| Quantification of GPCR internalization by single-molecule microscopy in living cells. | |
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MedLine Citation:
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PMID: 21541374 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Receptor internalization upon ligand stimulation is a key component of a cell's response and allows a cell to correctly sense its environment. Novel fluorescent methods have enabled the direct visualization of the agonist-stimulated G-protein-coupled receptors (GPCR) trafficking in living cells. However, it is difficult to observe internalization of GPCRs in vivo due to intrinsic autofluorescence and cytosolic signals of fluorescently labeled GPCRs. This study uses the superior positional accuracy of single-molecule fluorescence microscopy to visualize in real time the internalization of Dictyostelium discoideum cAMP receptors, cAR1, genetically encoded with eYFP. This technique made it possible to follow the number of receptors in time revealing that the fraction of cytosolic receptors increases after persistent agonist stimulation and that the majority of the receptors were degraded after internalization. The observed internalization process was phosphorylation dependent, as shown with the use of a phosphorylation deficient cAR1 mutant, cm1234-eYFP, or stimulation with an antagonist, Rp-cAMPS that does not induce receptor phosphorylation. Furthermore, experiments done in mound-stage cells suggest that intrinsic, phosphorylation-induced internalization of cAR1 is necessary for Dictyostelium wild type cells to progress properly through multicellular development. To our knowledge, this observation illustrates for the first time phosphorylation-dependent internalization of single cAR1 molecules in living cells and its involvement in multicellular development. This very sensitive imaging of receptor internalization can be a useful and universal approach for pharmacological characterization of GPCRs in other cell types. |
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Authors:
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Arnauld Sergé; Sandra de Keijzer; Freek Van Hemert; Mark R Hickman; Dale Hereld; Herman P Spaink; Thomas Schmidt; B Ewa Snaar-Jagalska |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-5-3 |
Journal Detail:
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Title: Integrative biology : quantitative biosciences from nano to macro Volume: - ISSN: 1757-9708 ISO Abbreviation: - Publication Date: 2011 May |
Date Detail:
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Created Date: 2011-5-4 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101478378 Medline TA: Integr Biol (Camb) Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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From Physics of Life Processes, Leiden Institute of Physics, Leiden University, P.O. Box 9504, Leiden, The Netherlands. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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