Highly conserved glutamic acid in the extracellular IV–V loop in rhodopsins acts as the counterion in retinochrome, a member of the rhodopsin family

Terakita, Akihisa; Yamashita, Takahiro; Shichida, Yoshinori

doi:10.1073/pnas.260349597

Cited by 107 publications

(122 citation statements)

References 29 publications

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“…Presence of Wat2a in these chloride-binding pigments is suggested by our Fourier transform IR study on the chicken red pigment (25). Moreover, it was recently shown that the E181Q mutation of bovine rhodopsin caused a 10-nm shift of its absorption maximum compared with the wild type and that Glu-181 can act as a direct counterion of the protonated Schiff base in the squid retinal photoisomerase retinochrome (26). These items of evidence support the idea that rearrangement of the residues and water molecules in site 2 of our current model most likely plays a crucial role in the spectral tuning of the diverse family of eukaryotic retinal proteins.…”

Section: Resultssupporting

confidence: 53%

Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography

Okada

Fujiyoshi

Silow

et al. 2002

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

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680

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Activation of G protein-coupled receptors (GPCRs) is triggered and regulated by structural rearrangement of the transmembrane heptahelical bundle containing a number of highly conserved residues. In rhodopsin, a prototypical GPCR, the helical bundle accommodates an intrinsic inverse-agonist 11-cis-retinal, which undergoes photo-isomerization to the all-trans form upon light absorption. Such a trigger by the chromophore corresponds to binding of a diffusible ligand to other GPCRs. Here we have explored the functional role of water molecules in the transmembrane region of bovine rhodopsin by using x-ray diffraction to 2.6 Å. The structural model suggests that water molecules, which were observed in the vicinity of highly conserved residues and in the retinal pocket, regulate the activity of rhodopsin-like GPCRs and spectral tuning in visual pigments, respectively. To confirm the physiological relevance of the structural findings, we conducted single-crystal microspectrophotometry on rhodopsin packed in our three-dimensional crystals and show that its spectroscopic properties are similar to those previously found by using bovine rhodopsin in suspension or membrane environment. C ell surface membrane receptors mediate a variety of biological signaling processes that are triggered by a multitude of diffusible molecules or light in the case of visual pigments. G protein-coupled receptors (GPCRs), including the rhodopsinlike family as a dominant subgroup, are the largest group of membrane receptors. Many of the members are considered as primary drug targets for various medical and pharmacological interventions. These receptors appear to be activated by a mechanism involving common heptahelical transmembrane architecture that undergoes major rearrangement upon signal reception, resulting in activation of the heterotrimeric G protein molecules.Rhodopsin from retinal rod cells mediates scotopic vision. It is a unique member among GPCRs in that it contains an intrinsic inverse-agonist, the 11-cis-retinal. Photon absorption results in retinal isomerization to the all-trans configuration, which drives the protein to the active metarhodopsin form (M II) (1). Visual pigments mediating color vision in cone cells share a common mechanism to evoke a cellular signaling cascade (2) through interaction between the M II state and the G protein. A view of the seven helices of bovine rhodopsin was provided by electron crystallography in 1993 (3), and an x-ray crystallographic study recently determined its structure at 2.8-Å resolution (4). This structure provided the template model at high resolution for the rhodopsin-like GPCRs and has been further refined at the same resolution (5).The 11-cis-retinal chromophore is covalently bound to Lys-296 in transmembrane helix VII by a protonated Schiff base linkage, which is stabilized by the negatively charged counterion Glu-113 in helix III. Disruption of this salt bridge (6) upon proton transfer is thought to trigger conformational changes in rhodopsin (7), which are necessary for G protein ...

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Section: Resultssupporting

confidence: 53%

Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography

Okada

Fujiyoshi

Silow

et al. 2002

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

Self Cite

680

899

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“…It has been suggested that invertebrate rhodopsins have the counterion glutamic acid at position 181 (Glu-181) and vertebrate rhodopsins acquired Glu-113 counterion during the molecular evolution (28,40). Parapinopsin has both Glu-113 and Glu-181, but the Glu-181 serves as the counterion.…”

