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 fluorescent protein (GFP) from jellyfish Aequorea victoria, combined with direct fluorescence detection techniques, has gained wide acceptance in protein-targeting research^4,5,6. GFP-based detection has the advantage of allowing the real-time imaging of living cells while avoiding fixation artifacts⁷. A series of versatile cloning vectors has been constructed to facilitate the expression of protein fusions to GFP in various cells^8,9. The pEGFP-N1 vector is a widely used and commercialized plasmid vector encoding an enhanced green fluorescent protein (EGFP), which has been optimized from wild-type GFP for brighter fluorescence and higher expression in mammalian cells. To observe the fluorescent signal of EGFP expressed in mammalian cells, the fluorescence microscopy should be conducted with the excitation at 488 nm and the emission at 507 nm, preferably with confocal microscopy.

Monitoring the subcellular localization and ligand-mediated internalization of receptors is a common technique to investigate the signal transduction and function of GPCRs^10,11. In most cases, internalization leads to desensitization of the GPCRs (the attenuation of the stimulus response in spite of the presence of agonists)^12,13. The internalized receptors can be incorporated into the lysosome and degraded, or they can be recycled back to the cell surface, depending on the nature of the receptors and the cell lines used in the experiments.

The gonadotropin-releasing hormone receptors (GnRHRs) belong to the GPCR family and have a typical GPCR protein structure, with an extracellular amino terminal, an intracellular -COOH terminal, and seven transmembrane (TM) domains¹⁴. The transfection of recombinant plasmid SjGnRHR-EGFP (with the GnRHR gene from Sepiella japonica) and the confocal microscopy detection of EGFP-tagged SjGnRHR (expressed in HEK293 cells) was reported previously, and GFP fluorescence was established as a reporter for transmembrane protein localization assays¹⁵. Now, the internalization of SjGnRHR, activated by S. japonica gonadotropin-releasing hormone (SjGnRH), was demonstrated in time- and dose-dependent manners by confocal microscopy.

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.
   4. After 2 - 4 h, check that the cells are attached. Replace the equilibrated growth medium and culture for another 24 - 48 h.
      NOTE: Changing the equilibrated growth medium is useful because it removes dimethyl sulfoxide (DMSO) and protects against cell damage.
2. Cell passage
   1. After 24 - 48 h, when the cells have grown to near confluence, discard the equilibrated growth medium and wash the cells with 2 mL of phosphate-buffered saline (PBS). Swirl the dish gently.
   2. Discard the PBS and add 1 mL of Trypsin-EDTA (saline, 0.25% trypsin, and 0.02% EDTA). To completely digest the cells, swirl the dish carefully and aspirate off the Trypsin-EDTA solution. Put the dish in the 37 °C water-jacketed CO₂ incubator for 3 min.
      NOTE: During this period, prepare three new 10-cm cell culture dishes and add 10 mL of equilibrated growth medium to each.
   3. Using a light microscope, determine if the cells have lifted from the bottom of the dish. Add 2 mL of equilibrated growth medium to the dish. Mix by pipetting and immediately pipet 0.6 mL of the cell suspension to each of the new dishes. Gently mix by pipetting and allow the cells to incubate at 37 °C and 5% CO₂.

2. Cell Seeding

1. After 24 - 48 h, when the passaged cells have grown to near confluence, discard the equilibrated growth medium and wash the cells with 2 mL of PBS. Swirl the dish gently.
2. Discard the PBS and add 1 mL of Trypsin-EDTA. To completely digest the cells, swirl the dish carefully and aspirate off the Trypsin-EDTA. Put the dish in the 37 °C water-jacketed CO₂ incubator for 3 min.
   NOTE: During this period, prepare a 6-well plate by adding 2 mL of equilibrated growth medium to each well.
3. Add 1 mL of equilibrated growth medium to the dish, if, under microscopic observation, the cells to have lifted from the dish bottom. Mix by pipetting and immediately pipet 0.1 mL of cell suspension (the volume is adjusted according to the cell growth density) to each well of the prepared 6-well plate (cell-less 6-well plate with 2 mL of equilibrated growth medium). Gently mix by pipetting and allow the cells to incubate at 37 °C and 5% CO₂.

