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Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229
Address all correspondence and requests for reprints to: Dr. David Puett, Department of Biochemistry and Molecular Biology, Life Sciences Building, University of Georgia, Athens, Georgia 30602-7229. E-mail: puett{at}bchiris.bmb.uga.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit of the G protein heterotrimer. This is followed by
dissociation of the G protein from the receptor and subsequent
dissociation to GTP and ß
; these components, in turn, interact
with regulatory enzymes and ion channels (2, 3). Endothelin-1 (ET-1), a 21-amino acid residue peptide with mitogenic activity to smooth muscle cells, is the most potent vasoconstrictor known and is capable of eliciting long lived responses (4). It exerts profound effects in the cardiovascular and central nervous systems (5) and is important developmentally (6, 7). These effects are mediated via two GPCRs, endothelin receptor subtypes A and B (ETAR and ETBR, respectively), that are coupled primarily to Gq and thus lead to the stimulation of phospholipase Cß, resulting in the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (5). ETAR, like a number of GPCRs associated with mitogenic signal transduction, has also been found to signal through the Ras/Raf activation pathway by way of receptor tyrosine phosphorylation (8, 9).
In addition to ligand dissociation and receptor desensitization mediated by phosphorylation, internalization of the receptor-bound ligand is a common method of signal termination for most peptide, polypeptide, and glycoprotein hormone ligands (10, 11). This pathway generally results in degradation of ligand and often of receptor as well, although in some instances the receptor is recycled (12). Therefore, internalization/degradation may be considered a protective mechanism to prevent a cell from becoming overstimulated. Internalization of GPCRs has been shown to occur via clathrin-coated pits (10) and perhaps caveolae as well for ETAR (13). It has been recently suggested that the ET-1-ETAR complex may continue to transduce a signal even after it has internalized, thereby accounting for the prolonged smooth muscle contraction characteristic of ET-1 (14). This hypothesis is supported by the detection of intact ET-1 in the intracellular fraction of Chinese hamster ovary (CHO) cells expressing ETAR (14) and in cellular lysates of smooth muscle A-10 cells (15) and neuroblastoma-glioma hybrid NG108–15 (16) up to 2 h after ET-1 binding. Therefore, receptor internalization may serve as a mechanism to terminate cell surface signaling as well as to potentially prolong the intracellular signal in some cases. In either event, these observations suggest that the receptor needs to be activated and capable of transducing a signal before it can be internalized. However, this requirement has not been directly tested with ETAR.
There is no consensus on the requirement of receptor signaling for the
internalization of GPCRs. A few studies with other
Gq-activating GPCRs, such as the m2 muscarinic
acetylcholine and TRH receptors, suggest the dependence upon
phospholipase C activation for endocytosis (17, 18). In contrast,
studies on the 
-opioid and ß2-adrenergic receptors
have shown independence of Gs-mediated adenylate cyclase
activation from endocytosis (19, 20). However, studies performed with
the LH receptor (LHR) suggest that receptor activation, but not
necessarily adenylate cyclase activation, is needed for efficient
internalization of the hormone-receptor complex (21, 22).
In this study we investigated the internalization of the ET-1-ETAR complex and the role, if any, of IP3 signaling. Our experimental approach involved two systems, both using human embryonic kidney (HEK) 293 cells expressing ETAR; one uses a combined concanavalin A (Con A)-sucrose gradient centrifugation technique that separates Con A-labeled plasma membranes from other cellular membranes, and the other is based on confocal microscopy with an antibody we raised to a synthetic peptide corresponding to the N-terminal region of ETAR. Using an ETAR chimera we developed, which binds ET-1 but does not signal, and an ETAR-selective antagonist, BQ123, internalization studies were performed with ET-1 and ETAR as a positive control. We conclude that internalization of the ET-1-ETAR complex does not require formation of IP3, and surprisingly, that the antagonist BQ123 also promotes ETAR endocytosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of chimeras and expression
The rat ETAR and LHR complementary DNAs (cDNAs),
provided by Drs. Shigetada Nakanishi (Institute of Immunology, Kyoto
University, Japan) and William Moyle (Robert Wood Johnson-Rutgers
Medical School, Piscataway, NJ), respectively, were cloned into the
pCDNA3 vector (Invitrogen Co., San Diego, CA). Silent mutations
incorporating restriction sites at the terminals of individual
extracellular, transmembrane, and intracellular domains were made by
PCR amplification using Pfu polymerase (Stratagene, La
Jolla, CA). All oligonucleotides were synthesized by Dr. Rudolf Werner
(University of Miami, Miami, FL). Each of the fragments was cloned into
the pCNTR vector (5-Prime 3-Prime, Boulder, CO), restriction digested,
and assembled in Bluescript SK- (Stratagene). The constructs were
verified by dideoxy chain termination sequencing (23) employing
Sequenase (U.S. Biochemical Corp., Cleveland, OH) and subsequently
cloned into pcDNA3 for transfection into COS-7 and HEK 293 cells.
