G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology
Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, beca...
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| Format: | Article |
| Language: | English |
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MDPI AG
2014-02-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | http://www.mdpi.com/1422-0067/15/2/2554 |
| _version_ | 1811295618506686464 |
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| author | Lenea Nørskov-Lauritsen Alex Rojas Bie Thomsen Hans Bräuner-Osborne |
| author_facet | Lenea Nørskov-Lauritsen Alex Rojas Bie Thomsen Hans Bräuner-Osborne |
| author_sort | Lenea Nørskov-Lauritsen |
| collection | DOAJ |
| description | Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers D-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3',5'-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries. |
| first_indexed | 2024-04-13T05:36:10Z |
| format | Article |
| id | doaj.art-d0442106ad4b4693aa58e8c3a385c5ef |
| institution | Directory Open Access Journal |
| issn | 1422-0067 |
| language | English |
| last_indexed | 2024-04-13T05:36:10Z |
| publishDate | 2014-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-d0442106ad4b4693aa58e8c3a385c5ef2022-12-22T03:00:17ZengMDPI AGInternational Journal of Molecular Sciences1422-00672014-02-011522554257210.3390/ijms15022554ijms15022554G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) TechnologyLenea Nørskov-Lauritsen0Alex Rojas Bie Thomsen1Hans Bräuner-Osborne2Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, Mailbox 10, Copenhagen DK-2100, DenmarkDepartment of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, Mailbox 10, Copenhagen DK-2100, DenmarkDepartment of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, Mailbox 10, Copenhagen DK-2100, DenmarkStudying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers D-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3',5'-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.http://www.mdpi.com/1422-0067/15/2/2554G protein-coupled receptor (GPCR)signaling pathwayhigh throughput screening (HTS)homogenous time-resolved Förster resonance energy transfer (HTRF®)D-myo-inositol 1-phosphate (IP1)cyclic adenosine 3',5'-monophosphate (cAMP)extracellular signal-regulated kinases (ERK1/2) |
| spellingShingle | Lenea Nørskov-Lauritsen Alex Rojas Bie Thomsen Hans Bräuner-Osborne G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology International Journal of Molecular Sciences G protein-coupled receptor (GPCR) signaling pathway high throughput screening (HTS) homogenous time-resolved Förster resonance energy transfer (HTRF®) D-myo-inositol 1-phosphate (IP1) cyclic adenosine 3',5'-monophosphate (cAMP) extracellular signal-regulated kinases (ERK1/2) |
| title | G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology |
| title_full | G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology |
| title_fullStr | G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology |
| title_full_unstemmed | G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology |
| title_short | G Protein-Coupled Receptor Signaling Analysis Using Homogenous Time-Resolved Förster Resonance Energy Transfer (HTRF®) Technology |
| title_sort | g protein coupled receptor signaling analysis using homogenous time resolved forster resonance energy transfer htrf r technology |
| topic | G protein-coupled receptor (GPCR) signaling pathway high throughput screening (HTS) homogenous time-resolved Förster resonance energy transfer (HTRF®) D-myo-inositol 1-phosphate (IP1) cyclic adenosine 3',5'-monophosphate (cAMP) extracellular signal-regulated kinases (ERK1/2) |
| url | http://www.mdpi.com/1422-0067/15/2/2554 |
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