Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin
George KhelashviliAnoop Narayana PillaiJoon LeeKalpana PandeyAlexander M. PayneZarek S. SiegelMichel A. CuendetTylor R. LewisVadim Y. ArshavskyJohannes BroichhagenJoshua LevitzAnant K. Menon
8
Citation
64
Reference
10
Related Paper
Citation Trend
Abstract:
Abstract Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.Keywords:
Opsin
HEK 293 cells
Opsin
Rabbit (cipher)
Cite
Citations (9)
Squid rhodopsin (lambda(max) 493 mmicro)-like vertebrate rhodopsins-contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (lambda(max) 500 mmicro) near neutrality, yellow (lambda(max) 380 mmicro) in alkaline solution. The two forms-acid and alkaline metarhodopsin-are interconverted according to the equation, Alkaline metarhodopsin + H(+) right harpoon over left harpoonacid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.
Opsin
Chromophore
Cite
Citations (315)
A protocol for the separation of phosphorhodopsin from phospho‐opsin has been developed. The method takes advantage of the finding that, while 0.5% N,N ‐dimethyldodecylamine‐ N ‐oxide completely solubilises membrane‐embedded phosphorhodopsin, at this concentration of detergent, phospho‐opsin is only sparingly soluble. Phosphorhodopsin solubilised in this manner may be freed from contaminant phospho‐opsin by chromatography on hydroxyapatite. Using this method, the rhodopsin‐kinase‐catalysed phosphorylation of photoexcited rhodopsin and native rhodopsin was studied in rod outer‐segment membranes at different levels of bleaching. Prior to analysis of the phosphorylation mixture, the phosphorylated form of photoexcited rhodopsin was converted into phospho‐opsin by treatment with NH 2 OH. It was found that, while at a 5% bleach level the amount of phosphorhodopsin produced was 15% that of phospho‐opsin, at 60% bleaching the phosphorhodopsin was less than 1% of phospho‐opsin. The phosphorylation reaction under different bleaching conditions was also studied in a completely soluble system (using 2% dodecyl maltoside) and the pattern of phosphate incorporation into rhodopsin versus opsin was identical to that in the membrane system. We have previously proposed that rhodopsin kinase normally exists in an inactive form and is only activated following interaction with photoexcited rhodopsin. The present work strengthens this conclusion and also shows that, following activation, the kinase preferentially phosphorylates photoexcited rhodopsin but can also act upon unbleached rhodopsin. Two possible mechanisms for the activation of the kinase are considered. From the distribution of phosphorhodopsin and phospho‐opsin at different bleaching levels, the relatives rates of the phosphorylation of photoexcited rhodopsin ( k R* ) and rhodopsin ( k R ) were calculated. K R* lk R values for the membrane system of 71 ± 20 and, for the solubilised system, of 80 ± 19 were obtained. The algebraic equation used to obtain these values highlights the fact that the ratio of the concentrations of the two substrates, photoexcited rhodopsin and rhodopsin, in a sample, determines the final distribution of phosphate between bleached and unbleached rhodopsin. This conclusion may contribute to the understanding of ‘high‐gain’ phosphorylation observed previously.
Opsin
Cite
Citations (19)
Opsin
Cite
Citations (4)
Opsin
Digitonin
Retinaldehyde
Visual phototransduction
Hydroxylamine
Cite
Citations (200)
Opsin
Photoreceptor cell
Cite
Citations (19)
Opsin
Chromophore
Retinaldehyde
Dark state
Cite
Citations (48)
The photochemical system upon which the excitation of rod vision depends has been analyzed, and all its component processes brought into free solution. The only action of light in this system is to convert rhodopsin to the highly unstable lumi-rhodopsin. This bleaches in the dark via the intermediate meta-rhodopsin to a mixture of the carotenoid, retinene1, and the colorless protein, opsin. Retinene1 is reduced to vitamin A1 by the coenzyme, reduced cozymase, acting in concert with the enzyme retinene reductase or alcohol dehydrogenase. These are the degradative processes in the rhodopsin system.The resynthesis of rhodopsin from these products is the basis of dark adaptation. A mixture of opsin and retinene1 forms rhodopsin spontaneously in the dark. The retinene reductase system, left to itself, reduces retinene1 to vitamin A1. In the presence of opsin, however, it oxidizes vitamin A1 to retinene1 as rapidly as retinene1, condenses with opsin to form rhodopsin.A mixture of four substances in solution performs all the reactions of the rhodopsin system: vitamin A1, cozymase, alcohol dehydrogenase, and opsin. Opsin is the only one of these specific to the retina.The synthesis of rhodopsin requires the presence of free sulfhydryl (−SH) groups in opsin. Conversely when rhodopsin bleaches, two such groups are liberated for each retinene1 formed. Based upon these changes, an artificial system has been devised in which the bleaching of rhodopsin results in an electrical variation. This may provide a model for the excitation process in rod vision.
Opsin
Retinaldehyde
Cite
Citations (26)
The photoreceptor protein, rhodopsin is a GPCR that has been proposed to exist as a dimer or even as higher order oligomers in native rod outer segment disk membranes. After activation by light, the dark-adapted form, rhodopsin is converted to the bleached form, opsin. Rhodopsin exhibits considerably greater thermal stability than opsin. This is reflected in the thermal denaturation temperature (Tm), which is almost 20 °C higher for rhodopsin than for opsin. In this study we investigate the effect of partial bleaching on the Tm and on the activation energy for thermal denaturation (Eact) of opsin and of rhodopsin to determine if opsin alters the stability of rhodopsin. If mixed oligomers are present we expect the Tm to be perturbed. Disk membranes were exposed to light to achieve between 0 and 100% bleaching. Differential scanning calorimetry (DSC) experiments were performed using a MicroCal VP-DSC microcalorimeter. Each sample was scanned at a rate of15, 30, 60 and 90°/hr. Because the protein transitions are irreversible, a second scan was used to determine the baseline. The Tm for rhodopsin and opsin were determined in the same DSC scan. The Tm remained constant for rhodopsin regardless of the level of bleaching while the Tm for opsin did not. The Eact was calculated from the dependence of the protein transition temperature (Tm) on the rate of the increase of temperature (scan rate). We conclude that in isolated disk membranes rhodopsin behaves as a monomer or it exhibits extraordinary cooperativity with respect to its thermal properties.
Opsin
Thermal Stability
Cite
Citations (0)
Opsin
Digitonin
Retinaldehyde
Hydroxylamine
Visual phototransduction
Cite
Citations (6)