From the
We demonstrate unusual features of the intracellular processing
of endothelin-1 (ET-1) and its receptor ET
Endothelin-1 (ET-1),
These diverse responses are mediated by
at least three distinct ET receptors which differ in their relative
binding affinities to the three ET isoforms, ET
Agonist-induced receptor desensitization is a common feature of
G-protein-coupled signal transduction systems (reviewed in Ref. 19).
One type of receptor desensitization is characterized by
phosphorylation of serine and threonine residues in the third
cytosol-facing loop and the carboxyl-terminal segment of the receptor,
reducing the ability of the receptor to interact with G proteins and
thus causing a diminished responsiveness to agonist stimulation. The
best-studied example is the
During receptor-mediated endocytosis, ligands
bound to their receptors are internalized via clathrin-coated pits and
vesicles (reviewed in Refs. 25 and 26). Most ligands are subsequently
degraded in lysosomes
(27, 28, 29, 30, 31) , whereas
the fate of the internalized receptors varies. Some, such as epidermal
growth factor receptors, are degraded together with their bound ligand
(29) while others are recycled back to the cell surface after
intracellular dissociation of their ligand
(32, 33, 34, 35, 36) . Following
binding of ligand,
Here, we demonstrate unusual features of
the intracellular processing of ET-1 and its receptor ET
Internalization of cell
surface ET
To confirm
the immunofluorescence results quantitatively and to obtain actual
rates of endocytosis, we directly determined the kinetics of
endocytosis of ET-1 and ET
In the first study, the
cell supernatant was brought to 60% ammonium sulfate and incubated on
ice for 30 min before filtration. Unbound
To
confirm the binding of ET-1 to the ET
Contraction and relaxation of smooth muscle cells that line
the arteries is one of the major ways by which blood pressure is
regulated, and these cells are affected by a number of competing
vasodilator and vasoconstrictor hormones. The long term vasopressor
effect of ET-1
(24) is one important aspect of this regulatory
system, and, therefore, it is necessary to have a molecular and
cellular understanding of how a single small injection of ET-1 into the
circulation of a rat increases its blood pressure for several hours.
This effect is likely to be mediated via ET
The most
important results of this paper are that binding of ET-1 to cell
surface ET
Interestingly,
The finding that a
significant amount of intact ET-1 is still bound to ET
Caveolae are
non-clathrin-coated invaginations of the plasma membrane. In different
cell types, caveolae contain different proteins that participate in
signal transduction by G protein-coupled and other receptors
(47) . We showed directly that ET
Plasma membrane
caveolae are thought to pinch off
(51) and form endosome-like
structures
(52) . The ET
CHO cells, either nontransfected or transfected
with ET
CHO cells, either nontransfected or transfected with
ET
We thank Drs. H. E. Ives, E. Lobo, D. Neumann, and U.
Liyanage for their invaluable discussions and N. Cohen for preparation
of plasmids.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, the receptor
subtype that mediates contraction of vascular smooth muscle cells.
First, we show that in stably transfected CHO cells expressing
ET
, binding of an ET-1 ligand induces rapid endocytosis of
cell surface ET
. Receptor endocytosis was measured both by
immunofluorescence and by radioiodinated antibodies specific for
ET
. Second, we demonstrate that ET-1 remains intact for up
to 2 h after endocytosis and, as judged by co-immunoprecipitation,
internalized
I-ET-1 remains bound to ET
receptors. We hypothesize that internalized ET-1, bound to
ET
receptors, continues to activate a signal-transducing G
protein, thus accounting for the prolonged period of contraction
induced in smooth muscle cells by a single administration of ET-1.
(
)
a 21-amino acid
peptide with two intramolecular disulfide bonds, is the most potent
vasopressor agent yet discovered
(1) . Initially isolated from
porcine aortic endothelial cells, it induces long-lasting hypertension;
a single injection (1 nmol/kg) into the circulation of a rat increases
blood pressure for several hours
(2) . ET-1 and its isoforms
ET-2 and ET-3 have numerous biological actions in vitro and
in vivo, including hemodynamic, cardiac, renal,
neuroendocrine, smooth muscle contraction, and protomitogenic effects
(reviewed in Refs. 3 and 4).
