A naturally occurring mutation of the opsin gene (T4R) in dogs affects glycosylation and stability of the G protein-coupled receptor.
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 Asn(2), whereas glycosylation at Asn(15) 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.
Department of Ophthalmology, University of Washington, 1957 NE Pacific St., Seattle, WA 98195-6485, USA.
Li
L
Zhu
Geeng-Fu
GF
Jang
Beata
B
Jastrzebska
Slawomir
S
Filipek
Susan E
SE
Pearce-Kelling
Gustavo D
GD
Aguirre
Ronald E
RE
Stenkamp
Gregory M
GM
Acland
Krzysztof
K
Palczewski
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004195:Disease Models, Animal; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015151:Immunoblotting; D007150:Immunohistochemistry; D008024:Ligands; D008027:Light; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000378:metabolism; D012160:Retina / Q000473:pathology; D012174:Retinitis Pigmentosa / Q000235:genetics; D012176:Retinoids / Q000378:metabolism; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry; D012374:Rod Cell Outer Segment; D017299:Rod Opsins / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014466:Ultraviolet Rays; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D003902:Detergents; D008024:Ligands; D010455:Peptides; D011506:Proteins; D043562:Receptors, G-Protein-Coupled; D012176:Retinoids; D017299:Rod Opsins; C013110:metarhodopsins; D012243:Rhodopsin; D020741:rho GTP-Binding Proteins