Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Dec 26;289(52):35918-28.
doi: 10.1074/jbc.M114.609958. Epub 2014 Oct 30.

Differential light-induced responses in sectorial inherited retinal degeneration

Eva Ramon  1 , Arnau Cordomí  2 , Mònica Aguilà  3 , Sundaramoorthy Srinivasan  1 , Xiaoyun Dong  1 , Anthony T Moore  4 , Andrew R Webster  4 , Michael E Cheetham  3 , Pere Garriga  5
Affiliations

Differential light-induced responses in sectorial inherited retinal degeneration

Eva Ramon et al. J Biol Chem. .

Abstract

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.

Keywords: G-protein-coupled Receptor; Light Exposure; Protein Stability; Protein Structure; Retinal Degeneration; Rhodopsin; Signal Transduction; Transducin; Visual Pigments.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Representative fundus images from patients with RHO p.N55K. Color fundus images of patient GC18466 showing no progression from years 41 (A) to 45 (B). C–H, autofluorescence images right (C, E, and G) and left eyes (D, F, and H) of GC18466 at 13 years (C and D), GC16563 at 39 years (E and F), and GC18466 at 45 years (G and H). OCT images of GC18466 at 13 years in left eye (I) and GC18466 at 45 years in right eye (J).
FIGURE 2.
FIGURE 2.
Representative images from patient with RHO p.M39R. Autofluorescence images, 55° of field of six patients, each heterozygous for RHO, p.M39R presented in ascending age. A, GC97/son, 28 years; B, GC19172 29 years; C, GC414 48 years; D, GC109 50 years; E, GC97/father 57 years; F, GC4740 68 years. Hyperfluorescent rings delineate normal retina in the two youngest individuals.
FIGURE 3.
FIGURE 3.
Environment of positions Met-391. 34 and Asn-551.50 in dark state and active (rhod)opsin crystal structures. A and B, lateral (A) and extracellular (B) views of the proposed entry/exit channel for retinal between TM1 and TM7, in the vicinity of Met-391.34. C, polar cluster at the cytoplasmic side of TM1, -2, and -7 close to Asn-551.50. Helices are displayed as schematics, and the relevant side chains are shown as sticks colored by atom type and crystallographic water molecules as spheres. Dark-state crystal structures (Protein Data Bank codes 1GZM, 1L9H, and 1U19) are shown in orange, and active-like structures (3DBQ, 2X72, and 3PQR) are in red. The side chains of Met/Arg-39 (A and B) and Asn/Lys-55 (C) are shown with a transparent surface. (Hydroxyethyloxy)tri(ethyloxy)octane, palmitoyl, and octyl glucoside (A) and retinal molecules (B) are shown as ball-and-sticks. The image was created using PyMOL (Schrodinger, LLC. The PyMOL Molecular Graphics System, Version 1.5).
FIGURE 4.
FIGURE 4.
Subcellular localization of WT, M39R, and N55K mutants in SK-N-SH cells. A and B, representative images of rod opsin WT, M39R, and N55K localization. SK-N-SH cells were transfected with WT and mutant rod opsins, and 24 h later, cells were fixed. A, Rh-1D4 primary antibody (green) was used to compare localization with BiP (red) following cell permeabilization with Triton X-100. B, Rh antibody to an extracellular epitope 4D2 was used on nonpermeabilized cells to show traffic to the cell surface. Images were taken using a Carl Zeiss LSM 710 laser-scanning confocal microscope. Scale bar, 10 μm. C, quantification of opsin localization. Cells were scored for the predominant opsin localization at the PM (open bars) or overlap with an ER marker (gray bars) in ∼400 cells. Error bars represent ± 2SE. D, Western blot of WT, N55K, and M39R transfected SK-N-SH cell lysates for rod opsin expression detected with Rh-1D4. 10 μg of soluble cell lysate protein (S) was compared with 10 μg of lysate digested with EndoH (E) or PNGase F (P). The asterisk highlights the position of deglycosylated rod opsin, and two asterisks indicate the position of opsin dimer. ER-retained opsin is sensitive to EndoH, whereas all opsin is sensitive to PNGase F and will tend to dimerize. The position of molecular mass markers is indicated on the left in kDa.
FIGURE 5.
FIGURE 5.
UV-visible characterization for the immunopurified WT and RP mutant pigment. UV-visible spectra of WT (top panel), M39R (middle panel), and N55K (bottom panel), in the basal state (solid line) and upon light stimulation (dashed line) are shown. Spectra were recorded in buffer C at 20 °C. N55K mutant presents lower chromophoric band compared with WT, with a slightly blue-shifted maximum (3 nm) according to UV-visible spectrophotometric measurements. Insets show the visible region for both dark and illuminated samples.
FIGURE 6.
FIGURE 6.
Chemical and thermal stability for WT and RP mutants. WT, M39R, and N55K in buffer C were treated with 50 mm hydroxylamine, pH 7.0, at 20 °C for the chemical stability assay. Thermal stability assay was carried out at 48 °C. A, chemical reagent stability was determined by monitoring the decrease of λmax over time (at 498 nm for WT and M39R and at 495 nm for N55K). B, thermal stability assay plot showing the decrease of absorbance at the λmax in the visible region over time.
FIGURE 7.
FIGURE 7.
MetaII decay for WT and RP mutants. WT, M39R, and N55K in Buffer C were illuminated, and the Trp fluorescence increase at 330 nm was monitored to follow ligand release (38). After fluorescence stabilization, 50 mm hydroxylamine, pH 7.0, was added to confirm complete retinal release. Fluorescence intensity spectra of MetaII decay for WT, M39R and N55K are shown.
FIGURE 8.
FIGURE 8.
Gt activation by WT and RP mutant opsin and Rh. WT and RP mutants opsin (open circles) and Rh (closed circles) activities were measured by means of a radionucleotide filter binding assay in COS-1 cell membranes in buffer Gt. The mean and standard error of three independent measurements are represented.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Petrs-Silva H., Linden R. (2014) Advances in gene therapy technologies to treat retinitis pigmentosa. Clin. Ophthalmol. 8, 127–136 - PMC - PubMed
    1. Berson E. L. (1993) Retinitis pigmentosa. The Friedenwald Lecture. Invest. Ophthalmol. Vis. Sci. 34, 1659–1676 - PubMed
    1. Palczewski K. (2006) G protein-coupled receptor rhodopsin. Annu. Rev. Biochem. 75, 743–767 - PMC - PubMed
    1. Katritch V., Cherezov V., Stevens R. C. (2013) Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 - PMC - PubMed
    1. Rosenbaum D. M., Rasmussen S. G., Kobilka B. K. (2009) The structure and function of G-protein-coupled receptors. Nature 459, 356–363 - PMC - PubMed

Publication types

LinkOut - more resources

-