,
ET
, and ET
(5, 6, 7, 8, 9, 10, 11, 12, 13) .
These receptors have seven putative membrane-spanning segments and are
coupled to G proteins. ET receptors are widely expressed but
distributed differently in tissues where they show pharmacological
effects
(6, 14, 15, 16) , such as the
vascular system, brain, kidney, lung, and adrenal glands
(5, 7, 8, 10, 11, 12, 13) .
Importantly, ET
but not ET
mRNA is expressed in
rat vascular smooth muscle A10 cells
(5) and on rat aorta
stripped of endothelial cells
(10, 11) . Thus, the
ET
receptor mediates vascular constriction, although in
some blood vessels, such as rabbit saphenous vein, ET
appears to mediate vasoconstriction
(17, 18) .
-adrenergic receptor
(20, 21) ; continued exposure of cells to
-agonists causes an attenuation of signal transduction
due to phosphorylation of the
-adrenergic receptor by
both the cAMP-dependent protein kinase
(22) and the
-adrenergic receptor kinase
(23) . This desensitization is
essentially complete within minutes. In contrast, activation of the
ET
receptor on smooth muscle cells causes contraction which
lasts over 2 h, resulting in a sustained increase in blood pressure
(24) . The cellular mechanisms which underlie the long-lasting
vasopressor effects of endothelins, however, are not understood. In
this paper, we suggest one mechanism for the protracted action of ET
which relates to the unusual intracellular processing of the receptor
following endocytosis.
-adrenergic receptors are
internalized by endocytosis
(20, 37) . However, the fate
of the ligand is unknown.
.
First, we show that ET-1 binding induces rapid endocytosis of
ET
. Second, we demonstrate that ET-1 remains intact for up
to 2 h after endocytosis, and that internalized ET-1 remains bound to
an ET
receptor. We hypothesize that internalized ET-1,
bound to ET
receptors, continues to activate a transducing
G protein, thus accounting for the prolonged period of contraction
induced in smooth muscle cells by a single administration of ET-1.
Materials
Rabbit anti-ET polyclonal
antibody 291, an antipeptide antibody specific for the extracellular
amino-terminal domain of ET
, was described previously
(38) .
Cell Culture and Transfection
The generation and
maintenance of stably transfected CHO cell lines expressing ET were described previously
(5) . Briefly, CHO cells were
co-transfected with pcDNA-3
(39) and the ET
cDNA in
the vector pcDNA-1
(5) using the calcium phosphate
precipitation technique. Two days after transfection, cells were plated
in selective medium containing 500 µg/ml G418 (Life Technologies,
Inc.). G418-resistant colonies were selected and amplified by
subcloning single colonies. Two clones were selected for further
studies. CHET-B
(5) was used for the experiments in Figs. 1 and
2. The other cell line, CHO ET
28, was used for the
experiments in Figs. 3 and 4 and Tables I and II. CHET-B or CHO
ET
28 cells express
10
cell surface ET
binding sites per cell
(5) .
(
)
Measurement of Internalization of Cell
Surface ET
Live CHO cells, either nontransfected or
stably transfected with the ET Receptors by
Immunofluorescence
cDNA, were grown on a
coverslip, washed twice with ice cold phosphate-buffered saline (PBS),
then once with HH/BSA buffer (Hank's balanced buffer also
containing 10 mM Hepes, pH 7.4, and 2% bovine serum albumin
(BSA)). The cells were then incubated successively on ice (1 h, with 3
washes between incubations) in HH/BSA with: (i) 20 µg/ml normal
goat IgG (Sigma or The Jackson Laboratory); (ii) 20 µg/ml
anti-ET
291 IgG; (iii) 50 µg/ml fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (The Jackson
Laboratory). The cells were then split into two sets. One set was
further incubated with 50 pM (unlabeled) ET-1 at 4 °C for
2 h overnight, followed by washing with PBS to remove excess ET-1. The
other set of cells was similarly treated except no ET-1 was added. The
cells were then warmed to 37 °C with 1 ml of PBS containing 11
mM glucose for various times (0-180 min), fixed in PBS
containing 2% paraformaldehyde for 20 min at room temperature, and
washed extensively with PBS. The fixed cells were mounted with
Slow-Fade antifade reagent (Molecular Probes) and viewed under a
Bio-Rad MRC600 confocal fluorescence microscope.
