title: Application of surface plasmon resonance imaging to monitoring G protein-coupled receptor signaling and its modulation in a heterologous expression system.
abstract: BACKGROUND
  G protein-coupled receptors (GPCRs) are ubiquitous surface proteins mediating various biological responses and thus, important targets for therapeutic drugs. GPCRs individually produce their own signaling as well as modulate the signaling of other GPCRs. Real-time observation of GPCR signaling and modulation in living cells is key to molecular study of biological responses and pharmaceutical development. However, fluorescence imaging, the technique widely used for this purpose, requires a fluorescent dye which may inhibit biological responses or a fluorescent-tagged target protein created through time-consuming genetic manipulation. In this study, we applied two-dimensional surface plasmon resonance (SPR) imaging to monitoring the translocation of protein kinase C (PKC), a major GPCR-coupled signaling molecule in the widely used HEK293 cell lines and examined whether the signaling of, and, modulation between heterologously expressed GPCRs can be measured without fluorescent labeling.
  
  
  RESULTS
  We cultured HEK293 cells on the gold-plated slide glass and evoked SPR at the interface between the cell's plasma membrane and the gold surface with incident light. The translocation of activated native PKC to the plasma membrane is expected to alter the incident angle-SPR extent relation, and this could be detected as a change in the intensity of light reflection from the specimen illuminated at a fixed incident angle. Direct activation of PKC with 12-O-tetradecanoylphorbol-13-acetate increased the reflection intensity. This increase indeed reported PKC translocation because it was reduced by a pre-treatment with bisindolylmaleimide-1, a PKC inhibitor. We further applied this technique to a stable HEK293 cell line heterologously expressing the GPCRs type-1 metabotropic glutamate receptor (mGluR1) and adenosine A1 receptor (A1R). (RS)-3,5-dihydroxyphenylglycine, a mGluR1 agonist, increased the reflection intensity, and the PKC inhibitor reduced this increase. A pre-treatment with (R)-N(6)-phenylisopropyladenosine, an A1R-selective agonist suppressed mGluR1-mediated reflection increase. These results suggest that our technique can detect PKC translocation initiated by ligand binding to mGluR1 and its modulation by A1R.
  
  
  CONCLUSIONS
  SPR imaging turned out to be utilizable for monitoring GPCR-mediated PKC translocation and its modulation by a different GPCR in a heterologous expression system. This technique provides a powerful yet easy-to-use tool for molecular study of biological responses and pharmaceutical development.
authors.affiliation: Laboratory for Medical Information Sensing, Graduate School of Science and Engineering, University of Toyama, 3190 Gokufu, Toyama, Toyama, 930-8555, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Nonobe
authors.affiliation: Laboratory for Medical Information Sensing, Graduate School of Science and Engineering, University of Toyama, 3190 Gokufu, Toyama, Toyama, 930-8555, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Yokoyama
authors.affiliation: Department of Pharmacology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kamikubo
authors.affiliation: Laboratory for Medical Information Sensing, Graduate School of Science and Engineering, University of Toyama, 3190 Gokufu, Toyama, Toyama, 930-8555, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: Laboratory for Medical Information Sensing, Graduate School of Science and Engineering, University of Toyama, 3190 Gokufu, Toyama, Toyama, 930-8555, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Hisajima
authors.affiliation: Laboratory for Bioelectronics, Graduate School of Innovative Life Science, University of Toyama, 3190 Gokufu, Toyama, Toyama, 930-8555, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Shinohara
authors.affiliation: Laboratory for Bioelectronics, Graduate School of Innovative Life Science, University of Toyama, 3190 Gokufu, Toyama, Toyama, 930-8555, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Shiraishi
authors.affiliation: Department of Pharmacology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Laboratory for Medical Information Sensing, Graduate School of Science and Engineering, University of Toyama, 3190 Gokufu, Toyama, Toyama, 930-8555, Japan. ttabata@eng.u-toyama.ac.jp.
authors.forename: Toshihide
authors.initials: T
authors.lastname: Tabata
mesh_terms: D057809:HEK293 Cells; D006801:Humans; D011493:Protein Kinase C / Q000032:analysis / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D011493:Protein Kinase C
keywords: Adenosine; G protein-coupled receptor; Glutamate; Heterologous expression; Neuron; Surface plasmon resonance imaging; Synaptic plasticity