Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor

Palczewski, Krzysztof; Kumasaka, Takashi; Hori, Tetsuya; Behnke, Craig A.; Motoshima, Hidemasa; Fox, Brian; Trong, I. Le; Teller, David C.; Okada, Tetsuji; Stenkamp, Ronald E.; Yamamoto, Masaki; Miyano, Masashi

doi:10.1126/science.289.5480.739

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“…[13] Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring,~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl.…”

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confidence: 98%

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

et al. 2010

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The immunomodulatory drug fingolimod (FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal metabolite myriocin), is phosphorylated in vivo by sphingosine kinases to produce (R)-FTY720-phosphate (FTY720-P). [1,2] FTY720-P activates sphingosine-1-phosphate (S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar concentrations and is inactive toward the S1P2 receptor.[1] The FTY720-P-mediated activation of the S1P1 receptor on lymphocytes induces receptor internalization, which attenuates T-cell response to S1P gradients, preventing their egress from secondary lymphoid tissues. [3] In addition to playing a role in the immune system, all S1P receptors except S1P4 are also found differentially expressed in the central nervous system [4] and on various tumor cell types. [5,6] Although the precise regulation of these receptors by locally released S1P remains unclear, S1P receptors are thought to play a role in such events as astrocyte migration, [7] oligodendrocyte differentiation, and cell survival [8,9] and neurogenesis. [10,11] To assess the relevance of individual S1P receptor subtypes for the activity of FTY720-P, selective agonists are required. Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, [12] we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC 50 = 270 nm), but has modest selectivity against S1P1 (EC 50 = 3140 nm) and S1P4 (EC 50 = 100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists.To guide the optimization process, homology models of all S1P receptors were built from a crystal structure of bovine rhodopsin (PDB ID: 1F88). [13] Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring,~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl. In the S1P5 receptor complex, the amide group forms a hydrogen bond with OG1-Thr120. The benzamide phenyl ring lies in a large hydrophobic pocket surrounded by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and Leu271. The aniline ring undergoes a T-shaped interaction with Phe116 and hydrophobic contacts with Leu271 and Leu292. The ortho-methyl substituents fill a small pocket formed by Tyr89, Val115, and Leu292 on one side, and sit at the face of Phe196 on the other side. Inspection of sequence alignments (Figure 2) revealed two positions, o...

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confidence: 98%

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

et al. 2010

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“…(2015) and this study (Figure 4). The amino acid replacements that have inference probabilities lower than 0.9 are underlined; replacements that occurred in known tuning sites (Table 3) are shown in bold; replacements that occurred in retinal surrounding sites (Palczewski et al., 2000) are asterisked. The phylogeny of the four flounders was depicted on the basis of Nelson (2006)…”

Section: Resultsmentioning

confidence: 99%

“…To survey amino acid residues causing the shift of SWS2A from blue sensitive to green sensitive, amino acid substitution Ala or Thr at position 275 was investigated, because this site is known to act as a spectral tuning site (Yokoyama & Tada, 2003; Yokoyama et al., 2007) and a retinal surrounding site (Palczewski, Kumasaka, Hori, & Behnke, 2000). In addition, the 275Thr codon (ACC) was common between V. moseri and V. variegatus , suggesting the nucleotide substitution was a single event in the Verasper genus ancestor.…”

Section: Resultsmentioning

confidence: 99%

Green‐shifting of SWS2A opsin sensitivity and loss of function of RH2‐A opsin in flounders, genus Verasper

