High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.
Identifieur interne : 001806 ( Main/Exploration ); précédent : 001805; suivant : 001807High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.
Auteurs : Ralph J. Garippa [États-Unis] ; Ann F. Hoffman ; Gabriele Gradl ; Achim KirschSource :
- Methods in enzymology [ 0076-6879 ] ; 2006.
English descriptors
- KwdEn :
- Algorithms, Animals, Arrestins (chemistry), Automation, Computational Biology (methods), Green Fluorescent Proteins (chemistry), Green Fluorescent Proteins (metabolism), Humans, Image Processing, Computer-Assisted, Ligands, Mice, Mice, Inbred C57BL, Microscopy, Confocal (instrumentation), Microscopy, Confocal (methods), Protein Transport, Receptors, G-Protein-Coupled (chemistry), Software.
- MESH :
- chemical , chemistry : Arrestins, Green Fluorescent Proteins, Receptors, G-Protein-Coupled.
- chemical , metabolism : Green Fluorescent Proteins.
- instrumentation : Microscopy, Confocal.
- methods : Computational Biology, Microscopy, Confocal.
- Algorithms, Animals, Automation, Humans, Image Processing, Computer-Assisted, Ligands, Mice, Mice, Inbred C57BL, Protein Transport, Software.
Abstract
Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.
DOI: 10.1016/S0076-6879(06)14007-0
PubMed: 17110189
Affiliations:
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Hoffman</name></author><author><name sortKey="Gradl, Gabriele" sort="Gradl, Gabriele" uniqKey="Gradl G" first="Gabriele" last="Gradl">Gabriele Gradl</name></author><author><name sortKey="Kirsch, Achim" sort="Kirsch, Achim" uniqKey="Kirsch A" first="Achim" last="Kirsch">Achim Kirsch</name></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2006">2006</date><idno type="doi">10.1016/S0076-6879(06)14007-0</idno><idno type="RBID">pubmed:17110189</idno><idno type="pmid">17110189</idno><idno type="wicri:Area/PubMed/Corpus">000478</idno><idno type="wicri:Area/PubMed/Curation">000478</idno><idno type="wicri:Area/PubMed/Checkpoint">000466</idno><idno type="wicri:Area/Ncbi/Merge">000411</idno><idno type="wicri:Area/Ncbi/Curation">000411</idno><idno type="wicri:Area/Ncbi/Checkpoint">000411</idno><idno type="wicri:doubleKey">0076-6879:2006:Garippa R:high:throughput:confocal</idno><idno type="wicri:Area/Main/Merge">001839</idno><idno type="wicri:Area/Main/Curation">001806</idno><idno type="wicri:Area/Main/Exploration">001806</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">High-throughput confocal microscopy for beta-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the Evotec Opera.</title><author><name sortKey="Garippa, Ralph J" sort="Garippa, Ralph J" uniqKey="Garippa R" first="Ralph J" last="Garippa">Ralph J. Garippa</name><affiliation wicri:level="2"><nlm:affiliation>Roche Discovery Technologies, Roche Inc., Nutley, NJ, USA.</nlm:affiliation><country xml:lang="fr">États-Unis</country><wicri:regionArea>Roche Discovery Technologies, Roche Inc., Nutley, NJ</wicri:regionArea><placeName><region type="state">New Jersey</region></placeName></affiliation></author><author><name sortKey="Hoffman, Ann F" sort="Hoffman, Ann F" uniqKey="Hoffman A" first="Ann F" last="Hoffman">Ann F. Hoffman</name></author><author><name sortKey="Gradl, Gabriele" sort="Gradl, Gabriele" uniqKey="Gradl G" first="Gabriele" last="Gradl">Gabriele Gradl</name></author><author><name sortKey="Kirsch, Achim" sort="Kirsch, Achim" uniqKey="Kirsch A" first="Achim" last="Kirsch">Achim Kirsch</name></author></analytic><series><title level="j">Methods in enzymology</title><idno type="ISSN">0076-6879</idno><imprint><date when="2006" type="published">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Algorithms</term><term>Animals</term><term>Arrestins (chemistry)</term><term>Automation</term><term>Computational Biology (methods)</term><term>Green Fluorescent Proteins (chemistry)</term><term>Green Fluorescent Proteins (metabolism)</term><term>Humans</term><term>Image Processing, Computer-Assisted</term><term>Ligands</term><term>Mice</term><term>Mice, Inbred C57BL</term><term>Microscopy, Confocal (instrumentation)</term><term>Microscopy, Confocal (methods)</term><term>Protein Transport</term><term>Receptors, G-Protein-Coupled (chemistry)</term><term>Software</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Arrestins</term><term>Green Fluorescent Proteins</term><term>Receptors, G-Protein-Coupled</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Green Fluorescent Proteins</term></keywords><keywords scheme="MESH" qualifier="instrumentation" xml:lang="en"><term>Microscopy, Confocal</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Computational Biology</term><term>Microscopy, Confocal</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Algorithms</term><term>Animals</term><term>Automation</term><term>Humans</term><term>Image Processing, Computer-Assisted</term><term>Ligands</term><term>Mice</term><term>Mice, Inbred C57BL</term><term>Protein Transport</term><term>Software</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>New Jersey</li></region></list><tree><noCountry><name sortKey="Gradl, Gabriele" sort="Gradl, Gabriele" uniqKey="Gradl G" first="Gabriele" last="Gradl">Gabriele Gradl</name><name sortKey="Hoffman, Ann F" sort="Hoffman, Ann F" uniqKey="Hoffman A" first="Ann F" last="Hoffman">Ann F. Hoffman</name><name sortKey="Kirsch, Achim" sort="Kirsch, Achim" uniqKey="Kirsch A" first="Achim" last="Kirsch">Achim Kirsch</name></noCountry><country name="États-Unis"><region name="New Jersey"><name sortKey="Garippa, Ralph J" sort="Garippa, Ralph J" uniqKey="Garippa R" first="Ralph J" last="Garippa">Ralph J. 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