Kinetics of Internalization of Cell Surface
ET
A10 cells and CHO cells stably
transfected with ET Receptors
were used for this study; each data
point was done in triplicate. Live cells, grown in 35-mm culture
dishes, were incubated successively in 4 °C (1 h each incubation)
with 20 µg/ml anti-ET
IgG followed by
I-labeled goat anti-rabbit IgG (0.2 µCi/ml in PBS,
Amersham), then washed again three times with PBS. The cells were
divided into two groups. One set was further incubated with 50
pM ET-1 at 4 °C for 2 h, and the other was similarly
incubated without ET-1. The medium was removed, and both sets of cells
were then incubated with 1 ml of PBS containing 11 mM glucose
at 37 °C for various times (0-180 min) to allow endocytosis.
After collecting the medium, the cells were incubated with 1 ml of 50
mM glycine, pH 2.5, three times for 10 min each at 4 °C,
and the acid solutions were pooled; this fraction is referred to as
surface-bound IgG. Consistently, in cells not incubated at 37 °C,
more than 95% of surface-bound IgG was removed by this acid wash
procedure. The cell pellet was dissolved in 1 ml of 1 N NaOH
at room temperature; this fraction contains the internalized IgG which
is resistant to the acid wash. The amount of
I-IgG from
the medium, cell surface, and cell pellet were measured using a liquid
scintillation counter.
Kinetics of Internalization of Cell Surface
ET-1
Cells were incubated with 50 pMI-ET-1 in PBS at 4 °C for 2 h to overnight, washed
with PBS, then incubated as above at 37 °C at various times
(0-180 min). The medium was collected. As above, the cells were
acid-washed and then dissolved in 1 N NaOH, yielding cell
surface and internalized
I-ET-1. The amount of
I-ET-1 from the medium, cell surface, and cell pellet was
measured.
Characterization of
CHO cells
transfected with ETI-ET-1 by High
Performance Liquid Chromatography (HPLC)
, grown in 35-mm tissue culture dishes,
were incubated with 50 pM
I-ET-1 in PBS at 4
°C for 2 h, then washed extensively with PBS. As above, the cells
were warmed to 37 °C for various times (0-180 min). Cell
surface-bound
I-ET-1 was removed by the acid-wash
procedure. The cells were then dissolved in 1 ml of 5% acetic acid, and
the cell supernatant, containing internalized
I-ET-1, was
lyophilized and redissolved in a 50 µl of 0.1% trifluoroacetic
acid.
I-ET-1 was then analyzed by HPLC using a C18 column
and a linear gradient of 100% solvent A (0.1% trifluoroacetic acid) to
100% solvent B (90% CH
CN in solvent A). Fractions were
collected every 30 s of the 50-min run, and the radioactivity of each
was measured.
Ammonium Sulfate Precipitation of Internalized
CHO cells either nontransfected or
transfected with ETI-ET-1
cDNA were labeled with 50 pM
I-ET-1 in PBS at 4 °C for 2 h, followed by incubation
at 37 °C (0-180 min) as above. Surface-bound
I-ET-1 was removed by the acid wash procedure. The cells
were solubilized in 50 mM Tris buffer, pH 7.4, containing 1%
Nonidet P-40 (Sigma), 1 mg/ml BSA, and 1 nM unlabeled ET-1.
