研究者詳細 - 横川　真梨子

論文 - 横川　真梨子

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NMR 1H, 13C, 15N backbone resonance assignments of 14-3-3ζ binding region of human FOXO3a (residues 1-284)

Enomoto S., Nakatsuka S., Kuwayama T., Kawatsu K., Yokogawa M., Osawa M.

Biomolecular NMR Assignments 18 （ 2 ） 275 - 283 2024年12月

研究論文（学術雑誌）, 査読有り, ISSN 18742718

In tumors, mutation in Ras proteins stimulates a signaling cascade through phosphorylation. Downstream of the cascade, many transcription and translation factors are up- or down-regulated by phosphorylation, leading to cancer progression. This phosphorylation cascade is sustained by 14-3-3ζ protein. 14-3-3ζ binds to its client proteins that are Ser/Thr-phosphorylated and prevents their dephosphorylation. One of those transcription factors is FOXO3a, whose transcriptional activity is suppressed in the phosphorylation cascade. FOXO3a binds to specific DNA sequences and activates the transcription of apoptosis-related proteins. In cancer cells, however, FOXO3a is phosphorylated, bound to 14-3-3ζ, and dissociated from the DNA, resulting in FOXO3a inactivation. To elucidate the mechanism of FOXO3a inactivation by the 14-3-3ζ binding, we aim to perform NMR analysis of the interaction between 14-3-3ζ and di-phosphorylated FOXO3a residues 1-284 (dpFOXO3a). Here, we report the backbone resonance assignments of dpFOXO3a, which are transferred from those of the N-terminal domain (NTD) and the DNA-binding domain (DBD) of dpFOXO3a.

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Ribonuclease inhibitor and angiogenin system regulates cell type–specific global translation

Stillinovic M., Sarangdhar M.A., Andina N., Tardivel A., Greub F., Bombaci G., Ansermet C., Zatti M., Saha D., Xiong J., Sagae T., Yokogawa M., Osawa M., Heller M., Keogh A., Keller I., Angelillo-Scherrer A., Allam R.

Science Advances （Science Advances） 10 （ 22 ） eadl0320 2024年05月

研究論文（学術雑誌）, 査読有り

Translation of mRNAs is a fundamental process that occurs in all cell types of multicellular organisms. Conventionally, it has been considered a default step in gene expression, lacking specific regulation. However, recent studies have documented that certain mRNAs exhibit cell type–specific translation. Despite this, it remains unclear whether global translation is controlled in a cell type–specific manner. By using human cell lines and mouse models, we found that deletion of the ribosome-associated protein ribonuclease inhibitor 1 (RNH1) decreases global translation selectively in hematopoietic-origin cells but not in the non–hematopoietic-origin cells. RNH1-mediated cell type–specific translation is mechanistically linked to angiogenin-induced ribosomal biogenesis. Collectively, this study unravels the existence of cell type–specific global translation regulators and highlights the complex translation regulation in vertebrates.

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Applying deep learning to iterative screening of medium-sized molecules for protein-protein interaction-targeted drug discovery

Shimizu Y., Yonezawa T., Bao Y., Sakamoto J., Yokogawa M., Furuya T., Osawa M., Ikeda K.

Chemical Communications （Chemical Communications） 59 （ 44 ） 6722 - 6725 2023年05月

研究論文（学術雑誌）, 査読有り, ISSN 13597345

We combined a library of medium-sized molecules with iterative screening using multiple machine learning algorithms that were ligand-based, which resulted in a large increase of the hit rate against a protein-protein interaction target. This was demonstrated by inhibition assays using a PPI target, Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2), and a deep neural network model based on the first-round assay data showed a highest hit rate of 27.3%. Using the models, we identified novel active and non-flat compounds far from public datasets, expanding the chemical space.

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TDP-43 N-terminal domain dimerisation or spatial separation by RNA binding decreases its propensity to aggregate

Miura M., Sakaue F., Matsuno H., Morita K., Yoshida A., Hara R.I., Nishimura R., Nishida Y., Yokogawa M., Osawa M., Yokota T.

