ETAR Case study

ETAR internalization assay development using QMCF/U2OS cell line 

Case study

Background

G protein coupled receptor family member endothelin receptor subtype A (ETAR) is expressed in vascular tissues and involved in regulation of blood pressure. Binding of its ligand Endothelin-1 (ET-1) to ETAR induces blood pressure elevation targeting ETAR and its signal pathway for a hypertension therapy development (1). There is also evidence indicating to involvement of ETAR induced proliferation and inhibition of apoptosis in cancer development processes. ETAR is easily traceable cell based assay target whereas ET-1 binding to ETAR induces receptor internalization into perinuclear area localizing recycling endosomes (2).

We have expressed human endothelin receptor subtype A (NM_001957.2) in U2OS based QMCF cell line and shown ligand induced receptor internalization.

References

  1. Schiffin EL (1995): Endothelin: potential role in hypertension and vascular hypertrophy. Hypertension, 1995 Jun; 25(6): 1135-43
  2. Bagnato A, Natali PG (2004): Endothelin receptors as novel targets in tumor therapy. Journal of Translational Medicine, 2 (1), 16

ETAR expression vector construction 

ETAR protein used for this assay is C-terminally fused with an epitope tag sequence derived from BPV type 1 E2 protein (aa sequence: SSTSSDFRDR) that can be detected by anti-5E11 tag mouse monoclonal antibody (Icosagen Cat No A1-100-100) and visualized by fluorescent label conjugated anti-mouse antibody. Several different configurations of expression cassettes were tested to find out optimal expression conditions in order to achieve maximal level of target positive cells and homogenous cell culture. Basic QMCF vector structure was used (Figure 1). 
 

Figure 1. QMCF expression vector structure.
Maintenance sequence – Epstein-Barr Virus Family of Repeats (FR); PyV core origin – murine polyomavirus origin of replication; SV40 pr – SV40 early promoter controlling expression of Neo resistance gene; Neo/Km – Neomycin/Kanamycin resistance marker; PolyA – polyadenylation sequence;  

Expression cassette – various expression cassette configurations, as listed below, were used:
    • pQMCF-1-ETAR-5E11_C#19: CMV-TK leader– hEF1α 5´intron – ETAR-5E11 – SV40 polyA
    • pFRG-ETAR-5E11_C 2#38: CMV-TK leader–ETAR-5E11 – rabbit β-globin 3´intron – TK polyA 
    • pQMCF-2-ETAR-5E11_C#22: hEF1α-HTLV leader – hEF1α 5´intron – ETAR-5E11 – SV40 polyA
    • pFR-2-ETAR-5E11_C#23: hEF1α-HTLV leader–ETAR-5E11 – rabbit β-globin 3´intron – bgh polyA
    • pQMCF-6-ETAR-5E11_C#63: hEF1α – hEF1α 5´intron – ETAR-5E11 – SV40 polyA 
    • pQMCF-5-ETAR-5E11_C#51: RSV-LTR – hEF1α 5´intron – ETAR-5E11 – SV40 polyA


Expression characterization

ETAR expression was analyzed by indirect immunofluorescence throughout continuous cultivation. Analysis to determine the percentage of cells expressing ETAR at detectable level (Figure 2) and the heterogeneity within populations ETAR expression (Figure 3) in case of different QMCF expression vector configurations was performed using different QMCF expression vector configurations.



Figure 2. Percentage of ETAR-5E11 expressing QMCF/U2OS cells during continuous cultivation under G418 selection (detected by indirect immunofluorescence)


ABC



DEF



Figure 3. Immunofluorescence analysis of ETAR-5E11 expression in the membrane of U2OSEBNALTC3 cell line. Images acquired after selection of stable ETAR-expressing population (3 weeks continuously cultured populations). 

A: pQMCF-6-ETAR-5E11_C#63; B: pQMCF-2-ETAR-5E11_C#22; C:pFR-2-ETAR-5E11_C#23; D: pQMCF-1-ETAR-5E11_C#19; E: pFRG-ETAR-5E11_C 2#38; F: pQMCF-5-ETAR-5E11_C#51


Expression cell bank characterization

3 weeks continuously under G418 selection cultivated pQMCF-6-ETAR-5E11_C#63 and pQMCF-2-ETAR-5E11_C#22 transfected QMCF/U2OS cells were cryopreserved for 4 months. The percentage of ETAR expressing cells in QMCF U2OS culture was determined by indirect immunofluorescence after 3 weeks of cultivation and from the resuscitated culture after 3 days of cultivation (Figure 4). 



Figure 4. Percentage of ETAR-5E11 expressing QMCF U2OS cells 48h after transfection, after 3 weeks continuous cultivation under G418 selection (before cryopreservation) and after cryopreservation.



Assay development 

ETAR localizes into plasma membrane in un-stimulated cells and is internalized into recycling endosomes upon ET-1 stimulation. Internalization can be quantified as an accumulation of fluorescent aggregates within perinuclear region and a decrease of ETAR specific signal within the plasma membrane region.

ETAR expressing QMCF/U2OS cell line was used for ETAR internalization assay development. 3.5x10^6 adherent QMCF/U2OS cells were transfected by electroporation with 800 ng ETAR expression vector pQMCF-6-ETAR-5E11_C#63. Cells were grown in DMEM supplemented with 10% FBS. 48 h after the transfection G418 at the final concentration 300 µg/ml, was added to the culture media. Subsequent cultivation was performed in the presence of G418. Expression cell bank was generated after 23 days of cultivation under G418 selection. ETAR expression was monitored by IF 48h after transfection, prior to expression cell bank generation following to culture expansion under G418 selection, and after resuscitation of stored expression bank.


Figure 2. Percentage of ETAR-5E11 expressing QMCF/U2OS cells 48h after transfection and before expression cell bank (ECB) generation (after 23 days of continuous cultivation under G418 selection). Detected by indirect immunofluorescence.


ETAR expressing QMCF U2OS cells were inoculated from cryo-preserved expression cell bank into DMEM supplemented with 10% FBS. 24h after resuscitation culture media was exchanged to fresh media containing G418 at the final concentration 300 µg/ml. One day prior to internalization induction 1.5x10^4 cells were inoculated per well onto 96-well plate. The dose-response curve was generated by incubating of ETAR expressing QMCF/U2OS cells in the presence of serially diluted ET-1 (0-100 nM). 3 h after incubation with the ligand, cells were fixed with 4% paraformaldehyde. C-terminally 5E11 epitope tag fused ETAR was visualized by IF using anti-5E11 tag mouse monoclonal antibody (Icosagen Cat No A1-100-100) and DyLight488 conjugated anti-mouse antibody (Thermo Scientific, 35502), the nuclei was stained with DAPI. The images were acquired on the Thermo Scientific ArrayScan VTI HCS Reader with 20x objective (Figure 6).

A: 0 nM ET-1B: 10 nM ET-1C: 15 nM ET-1



Figure 6. 20x objective was used for image acquisition. A false colour overlay of nuclear (blue), and ETAR (green) fluorescence is shown. ET-1 concentration dependent ETAR internalization can be observed as an accumulation of fluorescent aggregates within perinuclear region and a decrease of ETAR specific signal within the plasma membrane region.


For image analysis Cellomics software was used. An expanded nuclear region was monitored for detection internalized ETAR within the endosomes (Figure 7).


Figure 7. ETAR internalization in QMCF/U2OS cells monitored upon ligand ET-1 stimulation. The graph shows ET-1 dependent response curve resulting in an EC50 of 20 nM for QMCF U2OS-ETAR cells.