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Experimental Biology and Medicine 231:746-751 (2006)
© 2006 Society for Experimental Biology and Medicine

BASIC BIOLOGY

Differential Trafficking and Desensitization of Human ETA and ETB Receptors Expressed in HEK 293 Cells

Xiaoling Dai1 and James J. Galligan

Department of Pharmacology and Toxicology and the Neuroscience Program, Michigan State University, East Lansing, MI 48824

1To whom requests for reprints should be addressed at Department of Pharmacology and Toxicology, Life Science Building Room B308, Michigan State University, East Lansing, MI 48824. E-mail: daixiaol{at}msu.edu

Abstract

Endothelin-1 (ET-1) is a vasoconstrictor peptide that acts on ETA and ETB receptors on smooth muscle cells (SMCs). Because vascular SMCs can express both receptors, it is difficult to study the localization and properties of each subtype. Therefore, we investigated the localization and function of ETA and ETB receptors transfected into HEK 293 cells. Immunocytochemistry was used to examine colocalization of ET receptors with the plasma membrane marker, pan cadherin. In cells transfected with ETA receptors, 83 ± 2% of these receptors colocalized with pan cadherin. In ETB receptor–transfected cells, 54 ± 2% of the receptor colocalized with pan cadherin. When ETA and ETB receptors were cotransfected, 97 ± 1% of ETB receptors colocalized with ETA receptors and 84 ± 2% of ETB receptors colocalized with pan cadherin. ET-1 and sarafotoxin 6c (S6c, ETB receptor agonist) increased [Ca2+]i in cells transfected with ETA or ETB receptors; 100 nM of ET-1 and S6c caused maximal responses. When stimulated with ET-1, ETB receptors desensitized faster (t1/2 = 21 ± 1 sec) than ETA receptors (t1/2 = 48 ± 1 sec). S6c-induced increases in [Ca2+]i desensitized in cells expressing ETB receptors only (t1/2 = 17 ± 1 s). Desensitization was eliminated in cells cotransfected with ET receptors. We conclude that ETA receptors localize to the cell membrane, whereas ETB receptors are in the membrane and intracellular compartments. Coexpressed ET receptors are in the membrane. ETB receptors desensitize faster than ETA receptors, but receptor coexpression eliminates desensitization. Finally, ETA and ETB receptors interact to change receptor trafficking which may modify ET receptor function in vascular SMCs coexpressing these receptors.

Key Words: desensitization • endothelin receptor • trafficking

Introduction

The endothelins (ETs) are a family of 21 amino acid peptides generated primarily by endothelial cells to produce contraction of vascular smooth muscle cells (SMCs). ET-1 plays a critical role in blood pressure maintenance and has been implicated in multiple cardiovascular functions and diseases, including hypertension and heart failure (1, 2). ET-1 levels are elevated in some hypertensive patients, especially in blacks, and the vasoconstrictive effects of ET-1 suggest that it may contribute to hypertension in these patients (3).

ET-1 binds to two receptor subtypes, ETA and ETB receptors, which are G-protein–coupled receptors (GPCR), to cause vasoconstriction (4). Messenger RNA and protein for ET receptors are found in SMCs (5). ET receptors have different recycling pathways after ligand stimulation. After internalization via caveolae or clathrin-coated pits, the ETA receptor is recycled back to the cell surface (6, 7). In contrast, the ETB receptor is internalized via a clathrin-dependent pathway and transported to late endosomes and lysosomes (6–9).

Although the dimerization of ETA and ETB receptor has never been proven, some experimental data fit well with the now widely accepted view that GPCRs can exist as heterodimers (10–13). Atypical ligand binding has been observed in cells coexpressing ETA and ETB receptors. In these studies, the ETB receptor-selective ligand, sarafotoxin 6c (S6c), inhibits 125I-ET-1 binding only in the presence of the ETA receptor–selective antagonist, BQ123 in epithelial cells of the anterior pituitary (14). In astrocytes, ETA and ETB receptors cooperatively control ET-1 clearance, because only the combination of ETA and ETB receptor-selective antagonists, but not their individual application, increases ET-1 in the extracellular fluid (15).