FEBS Letters （FEBS Letters） 597 （ 12 ） 1667 - 1676 2023年

研究論文（学術雑誌）, 査読有り, ISSN 00145793

Aggregation of the 43 kDa TAR DNA-binding protein (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). RNA binding and TDP-43 N-terminal domain dimerisation has been suggested to ameliorate TDP-43 aggregation. However, the relationship between these factors and the solubility of TDP-43 is largely unknown. Therefore, we developed new oligonucleotides that can recruit two TDP-43 molecules and interfere with their intermolecular interactions via spatial separation. Using these oligonucleotides and TDP-43-preferable UG-repeats, we uncovered two distinct mechanisms for modulating TDP-43 solubility by RNA binding: One is N-terminal domain dimerisation, and the other is the spatial separation of two TDP-43 molecules. This study provides new molecular insights into the regulation of TDP-43 solubility.

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Paip2A inhibits translation by competitively binding to the RNA recognition motifs of PABPC1 and promoting its dissociation from the poly(A) tail.

Sagae T, Yokogawa M, Sawazaki R, Ishii Y, Hosoda N, Hoshino SI, Imai S, Shimada I, Osawa M

The Journal of biological chemistry （Journal of Biological Chemistry） 298 （ 5 ） 101844 2022年03月

研究論文（学術雑誌）, 査読有り, ISSN 0021-9258

Eukaryotic mRNAs possess a poly(A) tail at their 30-end, to which poly(A)-binding protein C1 (PABPC1) binds and recruits other proteins that regulate translation. Enhanced poly(A)dependent translation, which is also PABPC1 dependent, promotes cellular and viral proliferation. PABP-interacting protein 2A (Paip2A) effectively represses poly(A)-dependent translation by causing the dissociation of PABPC1 from the poly(A) tail; however, the underlying mechanism remains unknown. This study was conducted to investigate the functional mechanisms of Paip2A action by characterizing the PABPC1–poly(A) and PABPC1–Paip2A interactions. Isothermal titration calorimetry and NMR analyses indicated that both interactions predominantly occurred at the RNA recognition motif (RRM) 2–RRM3 regions of PABPC1, which have comparable affinities for poly(A) and Paip2A (dissociation constant, Kd = 1 nM). However, the Kd values of isolated RRM2 were 200 and 4 μM in their interactions with poly(A) and Paip2A, respectively; Kd values of 5 and 1 μM were observed for the interactions of isolated RRM3 with poly(A) and Paip2A, respectively. NMR analyses also revealed that Paip2A can bind to the poly(A)binding interfaces of the RRM2 and RRM3 regions of PABPC1. Based on these results, we propose the following functional mechanism for Paip2A: Paip2A initially binds to the RRM2 region of poly(A)-bound PABPC1, and RRM2-anchored Paip2A effectively displaces the RRM3 region from poly(A), resulting in dissociation of the whole PABPC1 molecule. Together, our findings provide insight into the translation repression effect of Paip2A and may aid in the development of novel anticancer and/or antiviral drugs.

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Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

Matsumura K., Shimomura T., Kubo Y., Oka T., Kobayashi N., Imai S., Yanase N., Akimoto M., Fukuda M., Yokogawa M., Ikeda K., Kurita J.i., Nishimura Y., Shimada I., Osawa M.

BMC Molecular and Cell Biology （BMC Molecular and Cell Biology） 22 （ 3 ） 3 2021年01月

研究論文（学術雑誌）, 共著, 査読有り

Background: Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Results: We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis. Conclusions: The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.

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Structural mechanism underlying G protein family-specific regulation of G protein-gated inwardly rectifying potassium channel

Kano H., Toyama Y., Imai S., Iwahashi Y., Mase Y., Yokogawa M., Osawa M., Shimada I.

Nature Communications 10 （ 1 ） 2019年12月

研究論文（学術雑誌）, 共著, 査読有り

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein βγ subunit (Gβγ), which is released from the heterotrimeric G protein (Gαβγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oβγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oβγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.

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Nanodiscs for structural biology in a membranous environment

Yokogawa M., Fukuda M., Osawa M.

