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Title

Endocytosis as a biological response in receptor pharmacology: Evaluation by fluorescence microscopy

AuthorsCampa, Víctor M.; Capilla, Almudena; Varela, María J.; Acanda de la Rocha, Arlet M.; Fernández-Troyano, Juan C.; Barreiro, R. Belén ; López-Giménez, Juan F.
Issue Date2015
PublisherPublic Library of Science
CitationPLoS ONE 10(4): e0122604 (2015)
AbstractThe activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT<inf>2C</inf> receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution.
DescriptionThis is an open access article distributed under the terms of the Creative Commons Attribution License.
Publisher version (URL)http://dx.doi.org/10.1371/journal.pone.0122604
URIhttp://hdl.handle.net/10261/130574
DOI10.1371/journal.pone.0122604
Identifiersdoi: 10.1371/journal.pone.0122604
issn: 1932-6203
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