Because vascular SMCs express both ET receptor subtypes, it is difficult to study the subcellular localization and functional properties of each subtype or interactions between receptors in SMCs. Therefore, we investigated the localization and function of ETA or ETB receptors transfected into HEK 293 cells. In these studies we attempted to determine the function and localization of ETA and ETB receptors when they are expressed alone and the changes in localization and function when ET receptors are coexpressed.

Materials and Methods

Cell Culture.
HEK 293 cells were obtained from American Type Culture Collection and maintained in advanced DMEM/F-12 medium supplemented with 10% fetal bovine serum and GluMax (Invitrogen, Carlsbad, CA) at 37°C in a 95% humidified air with 5% CO2 incubator. Cells were passaged once every 3 days when they reached 90% confluence.

Transient and Stable Expression of ET Receptors.
Plasmids (pcDNA3.1+, Invitrogen) containing the coding sequences of human ET receptors and human ET receptors with an N-terminal hemoagglutininepitope tag were purchased from University of Missouri, Rolla cDNA Resource Center (www.cdna.org). Cells growing on 35-mm Petri dishes at 90% confluency were transfected with 2.5 µg of plasmid DNA using 5 µl of the Lipofectamine transfection reagent (Invitrogen) following the manufacturer’s guidelines.

Immunocytochemistry.
Tissues were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.0 mM Na2HPO4, pH 7.4) for 20 mins at room temperature. Cells were blocked and permeabilized with 5% goat serum and 0.1% Triton X-100 in PBS and incubated with primary antibodies. Antibodies for the ETA receptor and ETB receptor were purchased from Alomone (Jerusalem, Israel) and the antibody for pan cadherin was obtained from Abcam (Cambridge, MA). Primary antibodies in PBS with 5% serum were applied and incubated at 37°C for 2 hr. Cells were rinsed in PBS and incubated with secondary antibodies conjugated with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (AMCA), fluorescein isothiocyanate (FITC), or Cy-3 (Jackson ImmunoResearch, West Grove, PA) in PBS for 1 hr. Cells then were rinsed in PBS, mounted on slides, and observed with a Nikon Eclipse fluorescence microscope. Images were captured using a Spot CCD camera (Sterling Heights, MI) at a resolution of 1024 x 1024 pixels as 8-bit color images. The colocalization of ET receptors with pan cadherin was analyzed using the image analysis software Metamorph software (Universal Imaging Corp., Downingtown, PA).

Measurement of Intracellular Calcium.
HEK cells transfected with ET receptors on coverslips were loaded with 2 mM Fluo 4/AM and Pluronic acid (1%) (Molecular Probes, Eugene, OR) in Opti-MEM medium (Invitrogen) for 40 mins at 37°C and placed in a chamber mounted on the stage of a Leica confocal microscope. Cells were flushed continuously with HEPES-Ringer solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES; pH 7.3) with or without drugs. The excitation and wavelength was 488 nm and a 500-nm long pass filter was used. Images were captured every 6 secs. Five baseline images were obtained and images were collected for 20 min during and after drug treatment. Changes in [Ca2+]i in cells expressing ETA or ETB receptors were represented by changes in fluorescence intensity in individual cells after agonist stimulation. Fluorescence intensity was quantitated using Leica confocal software. Cells were treated with ET-1 or S6c with or without pretreatment with ET receptor antagonists for 5 mins.

To obtain a concentration response relationship, cells transfected with ETA receptors were treated with ET-1 (1, 10, or 100 nM) for 2 mins. Other cells were treated with 10 nM ET-1 for 2 min after pretreatment with the ETA receptor antagonist BQ610 (100 nM) for 5 mins. Another group of cells transfected with ETB receptors was treated with 10 or 100 nM of S6c for 2 mins or with 10 nM ET-1 for 2 mins after pretreatment the ETB receptor antagonist BQ788 (100 nM) for 5 mins. Desensitization was studied in cells transfected with ETA or ETB receptors and treated with ET-1 or S6c (100 nM) for up to 20 mins. Additionally, blank HEK-293 cells treated with ionomycin (0.1 µM), a calcium ionophore, for up to 20 mins, were used as a control for fluorescence quenching or loss of Fluo-4 from treated cells.

