Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT

Kaori Esaki, Sosuke Yoshinaga, Tatsuichiro Tsuji, Etsuko Toda, Yuya Terashima, Takashi Saitoh, Daisuke Kohda, Toshiyuki Kohno, Masanori Osawa, Takumi Ueda, Ichio Shimada, Kouji Matsushima, Hiroaki Terasawa

    Research output: Contribution to journalArticle

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    Abstract

    The membrane-proximal C-terminal region (Pro-C) is important for the regulation of G-protein-coupled receptors (GPCRs), but the binding of the Pro-C region to a cytosolic regulator has not been structurally analyzed. The chemokine receptor CCR2 is a member of the GPCR superfamily, and the Pro-C region of CCR2 binds to the cytosolic regulator FROUNT. Studying the interaction between CCR2 Pro-C and FROUNT at an atomic level provides a basis for understanding the signal transduction mechanism via GPCRs. NOE-based NMR experiments showed that, when bound to FROUNT, CCR2 Pro-C adopted a helical conformation, as well as when embedded in dodecylphosphocholine micelles. A comparison of two types of cross-saturation-based NMR experiments, applied to a three-component mixture of Pro-C, FROUNT and micelles or a two-component mixture of Pro-C and micelles, revealed that the hydrophobic binding surface on Pro-C for FROUNT mostly overlapped with the binding site for micelles, suggesting competitive binding of Pro-C between FROUNT and micelles. Leu316 was important for both FROUNT and micelle binding. Phe319 was newly identified to be crucial for FROUNT binding, by NMR and mutational analyses. The association and dissociation rates of CCR2 Pro-C for lipid bilayer biomembranes were faster than those for FROUNT. We previously reported that FROUNT binding to CCR2 is detectable even in unstimulated cells and increases in response to chemokine stimulation. Taken together, these results support a model of CCR2 equilibrium: chemokine binding changes the conformational equilibrium of CCR2 toward the active state, and Pro-C switches its binding partner from the membrane to FROUNT.

    Original languageEnglish
    Pages (from-to)5552-5566
    Number of pages15
    JournalFEBS Journal
    Volume281
    Issue number24
    DOIs
    Publication statusPublished - Jan 1 2014

    Fingerprint

    Chemokine Receptors
    Micelles
    Membranes
    G-Protein-Coupled Receptors
    Nuclear magnetic resonance
    Chemokines
    Signal transduction
    Lipid bilayers
    Competitive Binding
    Lipid Bilayers
    Conformations
    Signal Transduction
    Experiments
    Binding Sites
    Switches
    Association reactions

    All Science Journal Classification (ASJC) codes

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    Esaki, K., Yoshinaga, S., Tsuji, T., Toda, E., Terashima, Y., Saitoh, T., ... Terasawa, H. (2014). Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT. FEBS Journal, 281(24), 5552-5566. https://doi.org/10.1111/febs.13096

    Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT. / Esaki, Kaori; Yoshinaga, Sosuke; Tsuji, Tatsuichiro; Toda, Etsuko; Terashima, Yuya; Saitoh, Takashi; Kohda, Daisuke; Kohno, Toshiyuki; Osawa, Masanori; Ueda, Takumi; Shimada, Ichio; Matsushima, Kouji; Terasawa, Hiroaki.

    In: FEBS Journal, Vol. 281, No. 24, 01.01.2014, p. 5552-5566.

    Research output: Contribution to journalArticle

    Esaki, K, Yoshinaga, S, Tsuji, T, Toda, E, Terashima, Y, Saitoh, T, Kohda, D, Kohno, T, Osawa, M, Ueda, T, Shimada, I, Matsushima, K & Terasawa, H 2014, 'Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT', FEBS Journal, vol. 281, no. 24, pp. 5552-5566. https://doi.org/10.1111/febs.13096
    Esaki, Kaori ; Yoshinaga, Sosuke ; Tsuji, Tatsuichiro ; Toda, Etsuko ; Terashima, Yuya ; Saitoh, Takashi ; Kohda, Daisuke ; Kohno, Toshiyuki ; Osawa, Masanori ; Ueda, Takumi ; Shimada, Ichio ; Matsushima, Kouji ; Terasawa, Hiroaki. / Structural basis for the binding of the membrane-proximal C-terminal region of chemokine receptor CCR2 with the cytosolic regulator FROUNT. In: FEBS Journal. 2014 ; Vol. 281, No. 24. pp. 5552-5566.
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    abstract = "The membrane-proximal C-terminal region (Pro-C) is important for the regulation of G-protein-coupled receptors (GPCRs), but the binding of the Pro-C region to a cytosolic regulator has not been structurally analyzed. The chemokine receptor CCR2 is a member of the GPCR superfamily, and the Pro-C region of CCR2 binds to the cytosolic regulator FROUNT. Studying the interaction between CCR2 Pro-C and FROUNT at an atomic level provides a basis for understanding the signal transduction mechanism via GPCRs. NOE-based NMR experiments showed that, when bound to FROUNT, CCR2 Pro-C adopted a helical conformation, as well as when embedded in dodecylphosphocholine micelles. A comparison of two types of cross-saturation-based NMR experiments, applied to a three-component mixture of Pro-C, FROUNT and micelles or a two-component mixture of Pro-C and micelles, revealed that the hydrophobic binding surface on Pro-C for FROUNT mostly overlapped with the binding site for micelles, suggesting competitive binding of Pro-C between FROUNT and micelles. Leu316 was important for both FROUNT and micelle binding. Phe319 was newly identified to be crucial for FROUNT binding, by NMR and mutational analyses. The association and dissociation rates of CCR2 Pro-C for lipid bilayer biomembranes were faster than those for FROUNT. We previously reported that FROUNT binding to CCR2 is detectable even in unstimulated cells and increases in response to chemokine stimulation. Taken together, these results support a model of CCR2 equilibrium: chemokine binding changes the conformational equilibrium of CCR2 toward the active state, and Pro-C switches its binding partner from the membrane to FROUNT.",
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    AU - Toda, Etsuko

    AU - Terashima, Yuya

    AU - Saitoh, Takashi

    AU - Kohda, Daisuke

    AU - Kohno, Toshiyuki

    AU - Osawa, Masanori

    AU - Ueda, Takumi

    AU - Shimada, Ichio

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