Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics
Understanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane rece...
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| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2011-01-01
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| Series: | The Scientific World Journal |
| Online Access: | http://dx.doi.org/10.1100/2011/690858 |
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| author | María S. Aymerich J. López-Azcárate J. Bonaventura G. Navarro D. Fernández-Suárez V. Casadó F. Mayor C. Lluís M. Valencia J. Artieda Rafael Franco |
| author_facet | María S. Aymerich J. López-Azcárate J. Bonaventura G. Navarro D. Fernández-Suárez V. Casadó F. Mayor C. Lluís M. Valencia J. Artieda Rafael Franco |
| author_sort | María S. Aymerich |
| collection | DOAJ |
| description | Understanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics. |
| format | Article |
| id | doaj-art-72624f424c8a4b6b82bc061a2f856a4c |
| institution | Kabale University |
| issn | 1537-744X |
| language | English |
| publishDate | 2011-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | The Scientific World Journal |
| spelling | doaj-art-72624f424c8a4b6b82bc061a2f856a4c2025-02-03T06:04:56ZengWileyThe Scientific World Journal1537-744X2011-01-01111995201010.1100/2011/690858690858Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor DynamicsMaría S. Aymerich0J. López-Azcárate1J. Bonaventura2G. Navarro3D. Fernández-Suárez4V. Casadó5F. Mayor6C. Lluís7M. Valencia8J. Artieda9Rafael Franco10Área de Neurociencias, CIMA, Universidad de Navarra, Avenida Pío XII 55, 31008 Pamplona, SpainÁrea de Neurociencias, CIMA, Universidad de Navarra, Avenida Pío XII 55, 31008 Pamplona, SpainCentro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, SpainCentro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, SpainÁrea de Neurociencias, CIMA, Universidad de Navarra, Avenida Pío XII 55, 31008 Pamplona, SpainCentro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, SpainDepartamento de Biología Molecular and Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), 28049 Madrid, SpainCentro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, SpainÁrea de Neurociencias, CIMA, Universidad de Navarra, Avenida Pío XII 55, 31008 Pamplona, SpainÁrea de Neurociencias, CIMA, Universidad de Navarra, Avenida Pío XII 55, 31008 Pamplona, SpainÁrea de Neurociencias, CIMA, Universidad de Navarra, Avenida Pío XII 55, 31008 Pamplona, SpainUnderstanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.http://dx.doi.org/10.1100/2011/690858 |
| spellingShingle | María S. Aymerich J. López-Azcárate J. Bonaventura G. Navarro D. Fernández-Suárez V. Casadó F. Mayor C. Lluís M. Valencia J. Artieda Rafael Franco Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics The Scientific World Journal |
| title | Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics |
| title_full | Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics |
| title_fullStr | Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics |
| title_full_unstemmed | Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics |
| title_short | Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics |
| title_sort | real time g protein coupled receptor imaging to understand and quantify receptor dynamics |
| url | http://dx.doi.org/10.1100/2011/690858 |
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