After low-speed centrifugation, the cell supernatant was incubated with
60% ammonium sulfate for 30 min on ice. The precipitated
I-ET-1 was collected on a Millipore filter (0.2 µm
pore size) followed by extensive washing with PBS, and the
radioactivity on the filters was measured.
Immunoprecipitation of Internalized
CHO cells either nontransfected or transfected
with ETI-ET-1 by an ET
Antibody
cDNA were labeled with 50 pM
I-ET-1 in PBS at 4 °C for 2 h and incubated
0-180 min at 37 °C, and the surface-associated ligand was
removed by an acid wash as above. The cells were solubilized at 4
°C with 1 ml of TBST (10 mM Tris, pH 8.0, 0.15 M
NaCl, 1% Triton X-100, 2 mM phenylmethylsulfonyl fluoride)
containing 60 mM octyl glucoside. After low-speed
centrifugation, the cell supernatant was incubated for 30 min at 4
°C with 50 µg/ml rabbit anti-ET
IgG. This rabbit
anti-ET
IgG had been reacted with 5 µg/ml Protein
A-Sepharose (Sigma) overnight before it was added to the cell
supernatant. The Protein A-Sepharose was collected by centrifugation
and washed four times with TBST. The radioactivity in the recovered
I-ET-1
ET
ET
antibody
complex was measured using a liquid scintillation counter.
ET-1 Induces Internalization of Cell Surface
ET
To determine whether cell surface ET undergoes endocytosis in CHO cells stably transfected with
ET
cDNA, we used polyclonal anti-peptide antibody 291
specific for the extracellular amino-terminal domain of ET
.
Antibody 291 is an excellent probe for receptor internalization because
it binds to receptors on live cells and does not inhibit binding of
radioiodinated ET-1 (data not shown; see below). We characterized
extensively the specificity of this antibody: as judged by
immunofluorescence it detected ET
receptors in
ET
-transfected, but not untransfected live,
nonpermeabilized, CHO cells, consistent with the proposed ectodomain
topology of the amino terminus. Moreover, the binding of Ab 291 to
ET
transfected cells was inhibited by 1 µg/ml
(100
) concentration of the immunizing peptide but not of
irrelevant peptides (data not shown).
was first monitored by immunofluorescence. To
assess internalization without ET-1 ligand, cells were incubated with
Ab 291 for 1 h at 4 °C, then with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG. Then the cells were
warmed to 37 °C for various times (0 to 180 min) to allow
endocytosis, fixed with paraformaldehyde, and visualized by confocal
fluorescence microscopy. Fig. 1 a shows the distribution
of ET
before incubation at 37 °C. The bright staining
of the cell periphery ( arrows) is indicative of cell surface
labeling. This pattern did not change up to 3 h at 37 °C,
suggesting that the labeled antibodies remain at or near the cell
surface (Fig. 1 b and data not shown). These data
indicate that, in the absence of ET-1, the ET
receptor is
not internalized.
Figure 1:
ET-1
induced internalization of cell surface ET. Live CHO cells,
stably transfected with ET
cDNA, were incubated at 4 °C
with anti-ET
IgG followed by fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (see ``Experimental
Procedures''). The cells were further incubated either without
ET-1 ( a and b) or with 50 pM ET-1 ( c and d) at 4 °C for 2 h, followed by extensive washing
to remove excess ET-1. Then the cells were warmed to 37 °C for 0
min ( a and c), 30 min ( b), and 10 min
( d) and then fixed and visualized by confocal fluorescence
microscopy. Shown here are the optical scans taken 4 µm above the
surface of the slide. Bar represents 5
µm.