Data Analysis and Statistics.
For each type of experiment, data were obtained from six to eight dishes of cells. Data are presented as mean ± SEM. When comparing two groups, Student’s t test was used. For multiple comparisons, an analysis of variance followed by the Bonferroni post hoc test was used. In all cases, a P value ≤ 0.05 was considered significant.

Results

ETA and ETB Receptors Have a Different Localization in Transfected HEK-293 Cells.
ETA and ETB receptors were localized using immunocytochemical techniques. It was found that immunoreactivity (ir) for ETA or ETB receptors was present in about 25% of cells, indicating a low transfection efficiency. However, pan cadherin ir (a plasma membrane marker) was detected in all cells (Fig. 1Go). It was also found that ETA and ETB receptors were colocalized with pan-cadherin in some cells (Fig. 1Go). In ETA receptor–transfected cells, 83 ± 2% of the receptors were colocalized with pan-cadherin. In cells transfected with ETB receptors, 54 ± 2% of the receptor was co-localized with pan-cadherin. These data indicate that ETA receptors were mainly localized on the cell membrane, whereas ETB receptors were equally distributed between the cell membrane and intracellular compartments.


Figure 1
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  		Figure 1. Immunocytochemistry for the ETA or ETB receptor costained with the cell membrane marker, pan cadherin. ETA (left) and ETB (right) receptors were transiently transfected into HEK 293 cells. ETA receptors are localized on the cell membrane, whereas ETB receptors are in the cell membrane and intracellular compartments. Most ETA receptors (green) are colocalized with pan-cadherin (red); ETB receptors (green) are colocalized with pan cadherin intracellularly.

 
The Distribution of ETB Receptors Changes When Coexpressed with ETA Receptors.
Because ET receptor antibodies are raised in the same host, cells cotransfected with ETA receptor and ETB-HA were used to examine colocalization using antibodies against the ETA receptor and against HA. When ETA and ETB receptors were transiently cotransfected, 97 ± 1% of ETB receptors colocalized with ETA receptors and 84 ± 2% of ETB receptors colocalized with pan-cadherin (Fig. 2Go).


Figure 2
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  		Figure 2. Immunocytochemical localization of ETA, and ETB receptors. The wild-type ETA receptor was colocalized with pancadherin and HA-tagged ETB receptors. Cells were costained with the cell membrane marker pan cadherin (red), ETA receptors (green), and hemoagglutinin (blue). Cells coexpressing ETA and ETB receptors are colocalized in the same compartment and may form a dimer.

 
ETA and ETB Receptors Transfected into HEK 293 Cells Are Functional.
ET-1 was used to stimulate cells transfected with ETA receptors; S6c was used to stimulate cells transfected with ETB receptors. Both treatments caused an increase in [Ca2+]i in a concentration-dependent manner. The fluorescence intensity for increases in [Ca2+]i for ETA receptor cells treated with 1, 10, or 100 nM ET-1 was 112 ± 6, 161 ± 5, and 158 ± 4 arbitrary fluorescence units, respectively. Fluorescence intensity for [Ca2+]i for ETB receptor cells treated with 10 or 100 nM ET-1 was 76 ± 3 and 104 ± 9 arbitrary fluorescence units, respectively. S6c did not increase [Ca2+]i in cells transfected with ETA receptors only. Cells transfected with ET receptors without or with a hemoagglutinin-epitope tag (ETA-HA or ETB-HA) were also used in this study. No difference in agonist-induced changes in [Ca2+]i was observed between cells transfected with wild-type ET receptors or hemoagglutinin-tagged ET receptors (data not shown). Changes in [Ca2+]i in cells transfected with ETA or ETB receptors were blocked by pretreatment with BQ610 or BQ788 (100 nM), antagonists of ETA and ETB receptors, respectively.

