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J. Biol. Chem., Vol. 279, Issue 51, 53828-53839, December 17, 2004

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A Naturally Occurring Mutation of the Opsin Gene (T4R) in Dogs Affects Glycosylation and Stability of the G Protein-coupled Receptor^*

From the Departments of ^aOphthalmology, ⁱPharmacology, ^bChemistry, ^gBiochemistry, and ^cBiological Structure and the ^hBiomolecular Structure Center, University of Washington, Seattle, Washington 98195-6485, the ^dInternational Institute of Molecular and Cell Biology, Warsaw PL-02109, Poland, the ^eJames A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401, and the ^fDepartment of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO^T4R/T4R dog retina, we found that the mutation abolished glycosylation at Asn², whereas glycosylation at Asn¹⁵ was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho^* lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G_t). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.

Received for publication, July 27, 2004 , and in revised form, September 29, 2004.

^* This work was supported in part by United States Public Health Service Grants EY01730, EY08061, EY13385, EY06855, EY13729, EY13132, GM63020, and EY13729 from the National Institutes of Health; an unrestricted grant from the Research to Prevent Blindness, Inc., New York (to the Department of Ophthalmology, University of Washington); a grant from the E. K. Bishop Foundation; the Van Sloun Fund for Canine Genetic Research; and Polish State Committee for Scientific Research Grant 3P05F02625. 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.

^j To whom correspondence should be addressed: Dept. of Ophthalmology, University of Washington, 1957 NE Pacific St., P. O. Box 356485, Seattle, WA 98195-6485. Tel.: 206-543-9074; Fax: 206-221-6784; E-mail: palczews{at}u.washington.edu.

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