3. Cell Transfection

1. Transfection method 1, with liposome formulations
   NOTE: Please see the Table of Materials for transfection reagent 1.
   1. The following day, ensure that cells should be 85 - 95% confluent.
   2. Add 3 µg of plasmid DNA (500 ng/µL) and 100 µL of reduced serum media to a sterile microcentrifuge tube, and mix gently. Incubate for 5 min at room temperature.
   3. Prepare 9 µL of Reagent 1 and 100 µL of reduced serum media in another microcentrifuge tube, and mix gently. Incubate for 5 min at room temperature.
   4. Combine the diluted plasmid mixture with diluted Reagent 1 (total volume = 215 µL). Mix gently and incubate for 20 min at room temperature.
   5. Aspirate medium off cells-cultured plate, and add 1.5 mL fresh DMEM without FBS.
   6. Add 215 µL of DNA-transfection reagent mixture to each well dropwise, and mix gently by swirling the plate.
   7. Incubate at 37 °C and 5% CO₂ for 4 - 8 h. Replace with fresh DMEM with 10% FBS.
   8. Incubate cells at 37 °C and 5% CO₂ for another 24 - 48 h before running any experiments.
2. Transfection method 2, with liposome formulations
   NOTE: Please see the Table of Materials for transfection reagent 2.
   1. Add 2 µg of plasmid DNA (500 ng/µL), 4 µL of Reagent 2, and 100 µL reduced serum media into a sterile microcentrifuge tube. Mix gently and incubate for 15 min at room temperature.
   2. Add the transfection mixture to the cells in a dropwise manner.
   3. After transfection, incubate cells for 24 - 48 h at 37 °C and 5% CO₂, before running any experiments.

4. Cell Culture on Cover Slips

1. Remove the growth medium from the culture 6-well plate.
2. Wash the cell monolayer with 1 mL of PBS without calcium and magnesium.
3. Add 0.5 mL Trypsin-EDTA. Swirl the dish carefully and aspirate off Trypsin-EDTA. Incubate the culture plate for 3 min at 37 °C and 5% CO₂. The actual incubation time varies with the cell line used. 
   NOTE: During this period, prepare a new 12-well plate and add 15 mm diameter sterile cover slips to each well. Cover slips coated with lysine, laminin, or collagen may improve cell attachment for HEK293 cells that easily detach.
4. When cells are detached, add 1 mL pre-warmed complete growth medium to each well. Mix by pipetting and immediately transfer  the required number of cells to the 12-well plate with cover slips. Add fresh complete growth medium to each  well to adjust the total volume to 1 mL
5. Incubate the cells for 16 - 24 h at 37 °C and 5% CO₂.
   NOTE: After 30 - 60 min, the majority of cells will have adhered to the sterile cover slips.

5. Confocal Microscopy Detection

1. Cellular localization
   1. After 16 - 24 h, when the cells have grown to near confluence on the sterile cover slips in the 12-well plate, add the membrane probe, DiI, to each well, giving a final concentration of 5 µM. Allow the cells to incubate for 30 min at 37 °C and 5% CO₂.
   2. After 30 min, remove the equilibrated growth medium and wash the cells twice in cold PBS (2 - 8 °C).
   3. Fix the cells by adding 1 mL of a 4% paraformaldehyde solution (in PBS) to each well. Gently agitate for 15 min.
      Caution: Paraformaldehyde is moderately toxic by skin contact or inhalation, and designated as a probable human carcinogen. Chemicals fume hoods, vented balance enclosures or other protective measures should be used during the weighing and handling of paraformaldehyde.
   4. Wash the fixed cells twice in PBS. Add 200-300 µL of DAPI (4',6-diamidino-2-phenylindole) solution (0.5 µg/mL) to each well and incubate for 10 min at room temperature.
   5. Wash the cells twice with PBS. Carefully remove the cover slips from the plate and wick off excess PBS. Invert onto microscope slides loaded with one drop of mounting medium (50% v/v glycerol/water). Remove excess mounting medium from the slides.
2. Cellular internalization
   1. After 16 - 24 h, when the cells have grown to near confluence on the sterile cover slips in the 12-well plate, remove the equilibrated growth medium and add fresh, warmed DMEM medium (37 °C). Incubate the cells for 1 h at 37 °C and 5% CO₂.
   2. Treat the cells with different concentrations (0, 10^-12, 10^-9, and 10^-6 M) of ligand for 30 min and treat them with 10^-6 M ligand for different times (0, 10, 30, and 60 min).
   3. Wash the cells twice in cold PBS (2 - 8 °C). Fix the cells by adding 1 mL of a 4% paraformaldehyde solution (in PBS) to each well. Gently agitate for 15 min.
   4. Wash the cells twice in PBS. Carefully remove the cover slips from the plate and wick off the excess PBS. Invert onto microscope slides loaded with one drop of mounting medium (50% v/v glycerol/water). Remove excess mounting medium from the slides.
3. Confocal microscopy
   1. Turn on the hardware of confocal microscope, including the mercury lamp power, PC microscope, scanner, and laser. Login to operating system account, launch the software, check the configuration and select laser. Turn on the different units of confocal microscopy system in the order according to instructions.
   2. Place the prepared slides on the microscope stage. Position the sample over the objective lens using the stage controller. Add one drop of cedar oil onto coverslip when the oil immersion lens is selected. Place the cover slips on slides facing the lens.
   3. Find the cells of interest under the fluorescent microscopy.
   4. Switch to scan mode. Choose the laser power and the spectral range of the emission fluorophore. Capture the high quality confocal images by tuning laser intensity and other parameters.

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