ETA Receptors Desensitize More Slowly Than ETB Receptors.
A maximum concentration of ET-1 and S6c (100 nM) was used in desensitization studies, in which cells were treated with agonists for 20 mins (Fig. 3AGo). When stimulated with ET-1, ETB receptors desensitized faster (t1/2 = 21 ± 1 sec) than ETA receptors (t1/2 = 48 ± 1 sec). S6c-induced increases in [Ca2+]i desensitized in cells expressing ETB receptors only (t1/2 = 17 ± 1 sec). There was no difference between cells transfected with wild-type or HA-tagged receptors (Table 1Go). To rule out variations coming from the decay of calcium indicator fluo-4/AM, control experiments were also done using the calcium ionophore, ionomycin, which caused a stable increase in the calcium signal for up to 20 mins (data not shown). The desensitization half-time (t1/2) in cells cotransfected with ETA and ETB receptors in response to either ET-1 or S6c activation was greater than 400 sec (Fig. 3BGo).


Figure 3
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  		Figure 3. Agonist-induced increases in [Ca2+]i in HEK-293 cells transiently transfected with ETA or ETB receptors. Maximal agonist concentrations (100 nM of ET-1 or S6c) were used in the desensitization study. A. When stimulated with ET-1, ETB receptors desensitized faster than ETA receptors. ET-1 and S6c-induced increases in [Ca2+]i desensitized in cells expressing ETB receptors only. B. In cells coexpressing ETA and ETB receptors the agonist induced desensitization t1/2 was >400 sec.

 

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  		Table 1. The t1/2 (sec) in Cells Transfected With ET Receptors or ET Receptors Tagged with Hemoagglutinin (HA)a
 
Discussion

We expressed human ETA or ETB receptors with or without a HA-epitope tag in HEK-293 cells and examined their cellular localization and function. We found that the ETA receptor was mainly localized on the cell membrane, whereas ETB receptors were not only localized on the cell membrane, but also in intracellular compartments. Our results also provide the first evidence that the distribution of ETB receptors changes when they are coexpressed with ETA receptors. We also show that ETA receptors desensitized more slowly than ETB receptors and that ET receptor desensitization is eliminated in cells coexpressing ETA and ETB receptors.

ET Receptor Localization.
ETB receptors have a different subcellular distribution than ETA receptors. We localized ET receptors in HEK-293 cells by costaining the receptors with pan cadherin, a cell membrane marker. We found that ETA receptors were found predominately in the cell membrane, whereas ETB receptors were distributed evenly between the cell membrane and intracellular compartments. Others have made similar observations in different cell lines transfected with green fluorescent protein-tagged ET receptors (6). These investigators found that, under steady-state conditions, ETA receptors are localized to the plasma membrane, whereas ETB receptors were localized to the cell membrane and to lysosomes where presumably they had been targeted for degradation. ETA and ETB receptors may have different subcellular localization because they are coupled to different signal transduction pathways in cell lines (16) and in isolated smooth muscle cells (17).

ET Receptor Function.
ETA and ETB receptors contribute to vasoconstriction of vascular SMCs by coupling to an increase in [Ca2+]i (18. 19). However, it is difficult to identify the contribution of each receptor in SMCs that constitutively coexpress ETA and ETB receptors. Studies using cell lines transfected with individual receptors provide an alternative approach for investigation of the function of individual receptors. We are the first to show that ETA and ETB receptors have different functional profiles when agonist-induced increases in [Ca2+]i are measured in cells transfected with ETA or ETB receptors alone. ETA receptors desensitized more slowly than ETB receptors. The differences in desensitization of ETA and ETB receptors are not agonist-dependent because ETB receptor cells desensitize similarly when activated with either ET-1 or S6c stimulation. We also show that there was little or no desensitization in cells coexpressing ETA and ETB receptors. ET receptors belong to GPCRs, in which the intracellular carboxy terminus of receptors contains sites for phosphorylation, and are used in the regulation of the receptor in internalization and desensitization (20). Coexpression of receptors may mask these phosphorylation sites and therefore impair receptor desensitization.