Kasagi

Mizusawa

Takahashi

2017

Ecology and Evolution

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We identified visual opsin genes for three flounder species, including the spotted halibut (Verasper variegatus), slime flounder (Microstomus achne), and Japanese flounder (Paralichthys olivaceus). Structure and function of opsins for the three species were characterized together with those of the barfin flounder (V. moseri) that we previously reported. All four flounder species possessed five basic opsin genes, including lws, sws1, sws2, rh1, and rh2. Specific features were observed in rh2 and sws2. The rh2‐a, one of the three subtypes of rh2, was absent in the genome of V. variegatus and pseudogenized in V. moseri. Moreover, rh2‐a mRNA was not detected in M. achne and P. olivaceus, despite the presence of a functional reading frame. Analyses of the maximum absorption spectra (λmax) estimated by in vitro reconstitution indicated that SWS2A of M. achne (451.9 nm) and P. olivaceus (465.6 nm) were blue‐sensitive, whereas in V. variegatus (485.4 nm), it was green‐sensitive and comparable to V. moseri (482.3 nm). Our results indicate that although the four flounder species possess a similar opsin gene repertoire, the SWS2A opsin of the genus Verasper is functionally green‐sensitive, while its overall structure remains conserved as a blue‐sensitive opsin. Further, the rh2‐a function seems to have been reduced during the evolution of flounders. λmax values of predicted ancestral SWS2A of Pleuronectiformes and Pleuronectidae was 465.4 and 462.4 nm, respectively, indicating that these were blue‐sensitive. Thus, the green‐sensitive SWS2A is estimated to be arisen in ancestral Verasper genus. It is suggested that the sensitivity shift of SWS2A from blue to green may have compensated functional reduction in RH2‐A.

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“…4). The cytoplasmic end of TMD2 in rhodopsin is the most buried by the other TMDs (26,27), suggesting perhaps that a similar configuration might constrain the accessibility of this region of Ste2.…”

Section: Ste2 Topologymentioning

confidence: 99%

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Choi

Konopka

2006

Biochemistry

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The yeast α-factor pheromone receptor (Ste2) belongs to the family of G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. To define the residues that are accessible to the cytoplasmic G protein, Cys scanning mutagenesis was carried out in which each of the residues that span the intracellular loops and the cytoplasmic end of transmembrane domain 7 were substituted with Cys. The 90 different Cys-substituted residues were then assayed for reactivity with MTSEAbiotin (2-([biotinoyl] amino) ethyl methanethiosulfonate), which reacts with solvent accessible sulfhydryl groups. As part of these studies we show that adding free Cys to stop the MTSEA-biotin reactions has potential pitfalls in that Cys can rapidly undergo disulfide exchange with the biotinylated receptor proteins at pH ≥7. The central regions of the intracellular loops of Ste2 were all highly accessible to MTSEA-biotin. Residues near the ends of the loops typically exhibited a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. Interestingly, these boundary residues were enriched in hydrophobic residues, suggesting that they may form a hydrophobic pocket for interaction with the G protein. Comparison with accessibility data from a previous study of the extracellular side of Ste2 indicates that the transmembrane domains vary in length, consistent with some transmembrane domains being tilted relative to the plane of the membrane as they are in rhodopsin. Altogether, these results define the residues that are accessible to the G protein and provide an important structural framework for the interpretation of the role of Ste2 residues that function in G protein activation.The S. cerevisiae α-factor pheromone receptor (Ste2) belongs to the large family of Gproteincoupled receptors (GPCRs) that transduce the signals for light, taste, olfaction, and many biomedically important hormones. Although GPCRs are quite diverse in sequence, they function in a similar manner to activate the α subunit of heterotrimeric G proteins to bind GTP and they also show similar structural architecture in that they are composed of a bundle of seven transmembrane domains (TMDs) connected by extracellular loops and intracellular loops (1,2). Ste2 shows overall similarity to many mammalian GPCRs in that the core region containing the TMDs is involved in signal transduction and the C terminal tail is a target for † This work was supported by National Institutes of Health Grant GM55107 awarded to J.B.K. post-translational modifications that regulate receptor desensitization and endocytosis (3). Ste2 activates a heterotrimeric G protein in which the α subunit shows about 45% identity with mammalian Gα proteins and is most closely related to the Gi subfamily (4). In addition, comparison of Ste2 with rhodopsin, a member of the large class A subfamily of mammalian GPCRs, indicates that there are similar microdomains in these divergent receptors (5). These results suggest that there are underlying ...

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Crystal Structure of Rhodopsin: A G Protein-Coupled Receptor

Cited by 5,257 publications

References 54 publications

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

Green‐shifting of SWS2A opsin sensitivity and loss of function of RH2‐A opsin in flounders, genus Verasper

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

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