Section: Discussionmentioning

confidence: 99%

The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Tsukamoto

Farrens

Koyanagi

et al. 2009

Journal of Biological Chemistry

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Light converts rhodopsin, the prototypical G protein-coupled receptor, into a form capable of activating G proteins. Recent work has shown that the light-activated state of different rhodopsins can possess different molecular properties, especially different abilities to activate G protein. For example, bovine rhodopsin is ϳ20-fold more effective at activating G protein than parapinopsin, a non-visual rhodopsin, although these rhodopsins share relatively high sequence similarity. Here we have investigated possible structural aspects that might underlie this difference. Using a site-directed fluorescence labeling approach, we attached the fluorescent probe bimane to cysteine residues introduced in the cytoplasmic ends of transmembrane helices V and VI in both rhodopsins. The fluorescence spectra of these probes as well as their accessibility to aqueous quenching agents changed dramatically upon photoactivation in bovine rhodopsin but only moderately so in parapinopsin. We also compared the relative movement of helices V and VI upon photoactivation of both rhodopsins by introducing a bimane label and the bimane-quenching residue tryptophan into helices VI and V, respectively. Both receptors showed movement in this region upon activation, although the movement appears much greater in bovine rhodopsin than in parapinopsin. Together, these data suggest that a larger conformational change in helices V and VI of bovine rhodopsin explains why it has greater G protein activation ability than other rhodopsins. The different amplitude of the helix movement may also be responsible for functional diversity of G protein-coupled receptors.Rhodopsin, the photosensitive G protein-coupled receptor (GPCR), 3 is responsible for transmitting a light signal into an intracellular signaling cascade through activation of G protein in visual and non-visual photoreceptor cells. Rhodopsin consists of a protein moiety (opsin, comprising seven transmembrane ␣-helical segments) combined with a chromophore (11-cis retinal) that acts as the light-sensitive ligand. Photoisomerization of the 11-cis retinal to the all-trans form induces structural changes in the protein moiety that then enable it to couple with and activate the G protein.The crystal structure of inactive bovine rhodopsin has been extensively investigated (1-3). Recently, a crystal structure of inactive invertebrate squid rhodopsin was also solved (4), and crystal structures of the inactive form of ␤-adrenergic receptors and A2 adenosine receptor have been reported (5-7). Remarkably, all of these crystal structures exhibit a very similar arrangement for the seven transmembrane helices (4, 8). Together, these facts suggest that the architecture for the inactive form is conserved among rhodopsin-like GPCRs.The structural features of an activated GPCR are much less defined. Thus, a variety of biochemical and biophysical methods, including cross-linking methods (9, 10) and site-directed spin and fluorescence labeling methods (10 -13), have been employed to identify the dynamic and stru...

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“…One hundred micrograms of the plasmid DNA were used for the transient expression in 10 dishes (100 mm in diameter) of HEK293s cells. The vector was cotransfected with the pRSV-Tag into HEK293s cells by the calcium-phosphate method according to the previous report (22,23). The transfected cells were harvested for 2 days and collected by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

Bistable UV pigment in the lamprey pineal

Koyanagi

Kawano

Kinugawa

et al. 2004

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

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Lower vertebrates can detect UV light with the pineal complex independently of eyes. Electrophysiological studies, together with chromophore extraction analysis, have suggested that the underlying pigment in the lamprey pineal exhibits a bistable nature, that is, reversible photoreaction by UV and visible light, which is never achieved by known UV pigments. Here we addressed the molecular identification of the pineal UV receptor. Our results showed that the long-hypothesized pigment is a lamprey homologue of parapinopsin, which exhibits an absorption maximum at 370 nm, in the UV region. UV light causes cis-trans isomerization of its retinal 2 chromophore, forming a stable photoproduct having an absorption maximum at 515 nm, in the green region. The photoproduct reverts to the original pigment upon visible light absorption, showing photoregeneration of the pigment. In situ hybridization showed that parapinopsin is selectively expressed in the cells located in the dorsal region of the pineal organ. We successfully obtained the hyperpolarizing responses with a maximum sensitivity of Ϸ380 nm from the photoreceptor cells at the dorsal region, in which the outer segment was clearly stained with anti-parapinopsin antibody. These results demonstrated that parapinopsin is the pineal UV pigment having photointerconvertible two stable states. The bistable nature of the parapinopsin can account for the photorecovery of the pineal UV sensitivity by background green light in the lamprey. Furthermore, we isolated the parapinopsin homologues from fish and frog pineal complexes that exhibit UV sensitivity, suggesting that parapinopsin is a common molecular basis for pineal UV reception in the vertebrate.

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Highly conserved glutamic acid in the extracellular IV–V loop in rhodopsins acts as the counterion in retinochrome, a member of the rhodopsin family

Cited by 107 publications

References 29 publications

Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography

Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography

The Magnitude of the Light-induced Conformational Change in Different Rhodopsins Correlates with Their Ability to Activate G Proteins

Bistable UV pigment in the lamprey pineal

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