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TheScientificWorldJOURNAL
Volume 11, Pages 1995-2010
http://dx.doi.org/10.1100/2011/690858
Research Article

Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

1Área de Neurociencias, CIMA, Universidad de Navarra, Avenida Pío XII 55, 31008 Pamplona, Spain
2Departamento de Bioquímica y Biología Molecular, Unversidad de Navarra, C/Irunlarrea s/n, 31008 Pamplona, Spain
3Centro 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, Spain
4Departamento de Biología Molecular and Centro de Biología Molecular “Severo Ochoa”, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), 28049 Madrid, Spain

Received 24 March 2011; Accepted 8 September 2011

Academic Editor: S. Ferré

Copyright © 2011 María S. Aymerich et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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.