Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Summary

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Abstract

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

Introduction

Cells possess cargo trafficking machinery to transport extracellular materials-such as ligands, microorganisms, nutrients, and transmembrane proteins-into the cell for information, energy, and other purposes^1,2. It is critical for cellular homeostasis, tissue function, and overall cell survival. The cell-based expression of fluorescent fusion proteins is a powerful way to study the localization and internalization of transmembrane receptors, such as G protein-coupled receptors (GPCR), in signaling pathways³.

The expression of fusion proteins tagged with green....

Protocol

1. Cell Preparation

1. Cell recovery
   1. Transfer the cryopreserved human embryonic kidney 293 (HEK293) cells from a liquid nitrogen tank to a 37 °C water bath and shake continuously for 1 - 2 min.
   2. Add 10 mL of equilibrated growth medium (Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin solution) to a 10-cm cell culture dish. Gently add the thawed cells to the equilibrated growth medium.
   3. Incubate the cells in a 37 °C water-jacketed CO₂ incubator with a humidified atmosphere containing 95% air and 5% CO₂ for 2 - 4 h.

Results

Figure 1 shows an example of a confocal microscope system. Figure 2 presents the expression of pEGFP-N1 in HEK293 cells. The GFP signal was detected in the cytoplasm and nucleus. Figure 3 shows the subcellular localization of GnRHR from S. japonica in HEK293 cells, consistent with our previously published results¹⁵. The fusion protein with the EGFP tag at the C-terminus of SjGnRHR (SjGnRH.......

Discussion

The protocol presented here provides an efficient and easily interpretable method to detect the subcellular localization and internalization of expressed GPCR fusion protein in HEK293 cells. The technique can be easily adapted for many different genes and cell types, such as for the cellular localization and internalization of the corazonin receptor from Bombyx mori in HEK293 and BmN cells¹⁶.

The successful application of this powerful assay relies on a few key.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEK293 cell line
DMEM/High glucose	Hyclone	SH30022.01	High glucose 4.0 mM L-glutamine
Fetal Bovine Serum (FBS)	PuFei	1101-100
Phosphate Buffered Saline (PBS)	Genom	GNM10010
Penicillin-Streptomycin	Genom	GNM15140
0.25% Trypsin & 0.02% EDTA	Genom	GNM25200
Opti-MEM (1x)	GIBCO, by Life Technologies	31985-062	reduced serum media
Lipofectamine 2000 Transfection Reagent	Invitrogen	11668027	Reagent 1
X-tremeGENE HP DNA Transfection Reagent	Roche	6366236001	Reagent 2
DAPI Staining Solution	Beyotime	C1006
DiI	Beyotime	C1036
Antifade Mounting Medium	Beyotime	P0126
Paraformaldehyde	Sinopharm Chemical Reagent Co.(SCRC)	80096618
100 mm cell culture	CORNING	430167
6-well plate	ExCell Bio	CS016-0092
12-well plate	ExCell Bio	CS016-0093
Cover Glass	NEST	801007
Microscope Slides	Citoglas	10127105P-G
Thermo Scientific Forma CO2 Incubator	Thermo	3111
TCS SP5II laser scanning confocal microscope	Leica	5100001311