Abstract:The adenosine A1 receptor (A1R) is a G protein-coupled receptor (GPCR) for adenosine, a ubiquitous neuromodulator, and thus regulates neuronal excitability, as well as arousal and sensitivity to pain. In addition, we have previously described a new mode of action for A1R: in cerebellar Purkinje cells, its activation attenuates neuronal responses to glutamate, as mediated by the type-1 metabotropic glutamate receptor (mGluR1). mGluR1 is also a GPCR, and elicits such responses as long-term depression of the post… Show more
“…This allowed us to focus on the signaling of heterologously expressed GPCRs. Once expressed in HEK293am cells, A1R and mGluR1 are thought to form heteromeric complexes as reported in the previous co-immunoprecipitation and Föster resonance energy transfer studies [ 14 , 16 ]. Fig.…”
Section: Resultsmentioning
confidence: 83%
“…For analysis of mGluR1- and A1R-mediated responses, we used HEK293am cells, a previously established stable cell line containing the tetracycline-controlled transcriptional activation system and mGluR1 and A1R genes [ 16 ]. This cell line was designed to express these GPCRs only when stimulated with a tetracycline derivative such as doxycycline.…”
Section: Resultsmentioning
confidence: 99%
“…This cell line was designed to express these GPCRs only when stimulated with a tetracycline derivative such as doxycycline. We used the inducible expression system because cell lines expressing the exogenous GPCRs under the control of constitutive promoters were often lost [ 16 ].…”
Section: Resultsmentioning
confidence: 99%
“…This cell line expressed these receptors only when stimulated with a tetracycline derivative such as doxycycline. We used the inducible expression system because cell lines expressing the exogenous GPCRs under the control of constitutive promoters were often lost [ 16 ]. For selection of HEK293am cells, the medium was supplemented with 15 μg/ml blasticidin S hydrochloride (KK-400, Kaken Pharmaceutical, Tokyo, Japan), 120 μg/ml hygromycin B (09287-84, Nacalai Tesque), and 480 μg/ml G418 (09380-44, Nacalai Tesque).…”
Section: Methodsmentioning
confidence: 99%
“…This signaling triggers long-term depression of Purkinje cell’s postsynaptic glutamate-sensitivity (glu-LTD), a cellular basis for cerebellar motor learning [ 15 ]. It is shown that mGluR1 forms complexes with A1R [ 13 , 14 , 16 ], which receives adenosine in the cerebrospinal fluid [ 17 , 18 ] and activates G i/o protein. We have revealed that A1R activation leads to suppression of glu-LTD [ 14 ].…”
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.
“…This allowed us to focus on the signaling of heterologously expressed GPCRs. Once expressed in HEK293am cells, A1R and mGluR1 are thought to form heteromeric complexes as reported in the previous co-immunoprecipitation and Föster resonance energy transfer studies [ 14 , 16 ]. Fig.…”
Section: Resultsmentioning
confidence: 83%
“…For analysis of mGluR1- and A1R-mediated responses, we used HEK293am cells, a previously established stable cell line containing the tetracycline-controlled transcriptional activation system and mGluR1 and A1R genes [ 16 ]. This cell line was designed to express these GPCRs only when stimulated with a tetracycline derivative such as doxycycline.…”
Section: Resultsmentioning
confidence: 99%
“…This cell line was designed to express these GPCRs only when stimulated with a tetracycline derivative such as doxycycline. We used the inducible expression system because cell lines expressing the exogenous GPCRs under the control of constitutive promoters were often lost [ 16 ].…”
Section: Resultsmentioning
confidence: 99%
“…This cell line expressed these receptors only when stimulated with a tetracycline derivative such as doxycycline. We used the inducible expression system because cell lines expressing the exogenous GPCRs under the control of constitutive promoters were often lost [ 16 ]. For selection of HEK293am cells, the medium was supplemented with 15 μg/ml blasticidin S hydrochloride (KK-400, Kaken Pharmaceutical, Tokyo, Japan), 120 μg/ml hygromycin B (09287-84, Nacalai Tesque), and 480 μg/ml G418 (09380-44, Nacalai Tesque).…”
Section: Methodsmentioning
confidence: 99%
“…This signaling triggers long-term depression of Purkinje cell’s postsynaptic glutamate-sensitivity (glu-LTD), a cellular basis for cerebellar motor learning [ 15 ]. It is shown that mGluR1 forms complexes with A1R [ 13 , 14 , 16 ], which receives adenosine in the cerebrospinal fluid [ 17 , 18 ] and activates G i/o protein. We have revealed that A1R activation leads to suppression of glu-LTD [ 14 ].…”
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.
Accumulating evidence suggests that G protein‐coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A1R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A1R of an A1R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A1R antagonists and showed evidence of covalent binding and simultaneous binding across an A1R homodimer. Greater selectivity for A1R over the adenosine A3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand.
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