Chemical and Pharmaceutical Bulletin 67 （ 4 ） 321 - 326 2019年

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00092363

The structures of many membrane proteins have been analyzed in detergent micelles. However, the environment of detergent micelles differs somewhat from that of the lipid bilayer, where membrane proteins exhibit physiological functions. Therefore, a more membrane-like environment has been awaited for structural analysis of membrane proteins. Nanodiscs are “hockey-puck”-shaped lipid bilayer particles that distribute in a monodispersed manner in aqueous solution. We review how nanodiscs or protein-reconstituted nanodiscs are prepared and how they are utilized to analyze protein structure, dynamics, and interactions with lipid molecules using solution NMR and cryo-electron microscopy.

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Structural basis for the ethanol action on G-protein-activated inwardly rectifying potassium channel 1 revealed by NMR spectroscopy.

Toyama Y, Kano H, Mase Y,Yokogawa M, Osawa M, Shimada I

Proc Natl Acad Sci U S A. 115 （ 15 ） 3858 - 3863 2018年03月

研究論文（学術雑誌）, 共著, 査読有り, ISSN 00278424

Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein-activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.

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Characterization of the multimeric structure of poly(A)-binding protein on a poly(A) tail.

Ryoichi Sawazaki, Shunsuke Imai, Mariko Yokogawa,Nao Hosoda, Shin-ichi Hoshino, Muneyo Mio, Kazuhiro Mio, Ichio Shimada, and Masanori Osawa

Sci. Rep. 8 （ 1 ） 1455 2018年01月

研究論文（学術雑誌）, 共著, 査読有り

Eukaryotic mature mRNAs possess a poly adenylate tail (poly(A)), to which multiple molecules of poly(A)-binding protein C1 (PABPC1) bind. PABPC1 regulates translation and mRNA metabolism by binding to regulatory proteins. To understand functional mechanism of the regulatory proteins, it is necessary to reveal how multiple molecules of PABPC1 exist on poly(A). Here, we characterize the structure of the multiple molecules of PABPC1 on poly(A), by using transmission electron microscopy (TEM), chemical cross-linking, and NMR spectroscopy. The TEM images and chemical cross-linking results indicate that multiple PABPC1 molecules form a wormlike structure in the PABPC1-poly(A) complex, in which the PABPC1 molecules are linearly arrayed. NMR and cross-linking analyses indicate that PABPC1 forms a multimer by binding to the neighbouring PABPC1 molecules via interactions between the RNA recognition motif (RRM) 2 in one molecule and the middle portion of the linker region of another molecule. A PABPC1 mutant lacking the interaction site in the linker, which possesses an impaired ability to form the multimer, reduced the in vitro translation activity, suggesting the importance of PABPC1 multimer formation in the translation process. We therefore propose a model of the PABPC1 multimer that provides clues to comprehensively understand the regulation mechanism of mRNA translation.

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Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses

Yuki Toyama, Hanaho Kano, Yoko Mase, Mariko Yokogawa, Masanori Osawa, and Ichio Shimada

Nat. Commun. 8 14523 2017年01月

研究論文（学術雑誌）, 共著, 査読有り

Nuclear magnetic resonance analysis of the conformational state of cancer mutant of fibroblast growth factor receptor 1 tyrosine kinase domain

Kobashigawa, Y.; Amano, S.; Yoza, K.; Himeno, R.; Amemiya, S.; Morioka, H.; Yokogawa, M.; Kumeta, H.; Schlessinger, J.; Inagaki, F.

Genes Cells 21 （ 4 ） 350 - 7 2016年04月

研究論文（学術雑誌）, 共著, 査読有り

Tyrosine kinases are key enzymes that play critical roles in growth signaling, the abnormal activation of which is associated with various human cancers. Activation of tyrosine kinases is mediated by tyrosine phosphorylation in the activation-loop, which transforms the catalytic domain to the active state conformation. Cancer mutations are supposed to transform the conformation of the catalytic domain into the active-form independent of the phosphorylation state of the activation-loop. Here, we report structural and biophysical analyses of cancer mutations of the tyrosine kinase domain of fibroblast growth factor receptor 1 (FGFR1). Based on the nuclear magnetic resonance analyses, phosphorylation of the activation-loop exhibited cooperative structural transition in the activation-loop, C-helix and P-loop regions, whereas cancer mutations induced structural transformation at either one or two of these regions.