Transient and stable transfections were performed using lipofectamine,
and the stable lines established in HEK 293 cells, according to
protocols from Life Technologies, were maintained in the presence of
400 µg/ml G418.
Binding assays
Specific surface binding of wild-type and individual chimeric
constructs as well as control mock transfections was determined 48
h after transient transfection of COS-7 cells by competitive binding
measurements, as described previously (24, 25, 26). Briefly, increasing
concentrations of unlabeled ET-1 were incubated in the presence of 50
pM [125I]ET-1 at 37 C for 2 h; excess
unlabeled hormone (1000-fold) was added to determine nonspecific
binding. The treated cells were solubilized with 1 N NaOH
and counted in a -counter.
Detergent-solubilized receptor binding of individual chimeric and
wild-type constructs transiently transfected in HEK 293 cells was
assayed using procedures similar to those described previously (27, 28). The cells were scraped and homogenized on ice in hypotonic buffer
(50 mM Tris-HCl, pH 7.4, and 10 mM EDTA) using
a Polytron (Ultra-Turax T125, Janke and Undel, Cincinnati, OH). The
homogenate was solubilized with 1.5%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and
20% glycerol; all undissolved material was discarded by centrifugation
at 1000 x g for 20 min. Total binding was assessed by
incubation of cell extracts with 100 nM
[125I]ET-1 for 18 h at 4 C in the presence or
absence of unlabeled ET-1. The extract was then passed through a
Whatman GF/B filter soaked in 0.3% polyethyleneimine (vol/vol) in 10
mM Tris-HCl (pH 9.1), followed by -counting of the
filters.
Saturation binding assays of mock-transfected cells and cells
expressing ETAR and ETARC (see Table 1
for designations) were performed as
described previously by adding increasing concentrations of
[125I]ET-1 (25). The results were analyzed by Prism
(GraphPad Software, San Diego, CA) and are shown as the mean ±
SEM.
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Internalization assay
The HEK 293 cell lines stably expressing ETAR and
the chimeric receptor, ETARC, were grown in 60-mm dishes
and pretreated with buffer A (HBSS containing 0.1% BSA, with or
without 0.1 mg/ml cycloheximide) at 37 C for 30 min. The
ETAR and ETARC cell lines were then incubated
with 0.1 nM [125I]ET-1 in buffer A with or
without 2 µM ET-1 for 1 h in an ice bath.
Subsequently, the cells were washed, and the dishes were incubated in
buffer A with or without 2 µM ET-1 for an additional 0,
5, 15, 30, 60, and 120 min at 37 C. Alternatively, 2 µM
ET-1 was added to buffer A at 37 C for the times indicated to determine
nonspecific binding.
