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

Zhu, Lida; Jang, Geeng Fu; Jastrzębska, Beata; Filipek, Sławomir; Pearce-Kelling, Susan E.; Aguirre, Gustavo D.; Stenkamp, Ronald E.; Acland, Gregory M.; Palczewski, Krzysztof

doi:10.1074/jbc.m408472200

Cited by 54 publications

(59 citation statements)

References 71 publications

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“…Although the introduction of a positive charge may destabilize this region in the case of T4K, as is proposed for the T4R dog (Zhu et al, 2004), our results obtained from the T4N mutant suggest that the underlying reason is loss of the ␤-sheet configuration.…”

Section: Discussionmentioning

confidence: 54%

“…Although a mouse model expressing human T17M rhodopsin exists (Li et al, 1998), localization of the mutant rhodopsin was not determined. In a naturally occurring dog model of RP, mutant T4R rhodopsin localizes to the ROS (Zhu et al, 2004). Our results indicate that nonglycosylated rhodopsin (N2S/N15S rhodopsin) was capable of targeting to and inserting into ROS disk membranes.…”

Section: Discussionmentioning

confidence: 72%

“…Using differential scanning calorimetry, it was determined that, although rat and frog rhodopsin are very similar in stability, bovine rhodopsin is significantly more thermally stable (⌬H ϭ 410, 416, and 630 kJ/mol, respectively; Td ϭ 340, 340, and 347°K, respectively) (Shnyrov and Berman, 1988). Bovine rhodopsin is more stable toward hydroxylamine bleaching than canine rhodopsin (Zhu et al, 2004), indicating that the chromophore pocket of canine rhodopsin is more accessible and perhaps adopts a less compact structure. Finally, there are multiple examples of bovine rhodopsin mutants that fold and bind chromophore in cultured cells, producing photo-labile pigments, whereas the corresponding human mutants do not (Nathans, 1990a,b;Sung et al, 1991;Min et al, 1993;Sung et al, 1993;.…”

Section: Discussionmentioning

confidence: 99%

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The Role of Rhodopsin Glycosylation in Protein Folding, Trafficking, and Light-Sensitive Retinal Degeneration

Tam¹,

Moritz²

2009

J. Neurosci.

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Several mutations in the N terminus of the G-protein-coupled receptor rhodopsin disrupt NXS/T consensus sequences for N-linked glycosylation (located at N2 and N15) and cause sector retinitis pigmentosa in which the inferior retina preferentially degenerates. Here we examined the role of rhodopsin glycosylation in biosynthesis, trafficking, and retinal degeneration (RD) using transgenic Xenopus laevis expressing glycosylation-defective human rhodopsin mutants. Although mutations T4K and T4N caused RD, N2S and T4V did not, demonstrating that glycosylation at N2 was not required for photoreceptor viability. In contrast, similar mutations eliminating glycosylation at N15 (N15S and T17M) caused rod death. Expression of T17M was more toxic than T4K to transgenic photoreceptors, further suggesting that glycosylation at N15 plays a more important physiological role than glycosylation at N2. Together, these results indicate that the structure of the rhodopsin N terminus must be maintained by an appropriate amino acid sequence surrounding N2 and may require a carbohydrate moiety at N15. The mutant rhodopsins were rendered less toxic in their dark inactive states, because RD was abolished or significantly reduced when transgenic tadpoles expressing T4K, T17M, and N2S/N15S were protected from light exposure. Regardless of their effect on rod viability, all of the mutants primarily localized to the outer segment and Golgi and showed little or no endoplasmic reticulum accumulation. Thus, glycosylation was not crucial for rhodopsin biosynthesis or trafficking. Interestingly, expression of similar bovine rhodopsin mutants did not cause rod cell death, possibly attributable to greater stability of bovine rhodopsin.

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Section: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 72%

Section: Discussionmentioning

confidence: 99%

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The Role of Rhodopsin Glycosylation in Protein Folding, Trafficking, and Light-Sensitive Retinal Degeneration

Tam¹,

Moritz²

2009

J. Neurosci.

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“…Several studies have indicated that loss of GPCR glycosylation can lead to improper folding and targeting, resulting in decreased function and compromised structure and stability (11). Loss of either N-terminal glycosylation site (Asn-2 or Asn-15) of rhodopsin is sufficient to cause loss of signal transduction despite no apparent change in localization or folding (21,22). Additionally, mutating out the glycosylation site of a mouse OR (mOR-EG) was found to abolish its ability to localize to the membrane (10), indicating that this modification may be necessary for OR function.…”

Section: Discussionmentioning

confidence: 99%

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

Cook

Steuerwald

Kaiser

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

131

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Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be Ϸ50% ␣-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices. BiacoreA100 ͉ detergent screen ͉ fos-choline 14 ͉ G protein-coupled receptor purification ͉ membrane protein A nimal noses have evolved the ability to rapidly detect a seemingly infinite array of odors at minute concentrations. The basis of this sensitivity are the olfactory (smell) receptors, a large class of G protein-coupled receptors (GPCRs) that function together combinatorially to allow discrimination between a wide range of volatile and soluble molecules (1, 2). As GPCRs, all olfactory receptors (ORs) are integral membrane proteins with 7 predicted transmembrane domains. To date, crystal structures exist for only 5 GPCR proteins (3). Despite the fact that ORs represent the largest class of known membrane proteins, no detailed structure exists for any OR, because the major obstacle to structural and functional studies on membrane proteins is the notorious difficulty involved in expressing and purifying the large quantities of receptor protein sample required for techniques such as X-ray crystallography. The first crucial step to enable such pivotal biochemical and structural analyses is to engineer systems with the capacity to produce and purify milligram quantities of an OR. hOR17-4 (alternately known as OR1D2) is of particular interest because, in addition to olfactory neurons, it is exp...

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“…We genetically eliminated the retinoid cycle, the process that regenerates the light-sensitive chromophore 11-cis-retinal (51,52). In contrast to adRP-causing opsin gene mutations P23H (29) and T4R (18,53), the E150K mutant opsin appears more stable under such conditions, inducing a milder retinal disease phenotype than these counterparts. This could be attributed to the profound oligomerization of opsin versus rhodopsin that could preferentially benefit the E150K mutant ROS.…”

Section: Figurementioning

confidence: 99%

Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization

et al. 2012

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The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin's stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higherorder organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration.

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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

Cited by 54 publications

References 71 publications

The Role of Rhodopsin Glycosylation in Protein Folding, Trafficking, and Light-Sensitive Retinal Degeneration

The Role of Rhodopsin Glycosylation in Protein Folding, Trafficking, and Light-Sensitive Retinal Degeneration

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization

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