Endocytosis of ET does occur following
addition of ET-1, as illustrated in Fig. 1 d. After
incubation of CHO cells, stably transfected with ET
with
Ab 291 and fluorescein isothiocyanate-conjugated secondary Ab, ET-1 was
added for 2 h at 4 °C. Subsequently, the cells were warmed to 37
°C for 0-180 min, fixed, and visualized by confocal
microscopy. Without incubation at 37 °C, the labeled antibody
remained on the cell surface (Fig. 1 c). However, after a
10-min incubation at 37 °C, a marked change in receptor
distribution was observed (Fig. 1 d). Cell surface
staining was dramatically reduced ( closed arrows), and
punctate staining of antigen appeared throughout the cytoplasm
( open arrows). These data indicate that ET-1 induces
internalization of cell surface ET
receptors.
using
I-labeled
goat anti-rabbit IgG (to monitor ET
) or
I-labeled ET-1 (to monitor ET-1) and an acid-stripping
procedure to distinguish surface-bound antibody or ET-1 from
internalized ones (see ``Experimental Procedures''). As shown
in Fig. 2 a, in the absence of ET-1, less than 10% of
I-labeled IgG bound to cell surface ET
receptors are internalized even after a 3-h incubation at 37
°C. Most of the
I-labeled IgG is recovered in the
medium, but we have not investigated the properties of this material.
On the other hand,
I-ET-1 is internalized rapidly
(Fig. 2 b). After 10 min of incubation at 37 °C,
35% of surface-bound
I-ET-1 has become internalized
rising to
80% by 1 h at 37 °C. We were concerned that
overexpression of ET
in stably transfected CHO cells might
somehow cause endocytosis of bound ET-1. However, Fig. 2 d shows the same rate and extent of endocytosis of surface-bound
I-ET-1 by vascular smooth muscle A10 cells, which express
the ET
receptor endogenously.
Figure 2:
Kinetics of internalization of
I-anti-ET
IgG and
I-ET-1. The
cells used were CHO cells transfected with ET
( a,
b, and c) or A10 cells ( d). They were
labeled at 4 °C with anti-ET
IgG followed by
I-goat anti-rabbit IgG ( a and c) or
with 50 pM
I-ET-1 ( b and d),
as described under ``Experimental Procedures.'' In a and c, an additional incubation (2 h, 4 °C) was
performed with ( c) or without ( a) 50 pM
unlabeled ET-1. The cells were then incubated at 37 °C up to 180
min. For each time point, the percentage of
I-labeled IgG
or ET-1 in the medium ( open circles), at the cell surface
( open triangles), and inside the cells ( closed
squares) was measured, using the acid-stripping procedure (see
``Experimental Procedures'') to differentiate surface-bound
from internalized radioactivity. Each point is the average of
triplicate measurements of two ( a, b, and c)
or three ( d) independent experiments. There was no more than
5-10% variation among experiments. a, internalization of
I-IgG bound to cell surface ET
in CHO cells
stably transfected with ET
. b, internalization of
I-ET-1 in CHO cells expressing ET
.
c, internalization of
I-IgG bound to cell
surface ET
in CHO cells expressing ET
, ET-1 was
present during incubation at 37 °C. d, internalization of
I-ET-1 in A10 cells.
A comparison of the
experiments in a and c of Fig. 2shows directly
that addition of unlabeled ET-1 causes the internalization of cell
surface ET. In the experiment in c, live CHO cells
transfected with ET
were incubated with Ab 291 at 4 °C
for 1 h and then, as with those used in a, with
I-IgG at 4 °C for 1 h. Unbound
I-IgG
was removed, and the cells were incubated with ET-1 (50 pM)
for 2 h at 4 °C. After excess unbound ET-1 was removed, the cells
were warmed to 37 °C. In contrast to the results in a,
endocytosis of
I-IgG complexes to cell surface ET
was rapid and efficient. Importantly, the rate and the maximal
extent of internalization of
I-IgG was almost identical
with the rate and the maximal extent of internalization of
I-ET-1 (compare b and c of
Fig. 2
). A comparison of a and c of
Fig. 2
establishes that ET-1 induces the internalization of cell
surface ET
. These experiments confirm the results of
Fig. 1
and demonstrate that ET-1 induces a rapid internalization
of cell surface ET
receptors. Over 80% of both cell surface
ET
receptors and surface-bound ET-1 become internalized
within a 1-h incubation at 37 °C.