Radioiodinated agonist-induced receptor endocytosis was analyzed by
separating a plasma membrane fraction and a total internal membrane
fraction with sucrose gradient centrifugation. An adapted protocol
based on Con A affinity to the plasma membrane of intact cells was
employed (29). The cells were placed on ice, washed, and incubated with
0.25 mg/ml biotin-conjugated Con A or Con A in isotonic buffer (1
mM Tris-HCl, pH 7.4, and 0.1 M NaCl) for 20
min. This was followed by two washes with the above hypotonic buffer,
mechanical removal, and homogenization of the cells. The homogenate was
layered onto a discontinuous sucrose cushion consisting of 1 ml 60%
and 8 ml 35% sucrose (wt/vol) and centrifuged at 100,000 x
g for 1 h in a Beckman SW-41 Ti rotor (Beckman, Palo
Alto, CA) to separate the internalized vesicles from the plasma
membrane fraction (30). One-milliliter fractions of the total 12 ml
were measured by detection of [125I]ET-1. The sum of
the radioactivity measured in fractions 1–12 represents the total
ligand bound at 0 C. The amounts of radioactivity measured in fractions
2–4 (0–35% sucrose interface) and 9–12 (35–60% sucrose interface)
were expressed as a percentage of the total ligand and represent the
fraction of internalized or cell surface-associated receptor,
respectively.
A microtiter plate assay for biotinylated Con A was performed using 0.1 ml from each centrifugation fraction. Streptavidin-horseradish peroxidase conjugate (1:5000 dilution; Amersham, Arlington Heights, IL) was used with a 3,3'5,5'-tetramethylbenzidine peroxidase substrate and a 0.18 N sulfuric acid stopping buffer following the protocol provided by Kirkegaard and Perry Laboratories (Gaithersburg, MD) to yield a colorimetric measurement, which was read at 450 nm.
Antibody generation and purification
The N-terminal 21-amino acid residue sequence of the rat
ETAR (DNPERYSTNLSNHVDDFTTFR) was synthesized on a multiple
antigen presentation resin (31) that was subsequently dissolved (200
µg/500 µl) in PBS and mixed with Freund’s complete adjuvant (1:1)
for injection into 18-week-old High Line W36 laying hens. This was
followed with a second injection of antigen plus Freund’s incomplete
adjuvant (1:1) after 2 weeks, and then a third injection of antigen
plus Freund’s complete adjuvant 2 weeks later (32). The eggs were
collected daily, and IgY was purified as recommended (personal
communication from Dr. Russell Malmberg, Department of Botany,
University of Georgia, Athens, GA). Briefly, 10 ml egg yolk were
diluted 4-fold in extraction buffer (10 mM Tris-HCl, pH
7.5, containing 0.1 M NaCl) for subsequent addition of
PEG-8000 at a final concentration of 35 mg/ml. The mixture was
centrifuged at 14,000 x g for 10 min, and the
supernatant was carefully filtered through a cotton-plugged funnel.
PEG-8000 (90 mg/ml) was then dissolved in the filtrate for a second
centrifugation at 14,000 x g for 10 min. The pellet
was resuspended in 10 ml extraction buffer for the addition of PEG-8000
(to 125 mg/ml) and centrifuged a third time at 14,000 x
g for 10 min. The purified IgY pellet was resuspended in 5
ml extraction buffer containing 0.02% (wt/vol) sodium azide for
antibody titer determination through horseradish peroxidase-based ELISA
and subsequent confocal microscopic analysis.
Confocal microscopy
For direct localization of Con A, stably transfected cells
expressing ETAR were grown on coverslips and treated with
0.2 mg/ml biotinylated Con A for 20 min on ice. The cells were then
washed with HBSS and fixed in 2% formalin. After washing with PBS,
cells were simultaneously permeabilized and blocked in PBS containing
0.2% saponin and 10% FBS for 10 min at 37 C. The cells were then
incubated with fluorescein isothiocyanate (FITC)-conjugated
streptavidin in permeabilizing block buffer for 30 min at 37 C and then
mounted and viewed with a Plan Apo x60 on a Nikon Diaphot microscope
(Nikon, Melville, NY) equipped with an argon/krypton confocal laser
(MRC-600, Bio-Rad Laboratories, Richmond, CA).