NMR Method for Characterizing Microsecond-to-Millisecond Chemical Exchanges Utilizing Differential Multiple-Quantum Relaxation in High Molecular Weight Proteins

Toyama, Y.; Osawa, M.; Yokogawa, M.; Shimada, I.

J Am Chem Soc 138 （ 7 ） 2302 - 11 2016年02月

研究論文（学術雑誌）, 共著, 査読有り

Chemical exchange processes of proteins on the order of microseconds (mus) to milliseconds (ms) play critical roles in biological functions. Developments in methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), which observes the slowly relaxing multiple quantum (MQ) coherences, have enabled the studies of biologically important large proteins. However, the analyses of mus to ms chemical exchange processes based on the methyl-TROSY principle are still challenging, because the interpretation of the chemical exchange contributions to the MQ relaxation profiles is complicated, as significant chemical shift differences occur in both (1)H and (13)C nuclei. Here, we report a new methyl-based NMR method for characterizing chemical exchanges, utilizing differential MQ relaxation rates and a heteronuclear double resonance pulse technique. The method enables quantitative evaluations of the chemical exchange processes, in which significant chemical shift differences exist in both the (1)H and (13)C nuclei. The versatility of the method is demonstrated with the application to KirBac1.1, with an apparent molecular mass of 200 kDa.

Structural basis for the regulation of enzymatic activity of Regnase-1 by domain-domain interactions

Yokogawa, M.; Tsushima, T.; Noda, N. N.; Kumeta, H.; Enokizono, Y.; Yamashita, K.; Standley, D. M.; Takeuchi, O.; Akira, S.; Inagaki, F.

Sci Rep 6 22324 2016年

研究論文（学術雑誌）, 共著, 査読有り

Regnase-1 is an RNase that directly cleaves mRNAs of inflammatory genes such as IL-6 and IL-12p40, and negatively regulates cellular inflammatory responses. Here, we report the structures of four domains of Regnase-1 from Mus musculus-the N-terminal domain (NTD), PilT N-terminus like (PIN) domain, zinc finger (ZF) domain and C-terminal domain (CTD). The PIN domain harbors the RNase catalytic center; however, it is insufficient for enzymatic activity. We found that the NTD associates with the PIN domain and significantly enhances its RNase activity. The PIN domain forms a head-to-tail oligomer and the dimer interface overlaps with the NTD binding site. Interestingly, mutations blocking PIN oligomerization had no RNase activity, indicating that both oligomerization and NTD binding are crucial for RNase activity in vitro. These results suggest that Regnase-1 RNase activity is tightly controlled by both intramolecular (NTD-PIN) and intermolecular (PIN-PIN) interactions.

Structural analysis of the mechanism of phosphorylation of a critical autoregulatory tyrosine residue in FGFR1 kinase domain

Kobashigawa, Y.; Amano, S.; Yokogawa, M.; Kumeta, H.; Morioka, H.; Inouye, M.; Schlessinger, J.; Inagaki, F.

Genes Cells 20 （ 10 ） 860 - 70 2015年10月

研究論文（学術雑誌）, 共著, 査読有り

Receptor and nonreceptor tyrosine kinases are enzymes that play important roles in regulating signal transduction pathways in a variety of normal cellular process and in many pathological conditions. Ordered phosphorylation is required for receptor tyrosine kinase (RTK) activation, a process mediated by transient dimer formation of the kinase domains. This process is triggered by the tyrosine phosphorylation in the activation loop. Here, we report structural and biochemical analyses of the tyrosine kinase domain interaction of fibroblast growth factor receptor 1 (FGFR1) required for the initial phosphorylation step. On the basis of nuclear magnetic resonance (NMR) analysis and covalent cross-linking experiments, we propose a parallel symmetric dimer model where specific contacts are formed between the N-lobes and C-lobes, respectively, in the FGFR1 kinase domains. Moreover, assignment of the contact sites between two FGFR1 kinase domains are supported by a trans-phosphorylation assay and by mutational analyses. The present report shows the molecular mechanism underlying the control of trans-phosphorylation of a critical auto-regulatory site in FGF receptors' catalytic domain.

Backbone resonance assignments for G protein alpha(i3) subunit in the GDP-bound state

Mase, Y.; Yokogawa, M.; Osawa, M.; Shimada, I.