Receptor Dimerization.
Several lines of evidence have suggested that different GPCRs can form heterodimers; receptors exhibiting this behavior include angiotensin AT1 receptor-bradykinin B2 receptor and ß2-adrenergic-{alpha}2 adrenergic receptor (21, 22). These studies showed that the activation of one receptor modulates the ligand binding, receptor trafficking, and intracellular signaling of the other receptor. This interaction can come about through a physical interaction such as occurs with GABAB1 and GABAB2 receptors. Fluorescence resonance energy transfer (FRET) analysis has revealed the potential for similar heterodimerization of ET receptors (23). In the present study, we showed that ETA and ETB receptors are closely colocalized in the membrane of cells coexpressing these receptors. Although we did not demonstrate a direct physical coupling of the two receptors, our data are consistent with heterodimer formation. Heterodimer formation is also supported by our functional studies as desensitization of ETA and ETB receptors was eliminated in cells coexpressing ETA and ETB receptors.

The molecular basis for ET receptor heterodimerization is unclear, but mounting evidence suggests that the dimerization of GPCRs is important for normal functioning of receptors. Heterodimerization of GPCRs is important during receptor synthesis and trafficking because dimerization masks specific retention signals that would otherwise keep the receptors in the endoplasmic reticulum, as shown for GABABRs (24). In some cases, heterodimerization between closely related subtypes is essential for the formation of functional receptors, such as GABABRs (24) and sweet (25) and L-amino acid (26) taste receptors. Another possibility is that the formation of an ETA/ETB dimer may occur in the presence of ET-1 as a bivalent ligand connecting two receptors (14, 27). However, our data show that in cells coexpressing ETA and ETB receptors the receptors are colocalized in the same subcellular compartments, suggesting that dimmer formation can occur in the absence of ET-1 binding (Fig. 3Go). ETA receptors have the motif for an internal PDZ (postsynaptic density-95/disc-large/zona occludens) domain that contributes to receptor recycling (28). PDZ domains are ubiquitous protein-protein interaction domains comprising 70–90 residues and the most prominent role of PDZ-containing proteins appears to be the assembly of protein complexes at the plasma membrane, where they bind to the C-termini of membrane proteins (29).

Significance of ET Receptor Dimerization: Implication in Arterial and Venous Differences.
ETA and ETB receptors influence contractility in mesenteric arteries and veins differently. ETA receptors are the predominant receptor mediating arterial contraction, whereas ETA and ETB receptors contribute to venous contraction. ET-1 elicits arterial contraction while the ETB receptor agonist, S6c, is inactive in arteries, whereas ET-1 and S6c cause venous constriction in veins (30–33). Veins are also more sensitive to the constrictor effects of ET-1 compared to arteries (34–36). In rat and murine mesenteric blood vessels, the ETB receptor antagonist BQ788 blocks S6c-induced venous contraction. However, ET-1–induced venous contraction is reduced by the ETA receptor antagonist BQ610, but not by BQ788, whereas the mixture of ETA and ETB receptor agonists further decreases venous contraction responses to ET-1 (36, 37). We speculate that the distinct arterial and venous responses to ET-1 may be due to the presence of the heterodimerization of ET receptors in veins, but not in arteries.

Our data supported that ET receptors transfected into HEK cells exist different trafficking and desensitization characteristics, which may or may not be different from the function of ET receptors in primary SMCs. Further studies of ET receptor trafficking and function need to be confirmed in vascular SMCs.

Footnotes

The study was funded by the National Institutes of Health grant P01HL70687 to J.J.G.

Received for publication September 26, 2005. Accepted for publication November 14, 2005.

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