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J. Biol. Chem., Vol. 279, Issue 16, 16278-16284, April 16, 2004

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Retinoids Assist the Cellular Folding of the Autosomal Dominant Retinitis Pigmentosa Opsin Mutant P23H^*

Syed M. Noorwez, Ritu Malhotra, J. Hugh McDowell, Karen A. Smith, Mark P. Krebs¶||, and Shalesh Kaushal**

From the Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, Florida 32610, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, and ^¶Department of Biological Sciences, Illinois State University, Normal, Illinois 61790

The clinically common mutant opsin P23H, associated with autosomal dominant retinitis pigmentosa, yields low levels of rhodopsin when retinal is added following induction of the protein in stably transfected HEK-293 cells. We previously showed that P23H rhodopsin levels could be increased by providing a 7-membered ring, locked analog of 11-cis-retinal during expression of P23H opsin in vivo. Here we demonstrate that the mutant opsin is effectively rescued by 9- or 11-cis-retinal, the native chromophore. When retinal was added during expression, P23H rhodopsin levels were 5-fold (9-cis) and 6-fold (11-cis) higher than when retinal was added after opsin was expressed and cells were harvested. Levels of P23H opsin were increased 3.5-fold with both compounds, but wild-type protein levels were only slightly increased. Addition of retinal during induction promoted the Golgi-specific glycosylation of P23H opsin and transport of the protein to the cell surface. P23H rhodopsins containing 9- or 11-cis-retinal had blue-shifted absorption maxima and altered photo-bleaching properties compared with the corresponding wild-type proteins. Significantly, P23H rhodopsins were more thermally unstable than the wild-type proteins and more rapidly bleached by hydroxylamine in the dark. We suggest that P23H opsin is similarly unstable and that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa and other protein conformational disorders are discussed.

Received for publication, November 4, 2003 , and in revised form, January 14, 2004.

^* This work was supported in part by a Career Development Award from the Foundation Fighting Blindness (to S. K.), funds from Research to Prevent Blindness (to S. K.), and the Department of Ophthalmology, College of Medicine, University of Florida (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Current address: Dept. of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL 32610.

^** To whom correspondence may be addressed: Dept. of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL 32610. Tel.: 352-846-2102; Fax: 352-392-7839; E-mail: skaushal{at}ufl.edu.

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