ET-1 Remains Intact 2 h after Endocytosis
To
determine whether ET-1 remains intact following receptor-mediated
endocytosis, I-ET-1 was incubated with
ET
-expressing CHO cells, and internalized
I-ET-1 was analyzed by HPLC (Fig. 3). Specifically,
I-ET-1 was allowed to bind to the cells at 4 °C;
after removal of unbound ligand, the cells were then warmed to 37
°C for various times. Surface-bound
I-ET-1 was
removed by an acid wash, then the cells were dissolved in 5% acetic
acid. The cell supernatant, containing internalized radiolabeled ET-1,
was lyophilized, redissolved in 0.1% trifluoroacetic acid, and analyzed
using a C18 HPLC column.
Figure 3:
Analysis of internalized
I-ET-1 by high performance liquid chromatography.
a,
I-ET-1 standard. b,
I-ET-1 bound to the surface of CHO cells transfected with
ET
. The cells were incubated with 50 pM
I-ET-1, dissolved, and analyzed by HPLC as described
under ``Experimental Procedures.'' c, internalized
I-ET-1 after 20 min at 37 °C.
ET
-expressing CHO cells were labeled as in b and
warmed to 37 °C for 20 min. After acid-stripping to remove the
surface-associated ligand, they were dissolved and analyzed by HPLC.
d, internalized
I-ET-1 after 120 min at 37
°C. The cells were treated as in c, except that the
incubation at 37 °C was for 120 min.
An I-ET-1 standard was run on
the same HPLC column; fractions 61 to 65 contained virtually all of the
radioactivity (Fig. 3 a, arrow) and therefore
represent intact
I-ET-1.
I-ET-1 bound to
cell surface receptors at 4 °C (without incubation at 37 °C or
an acid wash) migrated identically with the ET-1 standard
(Fig. 3 b, arrow). After a 20-min incubation at
37 °C,
90% of the internalized ( i.e. resistant to the
acid wash)
I radioactivity co-migrated with the ET-1
standard (Fig. 3 c, arrow). A small peak that
eluted earlier contained the remaining 10% of the radioactivity
(Fig. 3 c, open arrow). Two hours after warming
to 37 °C, more than 30% of the internalized
I-ET-1
co-migrated with the
I-ET-1 standard, indicating that
30% of internalized ET-1 remained intact (Fig. 3 d,
arrow). Several peaks of radioactive material eluted earlier
from the column (Fig. 3 d, open arrows),
indicating that some degradation had occurred. Therefore, internalized
ET-1 remained almost fully intact after 20 min at 37 °C
(Fig. 3 c), and, even after 2 h of internalization, more
than 30% of ET-1 was intact (Fig. 3 d).
ET-1 Remains Bound to ET
We utilized two techniques to show that the
majority of internalized ET-1 is still bound to ET 2 h after
Endocytosis
:
ammonium sulfate precipitation of receptor-ligand complexes
(), and immunoprecipitation of internalized
I-ET-1 by an antibody specific to ET
(). In both experiments, nontransfected (control)
and ET
-transfected CHO cells were incubated with 50
pM
I-ET-1 at 4 °C for 2 h, warmed to 37
°C for various times, then surface-bound
I-ET-1 was
removed by an acid wash. The cells were solubilized in a detergent
solution, and, after clarification by low-speed centrifugation,
receptor-ligand complexes were quantified.
I-ET-1 does not
precipitate under these conditions. However, as shown in ,
20 min after endocytosis,
50% of the total radioactivity from
internalized
I-ET-1 is recovered in the precipitate, and,
even 2 h after internalization, more than 30% of the internalized
I-ET-1 is precipitated. Thus, a significant fraction of
internalized ET-1 remains bound to some protein, presumably to the
ET
receptor, even 2 h after internalization. Note that, as
an additional control, no significant amount of
I-ET-1 is
recovered in extracts of control, nontransfected, CHO cells.
