Details

Autor(en) / Beteiligte

• Campa, Víctor M
• Capilla, Almudena
• Varela, María J
• de la Rocha, Arlet M Acanda
• Fernandez-Troyano, Juan C
• Barreiro, R Belén
• Lopez-Gimenez, Juan F
• Blenau, Wolfgang

Titel

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

Ist Teil von

• PloS one, 2015-04, Vol.10 (4), p.e0122604-e0122604

Ort / Verlag

United States: Public Library of Science

Erscheinungsjahr

2015

Quelle

Free E-Journal (出版社公開部分のみ）

Beschreibungen/Notizen

• The 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-HT2C 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.