Confocal microscopy was also used to determine ligand-mediated internalization of ETAR and ETARC. HEK 293 cells stably expressing ETAR were preincubated with 0.1 µM ET-1 or 1 µM BQ123, and those expressing ETARC were incubated with 0.1 µM ET-1 for 1 h at 0 C in the presence of 0.1 mg/ml cycloheximide. The cells were subsequently washed with HBSS and incubated in ligand-free medium containing cycloheximide (0.1 mg/ml) at 37 C for 0, 5, 15, 30, and 120 min before washing and fixation as described above. Then the cells were blocked and permeabilized in PBS containing 10% goat serum and 0.2% saponin for 10 min at 37 C. Cells were labeled with chicken-anti rat ETAR antibody by incubation in fresh blocking-permeabilizing buffer for 4 h at 37 C; then the cells were washed three times and incubated with FITC-conjugated rabbit antichicken secondary antibody (Pierce Chemical Co., Rockford, IL) for 1 h at 37 C, followed by three more washes. The slides were mounted in PBS and visualized on the confocal microscope.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As LHR is a GPCR that signals primarily through the
Gs-adenylyl cyclase-cAMP-dependent protein kinase pathway,
we chose regions of this receptor for replacement of comparable regions
of ETAR. For convenience, ETAR was divided into
three sections: the N-terminal extracellular domain, the transmembrane
helixes and connecting loops, and the C-terminal cytoplasmic tail.
Although not every possible combination was made, a variety of chimeras
were investigated that a priori may be candidates for having
ET-1 binding potential. Of the five prepared, only one bound
[125I]ET-1 to the same extent as ETAR. This
ETAR chimera, designated ETARC, has the
C-terminal cytoplasmic tail of ETAR replaced with that of
LHR. The other chimeras having replacements of the N-terminal
extracellular domain of ETAR with the 341-amino acid
residue extracellular domain of LHR, i.e.
LHR1/ETAR23 and LHR13/ETAR2, showed no binding
of [125I]ET-1, but exhibited [125I]hCG
binding in detergent-solubilized cells (data not shown). The importance
of the ETAR transmembrane region to ET-1 binding was
supported by the poor binding observed with the ETAR13/LHR2
and ETAR1/LHR23 constructs. Detergent-solubilized extracts
of the COS-7 cells transiently transfected with cDNAs to wild-type
ETAR and the ETAR chimeras were also evaluated
for [125I]ET-1 binding, and only cells expressing
wild-type ETAR and ETARC exhibited significant
binding; that for ETAR13/LHR2 was less than 10% of
wild-type receptor binding, and the other three chimeras bound less
than 1% relative to ETAR (data not shown).
Saturation binding was performed on the transiently transfected COS-7
cells expressing ETAR and ETARC, and average
receptor densities of 0.4 x 104 and 1 x 10
4 receptors/cells were obtained, respectively (results not
shown). To ensure that receptor number difference would not be a factor
in data interpretation of IP3 signaling and
internalization, clonal lines of HEK 293 cells were selected that
expressed a similar number of wild-type and chimeric receptors per cell
(
6.6 x 104 receptors/cell). Also, from saturation
binding of [125I]ET-1 to intact cells, a Kd
of 0.1 nM was determined for each clonal line, while
mock-transfected HEK 293 cells showed only minimal
[125I]ET-1 binding (Fig. 1).
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). Both stable clonal and nonclonal cell
lines exhibited similar results; moreover, as expected, there was no
increase in cAMP in either wild-type or chimera-expressing cells in
response to ET-1 measured 30 min after the addition of ligand in the
presence of 5 mM isobutylmethylxanthine (data not shown).
As LHR contains no tyrosine phosphorylation sites in its cytoplasmic
tail, ETARC can be assumed to be uncoupled from the Ras/Raf
signaling pathway as well.
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), corresponding to membranes of high
density. Moreover, to confirm that Con A association was limited to the
plasma membrane, biotinylated Con A was localized to the cell surface
when ETAR-expressing cells were incubated with
streptavidin-FITC for direct visualization by confocal microscopy (Fig. 3A, inset). Lastly, this lectin did not inhibit
[125I]ET-1 binding to ETAR-expressing HEK 293
cells or displace bound [125I]ET-1 from the cells (data
not shown).