receptor following
endocytosis, immunoprecipitation of the ligand-receptor complexes was
performed (). The cell supernatant was incubated with
antibody 291, specific for the ET
receptor, and
immunocomplexes were recovered using Protein A-Sepharose beads.
shows that 20 min after endocytosis
55% of the total
radioactivity from internalized
I-ET-1 is recovered in
the anti-ET
immunoprecipitate, and more than 25% of the
internalized
I-ET-1 is immunoprecipitated after 120 min
at 37 °C. At 120 min of endocytosis, 30% of internalized
I-ET-1 is intact (Fig. 3), and 25-30% of the
radioactivity from internalized
I-ET-1 is bound to
ET
receptors (Tables I and II). We conclude that, at this
time, all of the internalized, intact
I-ET-1 is bound to
an ET
receptor. It is possible that the intracellular
ET-1
ET
complexes continue to activate G proteins.
, the ET
receptor subtype expressed in vascular smooth muscle cells and thought
to play critical roles in regulating vascular resistance and the
distribution of blood flow
(5, 10, 11) . For
these reasons, we have studied the intracellular fate of ET
receptors, its dependence on ligand (ET-1), and the state of the
ET-1
ET
complexes after internalization.
receptors induces endocytosis of ET-1
ET
complexes, and that
30% of internalized ET-1
remains intact and tightly bound to ET
receptors even 2 h
after endocytosis. We hypothesize that internalized ET-1
ET
complexes might continue to activate a transducing G protein,
thus inducing prolonged contraction in smooth muscle cells. First, we
showed in transfected CHO cells that binding of an ET-1 ligand induces
rapid endocytosis of cell surface ET
. We measured receptor
endocytosis both by immunofluorescence and by radioiodinated antibodies
specific for ET
. Second, we demonstrated that
30% of
internalized
I-ET-1 remains intact for up to 2 h after
endocytosis, as judged by HPLC. Third, as shown by
co-immunoprecipitation, internalized, intact
I-ET-1
remains bound to an ET
receptor. A potential limitation of
transfected cells expressing a high number of receptors is that they
may not regulate receptors in the same way as smooth muscle cells.
Thus, we repeated these experiments in A10 cells which endogenously
express ET
. There was no difference between CHO and A10
cell lines in the kinetics of ET-1-induced ET
endocytosis
(Fig. 2 d). These experiments examine redistribution of
only cell surface receptors; thus, these findings suggest that
redistribution is mediated by an existing pool of cellular proteins,
not by newly synthesized receptors.
90% of
internalized ET-1 remains intact 20 min following endocytosis
(Fig. 3). At this time, however, only
50% of the
radioactivity from internalized
I-ET-1 is bound to the
ET
receptor, and, thus, a significant amount of
internalized ET-1 is free. Two hours following internalization,
however, about 30% of internalized ET-1 remains intact, and
25-30% of
I radioactivity from internalized
ET-1 is bound to ET
receptors (Tables I and II). Thus, at 2
h after internalization, all intact ET-1 is bound to ET
receptors, and all unbound ET-1 has been degraded. The slow
dissociation of internalized ET-1 from ET
receptors is
consistent with our kinetic studies of the binding of ET-1 to
ET
. At pH 7, the dissociation of
I-ET-1 from
cell surface ET
receptors, in transfected CHO cells, was
extremely slow, with a half-time of about 2 h at 4 °C.
Significantly, the rate of dissociation of
I-ET-1 from
ET
was the same at pH values as low as 4.0 (data not
shown). The internal pH of endosomes is thought to be between 5.5 and
6.0, and that of lysosomes about 4.5 to 5.5 (reviewed in Ref. 40).
Thus, at any pH value likely to be found in internal endosomes, ET-1
should remain bound to ET
receptors.
even
hours after endocytosis gives rise to the hypothesis that it may
transduce signals even after internalization. In such a case,
internalized ET-1
ET
complexes should be able to
interact with downstream components in the ET-1 signal transduction
pathway. This pathway involves activation of phospholipase C, protein
kinase C, Phospholipase A
, opening of non-selective
membrane cation channels, L-type Ca
channels, and cAMP-dependent protein kinase-dependent Cl channels
(41, 42, 43, 44, 45, 46) .
