drug design. The binding of an agonist to a GPCR causes a conformational change in the receptor that leads to its activated functional state. Rhodopsin, the membrane receptor responsible for photoreception in the...
G-protein coupled receptors (GPCRs) are transmembrane heptahelical receptors that constitute a large and widespread family of signal transduction proteins. A number of extracellular ligands, ranging from small molecules to GPCR-binding proteins, have been proposed as good candidates for drug design. The binding of an agonist to a GPCR causes a conformational change in the receptor that leads to its activated functional state. Rhodopsin, the membrane receptor responsible for photoreception in the vertebrate retina, is a prototypical GPCR and has been extensively used in structural, biochemical and biophysical studies of this class of receptors. Different small molecules have been described to be capable of binding to rhodopsin. In addition, mutations in rhodopsin have been associated with retinal diseases and efforts have been carried out in order to find potential ligands that can offset the effect of these mutations. Cyanidins, a group of flavonoids within the larger family of polyphenols, have been reported to stimulate chromophore regeneration of rhodopsin by means of the formation of regeneration intermediates.
The aim of the current study was to evaluate the effect of the flavonoid quercetin on the conformational properties of both native bovine rhodopsin and heterologously expressed recombinant rhodopsin. Rhodopsin was purified from bovine retinas by immunoaffinity chromatography, and photobleaching, thermal stability, metarhodopsin II decay and chromophore regeneration assays were carried out in the absence or in the presence of 1µM quercetin. For recombinant rhodopsin, a plasmid encoding wild-type opsin was transfected into mammalian COS-1 cells, in the absence or in the presence of 1µM quercetin, harvested, regenerated with 11-cis-retinal, or 9-cis-retinal, and subsequently purified in dodecyl maltoside solution. Western blot, photobleaching, thermal stability, metarhodopsin II decay and regeneration assays were carried out on the purified proteins in the absence and in the presence of quercetin.
No differences in photobleaching behavior, upon illumination, could be detected in the quercetin-containing samples compared to those in the absence of this flavonoid. In the case of rhodopsin, and the recombinant wild-type protein regenerated with 11-cis-retinal, quercetin did not significantly alter the thermal stability and rate of regeneration of the purified proteins under our experimental conditions. However, a two-fold increase in the thermal stability and a 40% increase in chromophore regeneration were observed for the recombinant wild-type protein regenerated with 9-cis-retinal in the presence of quercetin. In contrast, the presence of quercetin did not alter the electrophoretic and basic spectroscopic properties of rhodopsin, or those of the recombinant wild-type protein, suggesting no important structural alterations as a result of quercetin binding to the receptor. The positive effect of quercetin on the stability, and chromophore regeneration of rhodopsin, could be potentially used to counteract the effect of naturally-occurring misfolding mutations in rhodopsin. Therefore, a stabilizing effect can be predicted for quercetin, and other flavonoids, on the structure and stability of rhodopsin mutants associated with retinal diseases such as retinitis pigmentosa. To give further support to these observations we carried out a molecular modeling study aimed at understanding the way quercetin binds to rhodopsin. For this purpose we carried out a docking study of the ligand on the binding pocket of the crystallographic structure of rhodopsin (entry 1GZM). Among the favorable sites available for quercetin binding, we identified one that is compatible with the binding of 9-cis-retinal suggesting a complementary binding to the receptor, that is not compatible with 11-cis-retinal binding.