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for ETAR-expressing cells that had
been incubated with [125I]ET-1 at 0 C. The subsequent
incubation at 37 C in ligand-free medium at the zero time point shows
most of the radioactivity to be associated with the plasma membrane
fraction, whereas at 60 min most of the receptor-bound
[125I]ET-1 has been internalized.
Figure 4 illustrates one round of
ligand-mediated endocytosis at 37 C, depicting plasma
membrane-associated and intracellular membrane-associated
radioiodinated ligand as a percentage of the total ligand bound at 0 C.
Rapid [125I]ET-1 internalization was observed upon
incubation with the ETAR and ETARC clonal HEK
293 lines at 37 C (Fig. 4, A and B, respectively). The time at which
there was a half-maximal percentage of internalized ligand was taken as
t1/2, and the measured values were 5 and 15 min for
ETAR and ETARC, respectively. Preliminary
results show that incubation of cells with a protein kinase C
stimulator, the diacylglycerol analog, phorbol 12-myristate 13-acetate
(1 µM); a protein kinase C inhibitor, staurosporine (0.1
µM); and the phosphoinositol kinase inhibitor, wortmannin
(0.1 µM), had little or no effect on ETAR
internalization in the presence or absence of 0.1 µM ET-1
(data not shown). It should be noted that there was a background level
of, on the average, 10% of the total radioactivity not associated with
either vesicle fraction. This may result from shear forces associated
with centrifugation, thus dissociating free radiolabeled ligand that
would distribute throughout the sucrose gradient. To better quantify
and compare receptor compartmentalization kinetics, the first order
endocytic rate constant, ke, was determined
(34); these values were 0.1 and 0.03 min-1 for
ETAR and ETARC, respectively. Identical results
were obtained in the absence of cycloheximide (data not shown), which
was added in some experiments to minimize ETAR
biosynthesis.
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-amino group for radiolabeling, can lead to
internalization of ETAR. For these studies, ET-1 or BQ123
was incubated with cells for 1 h at low temperature, unbound
ligand was then removed, the cells were warmed to 37 C, and
internalization was monitored at various times between 0–120 min with
no additional ligand added. In the absence of ligand, receptors resided
predominantly on the cell surface, although in some cells hazy staining
could be observed within the cells, presumably reflecting constitutive
receptor turnover and receptor synthesis. Qualitatively, the use of
immunofluorescence to monitor internalization of ETAR and
ETARC gave results in accord with those obtained using
[125I]ET-1 and the Con A-based separation of plasma
membranes from other membranes via sucrose gradient centrifugation. For
example, upon warming the cells to 37 C, optical sections showed
cytoplasmic punctate staining in cells expressing ETAR and
ETARC by 5 and 30 min, respectively, which increased with
time (see Fig. 5 for
representative fields). After 120 min of incubation at 37 C, a general
decrease in labeling within the cell was noted, although complete
depletion of intracellular immunoreactive receptors was not observed.
Despite the intracellular staining distribution, the cell surface
remained labeled throughout the time course of the study; this was
attributed in large part to the use of nonsaturating concentrations of
ET-1 and perhaps to receptor recycling.
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, BQ123 promotes ETAR
internalization much like ET-1. Some cytoplasmic punctate staining was
apparent by 5 min at 37 C, and this is well defined at 15, 30, and 120
min. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The intracellular loops of GPCRs, including one formed by a conserved palmitoylation site on the cytoplasmic tail, have been suggested to be involved in G protein interaction (35). Our results support the proposal that the cytoplasmic tail can have an important role in the binding or activation of G proteins. This domain of LHR, like that of ETAR, contains serine/threonine phosphorylation sites and a palmitoylation site 18 amino acid residues from the plasma membrane interface; however, it lacks a tyrosine phosphorylation site. Interestingly, its substitution in ETARC resulted in diminished G protein-associated signaling and loss of the potential tyrosine phosphorylation site.
