Application of surface plasmon resonance imaging to monitoring G protein-coupled receptor signaling and its modulation in a heterologous expression system

Abstract

BackgroundG 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.ResultsWe 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)-N6-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.ConclusionsSPR 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.

A Sensitive and Stable Surface Plasmon Resonance Sensor Based on Monolayer Protected Silver Film

“…The surface plasmon wave was first reported by Wood in 1902 [ 1 ], and more than 60 years later, Kretschmann [ 2 ] and Otto [ 3 ] realized the surface plasmon resonance with the method of attenuated total reflection in 1968. Then, over the last decades, label-free, real-time and ultra-sensitive surface plasmon resonance (SPR) sensor has been rapidly developed for the detection of small and large molecules based on the monitoring of refractive index change of surrounding media [ 4 , 5 , 6 , 7 ], and it has been widely used in the field of drug screening [ 8 , 9 , 10 ] and other biomedical areas [ 11 ]. Practically, the stability of biosensor chips is significant for repeatable results.…”

G Protein-Coupled Glutamate and GABA Receptors Form Complexes and Mutually Modulate Their Signals

“…We previously showed that GBR activation leads to an increase in the ligand-sensitivity of mGluR1 in cerebellar Purkinje cells. , Here, we investigated whether GBR-mediated mGluR1 sensitization occurs in the non-neuronal cellular environment of HEKmg12 cells and dissected the mechanisms underlying this phenomenon, taking advantage of the manipulatable heterologous gene expression. In HEK293 cells, heterologously expressed mGluR1 can couple to the G q /G 11 protein–protein kinase C cascade . The G q /G 11 protein may also mediate signaling to intracellular Ca 2+ mobilization in this cell type .…”

“…GPCR complex formation and functional interaction play crucial roles in the regulation of neurotransmission . Previously, we showed complex formation and functional interaction between mGluR1 and the adenosine A1 receptor, which is another G i /G o -coupled receptor expressed in cerebellar Purkinje cells. ,, To further elucidate the functional interaction of GBR and mGluR1, we performed Ca 2+ imaging in HEKmg12 cells. This analysis showed that GBR activation increased mGluR1-mediated Ca 2+ signaling in the HEKmg12 cells (Figure ) as described previously in cerebellar Purkinje cells. , Pharmacological manipulation of GBR signaling showed that GBR-mediated mGluR1 signaling augmentation is independent of G i /G o protein, which is coupled to GBR (Figure A–F).…”

Recent Advances in Surface Plasmon Resonance Imaging Sensors

“…Notably, real-time monitoring of cellular pathways inside a living cell is an interesting area. For example, monitoring G protein-coupled receptor signaling and its modulation to detect PKC translocation initiated by the ligand binding to mGluR1 as well as A1R-mediated modulation of mGluR1-mediated PKC translocation in cultured kidney-derived HEK293 cell line cells on the SPRi sensing surface was used to study the cellular basis for cerebellar motor learning [146]. Also, secretory defects in vascularized micro-organs, known as the islets of Langerhans, could result in diabetes.…”