A Visualization Tool for Oligomerization and Internalization of Membrane Proteins in Living Cells: Coiled-Coil Labeling Method

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  • Yano Yoshiaki
    Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University
  • Kawano Kenichi
    Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University
  • Omae Kaoru
    Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University
  • Takeda Yuki
    Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University
  • Matsuzaki Sayaka
    Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University
  • Matsuzaki Katsumi
    Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University

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Other Title
  • 生細胞膜タンパク質の会合・内在化の可視化解析ツール：コイルドコイルラベル法
  • Symposium Review 生細胞膜タンパク質の会合・内在化の可視化解析ツール : コイルドコイルラベル法
  • Symposium Review ナマ サイボウマク タンパクシツ ノ カイゴウ ・ ナイザイカ ノ カシカ カイセキ ツール : コイルドコイルラベルホウ

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Abstract

  Genetic fusion of fluorescent/luminescent proteins to a target protein for specific labeling in living cells has been widely used to investigate the intracellular trafficking and oligomerization of the proteins. However, several limitations of fluorescent/luminescent proteins, such as considerable size, difficulty in controlling labeling ratio in multicolor labeling, can obscure true behaviors of the target proteins. To overcome these difficulties, post-translational labeling methods using pairs of small genetically-encodable ‘tags’ and synthetic ‘probes’ targeting the tags have been widely studied in recent years. We have developed a quick tag-probe labeling method using a high-affinity heterodimeric coiled-coil formation between the E3 tag (EIAALEK)3 attached to the target protein and the K4 probe (KIAALKE)4 labeled with a fluorophore. The labeling is cell-surface-specific and completed within 1 min, therefore suitable for monitoring oligomerization/internalization of membrane proteins on living cell surface. Taking advantage of easiness in multicolor labeling, we show that the oligomeric state of membrane proteins can be precisely analyzed based on fluorescence resonance energy transfer. By using this method, we found that β2 adrenergic receptors do not form constitutive homooligomers, and homooligomerization is not necessary for the receptor function. Furthermore, the degree of internalization of the β2 receptors following agonist stimulation was evaluated by ratiometric detection of pH decrease in endosomes.<br>

Journal

  • YAKUGAKU ZASSHI

    YAKUGAKU ZASSHI 134 (4), 501-506, 2014-04-01

    The Pharmaceutical Society of Japan

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