With the wild-type ETAR and its nonsignaling counterpart, ETARC, the role of signaling in ligand-mediated receptor internalization was examined. In studies such as this, it is important that ligand and receptor internalization be analyzed in concert. As the rate of internalization of ETAR and ETARC and their extended intracellular localization in the presence of ET-1, as visualized by confocal immunofluorescent microscopy, closely follow the internalization of [125I]ET-1 under identical conditions, our results demonstrate that the ligand internalizes and resides in the intracellular fraction for an extended period of time primarily complexed to its receptor. These results are consistent with previous reports by us and others that ET-1 remains intact for up to 2 h after internalization (14, 15, 16). Our results with [125I]ET-1 and ETAR also show that the initial cell surface-bound ligand is never completely depleted or, alternatively, that there is some recycling of the internalized ligand-receptor complex to the cell surface during the course of the experiments. Whether the long acting effect of ET-1 can be ascribed to a cell surface fraction of the receptor-bound ligand that has escaped desensitization (36) and/or internalization or, as has been suggested, to internalized receptor-bound ligand (14) cannot be determined from our results.
Recent studies indicate that normal kidney function is modulated by the binding of ET-1 to ETAR, which is abundant in mature renal tissue (37). Elevated levels of circulating ET-1 are measured in diabetics and patients with renal insufficiency (37, 38), and it has been suggested that BQ123 and other ETAR antagonists have beneficial effects (38). However, it is unclear how BQ123, with a 10-fold lower affinity to ETAR than ET-1 (25), is able to successfully overcome the localized high concentrations of ET-1 in these patients. Therefore, our observation in embryonic kidney cells that BQ123 promotes ETAR internalization with kinetics not substantially different qualitatively from those produced by ET-1 is of considerable surprise and potential clinical significance. We anticipate that the potent down-regulatory capabilities of BQ123 prevent ET-1 displacement of the antagonist from its receptor in kidney cells.
Our findings suggest that occupancy of ETAR by agonist (ET-1) or antagonist (BQ123) is sufficient to promote rapid endocytosis. Interestingly, antagonists of GnRH and cholecystokinin were also found to promote receptor internalization (39, 40). Presumably, a conformational change occurs in the ETAR receptor, but signaling is not required, as evidenced by the results with ET-1-ETARC and with BQ123-ETAR. This, in turn, implies that at least two conformational states or modified states, e.g. via phosphorylation, exist for the ligand-occupied receptor: one directs the complex for endocytosis, and the other leads to activation of Gq. The agonist ET-1 can promote both forms, presumably in a sequential fashion of signaling and then internalization; the antagonist BQ123 can promote only the latter. It is possible, of course, that the antagonist is internalized by a pathway different from that of ET-1. If this concept or some modification of it is correct, it will be interesting to determine at which step the GPCR kinases are functional and the nature of the form of ET-1 that is protected from degradation. In this vein, there is the possibility of two populations of the same receptor within a cell, one that internalizes rapidly concomitant with ligand binding and one that does not.
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Acknowledgments |
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Footnotes |
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Received December 12, 1997.
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References |
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opioid receptor. Role of the C terminus
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S. L. Ferrari, V. Behar, M. Chorev, M. Rosenblatt, and A. Bisello Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves beta -Arrestin2. REAL-TIME MONITORING BY FLUORESCENCE MICROSCOPY J. Biol. Chem., October 15, 1999; 274(42): 29968 - 29975. [Abstract] [Full Text] [PDF]
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 N. Bhowmick, P. Narayan, and D. Puett Identification of Ionizable Amino Acid Residues on the Extracellular Domain of the Lutropin Receptor Involved in Ligand Binding Endocrinology, October 1, 1999; 140(10): 4558 - 4563. [Abstract] [Full Text]
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R. Yu and P. M. Hinkle Signal Transduction and Hormone-dependent Internalization of the Thyrotropin-releasing Hormone Receptor in Cells Lacking Gq and G11 J. Biol. Chem., May 28, 1999; 274(22): 15745 - 15750. [Abstract] [Full Text] [PDF]
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) and cells expressing ETAR
(
) and ETARC (
). The number of receptors is
normalized to cell number and presented as the mean ±
SEM of two experiments, each performed in duplicate.
