Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy

Mohammed M. Nooh, Suleiman Bahouth

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β1-adrenergic receptor (β1-AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β1-AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc.
Pages67-78
Number of pages12
DOIs
StatePublished - Jan 1 2017

Publication series

NameMethods in Cell Biology
Volume142
ISSN (Print)0091-679X

Fingerprint

G-Protein-Coupled Receptors
Confocal Microscopy
Recycling
Adrenergic Receptors
Epitopes
Cell Membrane
Antibodies
Drug Industry
Fluorescence Microscopy
Fluorescent Antibody Technique
Pharmaceutical Preparations
Proteins

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

Nooh, M. M., & Bahouth, S. (2017). Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. In Methods in Cell Biology (pp. 67-78). (Methods in Cell Biology; Vol. 142). Academic Press Inc.. https://doi.org/10.1016/bs.mcb.2017.07.010

Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. / Nooh, Mohammed M.; Bahouth, Suleiman.

Methods in Cell Biology. Academic Press Inc., 2017. p. 67-78 (Methods in Cell Biology; Vol. 142).

Research output: Chapter in Book/Report/Conference proceedingChapter

Nooh, MM & Bahouth, S 2017, Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. in Methods in Cell Biology. Methods in Cell Biology, vol. 142, Academic Press Inc., pp. 67-78. https://doi.org/10.1016/bs.mcb.2017.07.010
Nooh MM, Bahouth S. Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. In Methods in Cell Biology. Academic Press Inc. 2017. p. 67-78. (Methods in Cell Biology). https://doi.org/10.1016/bs.mcb.2017.07.010
Nooh, Mohammed M. ; Bahouth, Suleiman. / Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy. Methods in Cell Biology. Academic Press Inc., 2017. pp. 67-78 (Methods in Cell Biology).
@inbook{c0653381fc91421d8e19890d46af1885,
title = "Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy",
abstract = "G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40{\%} of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β1-adrenergic receptor (β1-AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β1-AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review.",
author = "Nooh, {Mohammed M.} and Suleiman Bahouth",
year = "2017",
month = "1",
day = "1",
doi = "10.1016/bs.mcb.2017.07.010",
language = "English (US)",
series = "Methods in Cell Biology",
publisher = "Academic Press Inc.",
pages = "67--78",
booktitle = "Methods in Cell Biology",
address = "United States",

}

TY - CHAP

T1 - Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy

AU - Nooh, Mohammed M.

AU - Bahouth, Suleiman

PY - 2017/1/1

Y1 - 2017/1/1

N2 - G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β1-adrenergic receptor (β1-AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β1-AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review.

AB - G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β1-adrenergic receptor (β1-AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β1-AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review.

UR - http://www.scopus.com/inward/record.url?scp=85058199982&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85058199982&partnerID=8YFLogxK

U2 - 10.1016/bs.mcb.2017.07.010

DO - 10.1016/bs.mcb.2017.07.010

M3 - Chapter

T3 - Methods in Cell Biology

SP - 67

EP - 78

BT - Methods in Cell Biology

PB - Academic Press Inc.

ER -