Constitutively active mutants of rhodopsin

Robinson, Phyllis R.; Cohen, George B.; Zhukovsky, Eugene A.; Oprian, Daniel D.

doi:10.1016/0896-6273(92)90034-b

Cited by 504 publications

(369 citation statements)

References 31 publications

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“…Rhodopsin is primarily absent in RPE65 Ϫ/Ϫ mice because of a defect in the visual cycle to regenerate 11-cis-retinal, but the presence of the opsin apoprotein preserves rod outer segment structure (Redmond et al, 1998). Opsin in RPE65 Ϫ/Ϫ mice is constitutively phosphorylated (Ablonczy et al, 2002;Van Hooser et al, 2002), probably as a consequence of the weak constitutive activity of opsin described in vitro (Robinson et al, 1992;Surya et al, 1995;Buczylko et al, 1996). In RPE65 Ϫ/Ϫ mice, opsin appeared to signal constitutively for arrestin translocation to rod outer segments, regardless of the lighting conditions.…”

Section: Discussionmentioning

confidence: 99%

“…This result indicates that opsin alone, without the retinal chromophore, has some signaling capacity to trigger arrestin translocation. Opsin has been reported to have a small but measurable catalytic activity in vitro (Robinson et al, 1992;Surya et al, 1995;Buczylko et al, 1996). This weak constitutive activity, however, is apparently not sufficient to trigger Tr ␣ translocation to proximal compartments in RPE65 Ϫ/Ϫ mice, because Tr ␣ immunoreactivity is restricted to the outer segments in both darkand light-adapted retinas (Fig.…”

Section: Light-regulation Of Arrestin Movement Is Lost In Rpe65 ؊/؊ Micementioning

confidence: 99%

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Light-Dependent Translocation of Arrestin in the Absence of Rhodopsin Phosphorylation and Transducin Signaling

2003

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Visual arrestin plays a crucial role in the termination of the light response in vertebrate photoreceptors by binding selectively to lightactivated, phosphorylated rhodopsin. Arrestin localizes predominantly to the inner segments and perinuclear region of dark-adapted rod photoreceptors, whereas light induces redistribution of arrestin to the rod outer segments. The mechanism by which arrestin redistributes in response to light is not known, but it is thought to be associated with the ability of arrestin to bind photolyzed, phosphorylated rhodopsin in the outer segment. In this study, we show that light-driven translocation of arrestin is unaffected in two different mouse models in which rhodopsin phosphorylation is lacking. We further show that arrestin movement is initiated by rhodopsin but does not require transducin signaling. These results exclude passive diffusion and point toward active transport as the mechanism for lightdependent arrestin movement in rod photoreceptor cells.

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Section: Discussionmentioning

confidence: 99%

Section: Light-regulation Of Arrestin Movement Is Lost In Rpe65 ؊/؊ Micementioning

confidence: 99%

Light-Dependent Translocation of Arrestin in the Absence of Rhodopsin Phosphorylation and Transducin Signaling

2003

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“…Several highly conserved motifs in GPCRs are also present in the MOR42 subfamily, including the (D/E)R(Y/W) motif located near the cytoplasmic end of TMIII and the NPXXY motif in TMVII. These motifs are thought to form a hydrogen bond network important for inter-helical interactions (37) and may play a role in receptor transformation from an inactive form to an active, G protein-coupled conformation (38). Conserved prolines that produce kinks in the helix structure of MOR42-3 are Pro-162 (in TMIV, equivalent to Pro-170 in rhodopsin) and Pro-303 (in TMVII, position 291 in rhodopsin).…”

Section: Initial Mutation Screen Of Mor42-3-mentioning

confidence: 99%

The Molecular Basis for Ligand Specificity in a Mouse Olfactory Receptor

Abaffy

Malhotra

Luetje

2007

Journal of Biological Chemistry

103

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Sequence differences between members of the mouse olfactory receptor MOR42 subfamily (MOR42-3 and MOR42-1) are likely to be the basis for variation in ligand binding preference among these receptors. We investigated the specificity of MOR42-3 for a variety of dicarboxylic acids. We used site-directed mutagenesis, guided by homology modeling and ligand docking studies, to locate functionally important residues. Receptors were expressed in Xenopus oocytes and assayed using high throughput electrophysiology. The importance of the Val-113 residue, located deep within the receptor, was analyzed in the context of interhelical interactions. We also screened additional residues predicted to be involved in ligand binding site, based on comparison of ortholog/paralog pairs from the mouse and human olfactory receptor genomes (Man, O., Gilad, Y., and Lancet, D. (2004) Protein Sci. 13, 240 -254). A network of 8 residues in transmembrane domains III, V, and VI was identified.These residues form part of the ligand binding pocket of MOR42-3. C12 dicarboxylic acid did not activate the receptor in our functional assay, yet our docking simulations predicted its binding site in MOR42-3. Binding without activation implied that C12 dicarboxylic acid might act as an antagonist. In our functional assay, C12 dicarboxylic acid did indeed act as an antagonist of MOR42-3, in agreement with molecular docking studies. Our results demonstrate a powerful approach based on the synergy between computational predictions and physiological assays.

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“…The high pH opsin conformation may thus be similar to inactive Meta I. [92,93]. From proton uptake measurements on the E134Q mutant Glu134 was suggested as the possible proton acceptor group [94].…”

Section: ____________________________________________________________mentioning

confidence: 99%

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Park

Scheerer

Hofmann

et al. 2008

Nature

851

925

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Compared with the known structure of inactive rhodopsin, opsin displays prominent structural changes in the conserved E(D)RY and NPxxY(x) 5,6 F regions and TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, of which some are attributed to an active GPCR state, reorganize the empty retinal binding pocket to disclose two openings for exit and entry of retinal. The opsin structure thus sheds new light on binding of hydrophobic ligands to GPCRs, GPCR activation and signal transfer to the G protein.

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Constitutively active mutants of rhodopsin

Cited by 504 publications

References 31 publications

Light-Dependent Translocation of Arrestin in the Absence of Rhodopsin Phosphorylation and Transducin Signaling

Light-Dependent Translocation of Arrestin in the Absence of Rhodopsin Phosphorylation and Transducin Signaling

The Molecular Basis for Ligand Specificity in a Mouse Olfactory Receptor

Crystal structure of the ligand-free G-protein-coupled receptor opsin

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