It is not known which, if any, of these proteins co-localize to
endosomes containing internalized ET-1
ET
complexes.
Some insight into this issue derives from our recent demonstration that
plasma membrane ET
receptors, together with bound ET-1
ligand, are localized to caveolae
(38) .
endothelin
receptors, together with its bound ligand, are found in plasma membrane
caveolin-containing complexes
(38) . In capillary endothelial
cells and visceral smooth muscle cells, two signal-transducing proteins
activated by binding of ET-1 to the ET
receptor, a calcium
channel and an IP
receptor, are enriched in plasma membrane
caveolae
(48, 49) . Caveolae isolated from tissues and
cultured cells are enriched in both G
and G
subunits of heterotrimeric GTP-binding proteins
(50) . In
particular, G
, the subunit of the heterotrimeric G
protein that is coupled to ET
, is also present in caveolae
(47) . Taken together, these results strongly indicate that the
endothelin receptor binds its ligand and generates an intracellular
signal while localized in plasma membrane caveolae.
receptor with its bound
ET-1 ligand is likely be incorporated in these vesicles. Other
caveolar-localized proteins that participate in signal transduction,
such as G
, might also be incorporated into these
vesicles. G proteins are associated with a variety of intracellular
membranes (reviewed in Refs. 53 and 54). In polarized epithelial
LLC-PK-1 cells, the G
subunit is localized to the
cytoplasmic face of Golgi cisternae; it was distributed across the
entire Golgi stack
(55, 56) . In polarized Madin-Darby
canine kidney cells, both the
and
subunits of G
function to control vesicular transport
(57, 58) .
Although evidence is lacking, G
might also be found in
internalized caveolae. G
could be a resident protein
in these internal organelles or it could move to them from the plasma
membrane along with ET
receptors. Little is known of the
nature of internal membranes that contain caveolin and other proteins
characteristic of plasma membrane caveolae, and, at this time, we are
unsure of the nature of the internal organelles that contain the
ET-1
ET
complexes. Nonetheless, it is reasonable to
suppose that signal transduction by the ET receptor occurs in
internalized caveolae, accounting for the long-lasting elevation of
cytosolic Ca
and contraction of smooth muscle cells
that occurs following a single addition of ET-1.
Table:
Ammonium sulfate precipitation of
I-ET-1
cDNA (CHO ET
), were incubated with
I-ET-1 (2 h, 4 °C) and warmed to 37 °C for 20 or
120 min. After stripping surface ET-1 by an acid wash, the cells were
dissolved in detergent. The cell extract were centrifuged at low speed,
then the radioactivity in a portion of the cell supernatant was
measured (before precipitation). The remaining cell supernatant was
incubated with 60% ammonium sulfate for 30 min on ice, and the
precipitated
I-ET-1 was collected and counted (after
precipitation).
Table:
Immunoprecipitation of
I-ET-1
ET
complex with anti-ET
Ab
cDNA (CHO ET
), were incubated with
I-ET-1 (2 h, 4 °C) and warmed to 37 °C for 20 min
or 120 min. After stripping surface ET-1 by the acid wash procedure
(see ``Experimental Procedures''), the cells were dissolved
in a buffer containing 1% Triton X-100 and 60 mM octyl
glucoside. ET
was precipitated by anti-ET
antibody and Protein A-Sepharose (see ``Experimental
Procedures''). The immunocomplexes were recovered by
centrifugation, washed, and counted. In control experiments, less than
1% of
I-ET-1 was recovered in the ET
immunoprecipitates.
, endothelin receptor subtype
A; ET
, endothelin receptor subtype B; ET
,
endothelin receptor subtype C; G proteins, heterotrimeric GTP-binding
signal-transducing proteins; HPLC, high performance liquid
chromatography; PBS, phosphate-buffered saline; BSA, bovine serum
albumin; Ab, antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.