Enhanced Shutoff of Phototransduction in Transgenic Mice Expressing Palmitoylation-deficient Rhodopsin*♦

Wang, Zhongyan; Wen, Xiaohong; Crouch, Rosalie K.; Makino, Clint L.; Lem, Janis

doi:10.1074/jbc.m502588200

Cited by 39 publications

(46 citation statements)

References 49 publications

(37 reference statements)

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“…Therefore, the shutoff of rhodopsin activation by phosphorylation would be less in the mutant. We tried to experimentally assess the phosphorylation efficiency of the E122Q mutant rhodopsin in knock-in mouse ROS, because phosphorylation efficiency affects the rod photosensitivity (37). Preliminary results by using the methods described in Ref.…”

Section: Resultsmentioning

confidence: 99%

Molecular Properties of Rhodopsin and Rod Function

Imai

Kefalov

Sakurai

et al. 2007

Journal of Biological Chemistry

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Signal transduction in rod cells begins with photon absorption by rhodopsin and leads to the generation of an electrical response. The response profile is determined by the molecular properties of the phototransduction components. To examine how the molecular properties of rhodopsin correlate with the rod-response profile, we have generated a knock-in mouse with rhodopsin replaced by its E122Q mutant, which exhibits properties different from those of wild-type (WT) rhodopsin. Knock-in mouse rods with E122Q rhodopsin exhibited a photosensitivity about 70% of WT. Correspondingly, their single-photon response had an amplitude about 80% of WT, and a rate of decline from peak about 1.3 times of WT. The overall 30% lower photosensitivity of mutant rods can be explained by a lower pigment photosensitivity (0.9) and the smaller single-photon response (0.8). The slower decline of the response, however, did not correlate with the 10-fold shorter lifetime of the meta-II state of E122Q rhodopsin. This shorter lifetime became evident in the recovery phase of rod cells only when arrestin was absent. Simulation analysis of the photoresponse profile indicated that the slower decline and the smaller amplitude of the single-photon response can both be explained by the shift in the meta-I/ meta-II equilibrium of E122Q rhodopsin toward meta-I. The difference in meta-III lifetime between WT and E122Q mutant became obvious in the recovery phase of the dark current after moderate photobleaching of rod cells. Thus, the present study clearly reveals how the molecular properties of rhodopsin affect the amplitude, shape, and kinetics of the rod response.Light absorption by rhodopsin in rod photoreceptor cells results in the activation of a G protein-mediated signal transduction cascade that eventually generates an electrical response (1). The key proteins in this cascade have been identified, and their molecular properties as well as interactions with each other have been extensively investigated (2, 3). A current question is how well these properties and interactions correlate with the response profile of the photoreceptor cells. Although the gene knock-out approach has been very useful in addressing this question for the proteins rhodopsin kinase and arrestin (4, 5), this strategy is less appropriate for rhodopsin and G protein, because the deletions of these proteins eliminated the light response (6, 7). The gene knock-in approach is an alternative way, with a mutant protein replacing the wild-type (WT) 8 version. The maintenance of the same expression level of the protein in this procedure is important, because the interpretation can be difficult otherwise. For example, the photoresponse profile is altered when the rhodopsin content in rods is halved (8, 9).Our past work on comparing rhodopsin and cone pigments (10) has shown that their photosensitivities are not so different, but the meta-II (the G protein-activating state), as well as the subsequent meta-III, intermediates of cone pigments exhibit faster decay than those of rhodopsi...

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Section: Resultsmentioning

confidence: 99%

Molecular Properties of Rhodopsin and Rod Function

Imai

Kefalov

Sakurai

et al. 2007

Journal of Biological Chemistry

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“…Depalmitoylation of rhodopsin by chemical treatment with hydroxylamine was extensively used to elucidate the role of palmitoylation (6,10,(25)(26)(27), but the results should be interpreted with caution, especially because high concentrations of this strong nucleophile can produce off-target effects on membrane structures. Expressed in COS cells, bovine opsin mutants containing a serine substitution at Cys-323 showed reduced lightdependent phosphorylation by rhodopsin kinase (9), but subsequent studies employing Cys-322-Thr and Cys-323-Ser knockin mice revealed that, after exposure to light, rhodopsin became phosphorylated at a faster rate in mutant than in WT retinas (11). Conformational changes in the C terminus were observed upon depalmitoylation in biochemical assays (25).…”

Section: Discussionmentioning

confidence: 99%

“…Initial characterization of these Palm −/− mice showed that the mutant rhodopsin became hyperphosphorylated upon light activation, thereby enhancing "shut-off" of rhodopsin (11). Palm −/− mice manifested differences in the rate of transducin activation by rhodopsin and single-molecule force microscopy revealed changes in the force needed to unfold the last stable structural segment of rhodopsin at its carboxyl terminal end (12).…”

Section: Gnat1mentioning

confidence: 99%

“…To circumvent secondary effects on receptor structure and function, a knock-in mouse model expressing palmitoylationdeficient rhodopsin was generated wherein Cys residues at positions 322 and 323 were mutated to Thr and Ser, respectively (11). Initial characterization of these Palm −/− mice showed that the mutant rhodopsin became hyperphosphorylated upon light activation, thereby enhancing "shut-off" of rhodopsin (11).…”

Section: Gnat1mentioning

confidence: 99%

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Palmitoylation stabilizes unliganded rod opsin

Maeda

Okano²,

Park

et al. 2010

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

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S-palmitoylation is a conserved feature in many G protein–coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient ( Palm −/− ) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm −/− mice were crossed with Lrat −/− mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm −/− Lrat −/− mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm −/− Gnat1 −/− mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.

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“…Similar strategies to evaluate the in vivo importance of specific cysteine oxidation targets are largely lacking, and only a few knock-in mice with mutated cysteine knock-ins have been reported. Rhodopsin is a target for palmitoylation at cysteine residues 322 and 323, and knock-in mice in which these sites were mutated to threonine and serine residues were reported to have enhanced shut-off phototransduction [238]. A mouse containing a single amino acid substitution (Cys249Trp) in the human Crumbs homolog-1 gene displayed in retinal degeneration [239].…”

Section: Assessment Of the Functional Importance Of Cysteine Oxidatiomentioning

confidence: 99%