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H. Zhang, J. Fan, S. Li, S. Karan, B. Rohrer, K. Palczewski, J. M. Frederick, W. Baehr, R. K. Crouch; 11-Cis-Retinal Is Required for Normal Trafficking of Membrane-Associated Proteins to Cone Photoreceptor Outer Segments. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4003. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In Lrat-/- or Rpe65-/- mice, the retinoid cycle is disrupted, 11-cis retinal is absent, cone pigments mislocalize and cone photoreceptors degenerate. In contrast, rhodopsin localizes normally to outer segments and rods are relatively stable. We analyzed expression and trafficking of membrane-associated proteins to determine factors causative for rapid degeneration of mutant cones.
Retinas of Lrat-/-, Rpe65-/- and wildtype mice were examined for pigment content, rhodopsin phosphorylation, histology, and distribution/content of peripheral membrane proteins by immunocytochemistry/immunoblotting using monospecific antibodies. Selected transcript levels were determined by quantitative RT/PCR.
Rod opsins (Rho) of either Lrat-/- or Rpe65-/- mice regenerate pigment with 11-cis retinal similar to wildtype levels, transport to the outer segment in the absence of chromophore and are phosphorylated. In contrast, OPN1SW and OPN1MW (S-opsin and M/L-opsin, respectively), GUCY2E (guanylate cyclase 1), GNAT2 (cone transducin α-subunit), PDE6C (cone PDE6α’-subunit) and GRK1 (rhodopsin kinase) fail to traffic to the outer segment. Aberrant localization was evident at P16, before onset of cone degeneration. Immunoblots of P16 retina lysates indicate that protein levels of GNAT2 and PDE6C are reduced, while qRT/PCR shows normal Gnat2 and Pde6c transcript levels suggesting posttranslational downregulation. Correct trafficking of cone pigments could be restored partially in both models upon administration of 11-cis-retinal. In a Rpe65-/-:Rho-/- double knockout model, 11-cis-retinal administration aslo restores trafficking of both cone pigments and membrane-associated phototransduction components to the cone outer segments.
Our results suggest that the cone opsins must be present in the inactive conformation, containing an inverse agonist as the ligand, to transport to the outer segments. The inactive conformation appears essential to direct post-synthesis vesicular trafficking of membrane-associated cone phototransduction proteins.
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