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

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 function...

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Published in:PloS one 2015-04, Vol.10 (4), p.e0122604-e0122604
Main Authors: 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
Format: Article
Language:English
Subjects:
Activation
Algorithms
Analysis
Biological effects
Biotechnology
Cells (biology)
Cytoplasm
Endocytosis
Endocytosis - drug effects
Endosomes
Enkephalin, Ala-MePhe-Gly- - pharmacology
Enkephalins
Fluorescence
Fluorescence microscopy
Half-Life
HEK293 Cells
HeLa Cells
Humans
Image analysis
Image processing
Kinetics
Ligands
Methadone
Microscopy
Microscopy, Fluorescence
Morphine
Narcotics
Opioid receptors (type mu)
Pharmacology
Plasma
Protein Transport
Proteins
Receptor, Serotonin, 5-HT2C - metabolism
Receptors
Serotonin
Serotonin - pharmacology
Serotonin 5-HT2 Receptor Agonists - pharmacology
Serotonin S2 receptors
Signal transduction
Temporal resolution
Translocation
Transport Vesicles - metabolism
Yellow fluorescent protein
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cited_by cdi_FETCH-LOGICAL-c692t-b3dd805dd77c0ceb3503050985a76797081d121c4713586a91871296c04993143
cites cdi_FETCH-LOGICAL-c692t-b3dd805dd77c0ceb3503050985a76797081d121c4713586a91871296c04993143
container_end_page e0122604
container_issue 4
container_start_page e0122604
container_title PloS one
container_volume 10
creator 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
description 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.
doi_str_mv 10.1371/journal.pone.0122604
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subjects Activation
Algorithms
Analysis
Biological effects
Biotechnology
Cells (biology)
Cytoplasm
Endocytosis
Endocytosis - drug effects
Endosomes
Enkephalin, Ala-MePhe-Gly- - pharmacology
Enkephalins
Fluorescence
Fluorescence microscopy
Half-Life
HEK293 Cells
HeLa Cells
Humans
Image analysis
Image processing
Kinetics
Ligands
Methadone
Microscopy
Microscopy, Fluorescence
Morphine
Narcotics
Opioid receptors (type mu)
Pharmacology
Plasma
Protein Transport
Proteins
Receptor, Serotonin, 5-HT2C - metabolism
Receptors
Serotonin
Serotonin - pharmacology
Serotonin 5-HT2 Receptor Agonists - pharmacology
Serotonin S2 receptors
Signal transduction
Temporal resolution
Translocation
Transport Vesicles - metabolism
Yellow fluorescent protein
title Endocytosis as a biological response in receptor pharmacology: evaluation by fluorescence microscopy
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