Biomol NMR Assign 8 （ 2 ） 237 - 41 2014年10月

研究論文（学術雑誌）, 共著, 査読有り

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein alpha subunit with GDP (Galpha.GDP) and the G protein betagamma subunit (Gbetagamma). Ligand binding to GPCRs promotes the GDP-GTP exchange on Galpha, leading to the dissociation of the GTP-bound form of Galpha (Galpha.GTP) and Gbetagamma. Then, Galpha.GTP and Gbetagamma bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Galpha hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gbetagamma. The G proteins are classified with four major families based on the amino acid sequences of Galpha: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Galphai3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Galpha(i3) in complex with a GTP-analogue, GTPgammaS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPgammaS-bound states, as indicated in the crystal structures. The assignments of Galpha(i3).GDP would be useful for the analyses of the dynamics of Galpha(i3) and its interactions with various target molecules.

Large Protein Complexes Revealed by Solution-State NMR: G Proteins and G Protein-Activated Inwardly Rectifying Potassium Ion Channel.

Osawa M, Mase Y, Yokogawa M, Takeuchi K, and Shimada I.

Advances in Biological Solid-State NMR （Royal Society of Chemistry） 501 - 532 2014年01月

研究論文（学術雑誌）, 共著, 査読有り

NMR analyses of the interaction between the FYVE domain of early endosome antigen 1 (EEA1) and phosphoinositide embedded in a lipid bilayer

Yokogawa, M.; Kobashigawa, Y.; Yoshida, N.; Ogura, K.; Harada, K.; Inagaki, F.

J Biol Chem 287 （ 42 ） 34936 - 45 2012年10月

研究論文（学術雑誌）, 共著, 査読有り

Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment.

Backbone resonance assignments for G protein alpha(i3) subunit in the GTP-bound state

Mase, Y.; Yokogawa, M.; Osawa, M.; Shimada, I.

Biomol NMR Assign 6 （ 2 ） 217 - 20 2012年10月

研究論文（学術雑誌）, 共著, 査読有り

Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein alpha subunit (Galpha(GDP)) and G protein betagamma subunit (Gbetagamma). Ligand binding to GPCRs promotes the GDP-GTP exchange on Galpha, leading to the dissociation of the GTP-bound form of Galpha (Galpha(GTP)) and Gbetagamma. Then, Galpha(GTP) and Gbetagamma bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Galpha hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gbetagamma. G proteins are classified with four major families based on the amino acid sequences of Galpha: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Galpha(i3), a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPgammaS.

Structural basis for modulation of gating property of G protein-gated inwardly rectifying potassium ion channel (GIRK) by i/o-family G protein alpha subunit (Galphai/o)

Mase, Y.; Yokogawa, M.; Osawa, M.; Shimada, I.

J Biol Chem 287 （ 23 ） 19537 - 49 2012年06月

研究論文（学術雑誌）, 共著, 査読有り

G protein-gated inwardly rectifying potassium channel (GIRK) plays a crucial role in regulating heart rate and neuronal excitability. The gating of GIRK is regulated by the association and dissociation of G protein betagamma subunits (Gbetagamma), which are released from pertussis toxin-sensitive G protein alpha subunit (Galpha(i/o)) upon GPCR activation in vivo. Several lines of evidence indicate that Galpha(i/o) also interacts directly with GIRK, playing functional roles in the signaling efficiency and the modulation of the channel activity. However, the underlying mechanism for GIRK regulation by Galpha(i/o) remains to be elucidated. Here, we performed NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Galpha(i3) in the GTP-bound state. The NMR spectral changes of Galpha upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated their direct binding mode, where the K(d) value was estimated as approximately 1 mm. The TCS experiments identified the direct binding sites on Galpha and GIRK as the alpha2/alpha3 helices on the GTPase domain of Galpha and the alphaA helix of GIRK. In addition, the TCS and paramagnetic relaxation enhancement results suggested that the helical domain of Galpha transiently interacts with the alphaA helix of GIRK. Based on these results, we built a docking model of Galpha and GIRK, suggesting the molecular basis for efficient GIRK deactivation by Galpha(i/o).

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横川 真梨子

論文 - 横川 真梨子

横川　真梨子

論文 - 横川　真梨子