Growth Hormone Receptor Ubiquitination Coincides with Recruitment
to Clathrin-coated Membrane Domains*
Peter
van Kerkhof,
Martin
Sachse,
Judith
Klumperman, and
Ger J.
Strous
From the Department of Cell Biology, University Medical Center
Utrecht and Institute of Biomembranes, 3584CX Utrecht,
The Netherlands
Received for publication, August 11, 2000, and in revised form, October 13, 2000
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ABSTRACT |
Endocytosis of the growth hormone receptor (GHR)
depends on a functional ubiquitin conjugation system. A 10-amino acid
residue motif within the GHR cytosolic tail (the
ubiquitin-dependent endocytosis motif) is involved
in both GHR ubiquitination and endocytosis. As shown previously,
ubiquitination of the receptor itself is not required. In this paper
ubiquitination of the GHR was used as a tool to address the question of
at which stage the ubiquitin conjugation system acts in the process of
GHR endocytosis. If potassium depletion was used to interfere with
early stages of coated pit formation, both GHR endocytosis and
ubiquitination were inhibited. Treatment of cells with
methyl-
-cyclodextrin inhibited endocytosis at the stage of
coated vesicle formation. Growth hormone addition to
methyl-
-cyclodextrin-treated cells resulted in an accumulation of
ubiquitinated GHR at the cell surface. Using immunoelectron microscopy,
the GHR was localized in flattened clathrin-coated membranes. In
addition, when clathrin-mediated endocytosis was inhibited in HeLa
cells expressing a temperature-sensitive dynamin mutant, ubiquitinated
GHR accumulated at the cell surface. Together, these data show that the
GHR is ubiquitinated at the plasma membrane, before endocytosis occurs,
and indicate that the resident time of the GHR at the cell surface is
regulated by the ubiquitin conjugation system together with the
endocytic machinery.
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INTRODUCTION |
Clathrin-mediated endocytosis involves the formation of
clathrin-coated vesicles from coated pits at the plasma membrane. Recruitment of membrane proteins into clathrin-coated pits is mediated
by specific amino acid sequences within their cytoplasmic domain (for
review, see Refs. 1, 2). The best-defined coated pit localization
signals are the tyrosine-based motifs NPXY as described for,
e.g. the low-density lipoprotein receptor (3), and
YXX
(where
is an amino acid with a bulky hydrophobic
group) found in, e.g. the transferrin receptor (4).
Alternatively, internalization of the insulin and
2-adrenergic
receptor is mediated by a dileucine-containing motif (5, 6). Many
receptors, including the low-density lipoprotein receptor and the
transferrin receptor, are clustered in coated pits and
internalized constitutively, independent of ligand occupancy. The
heterotetrameric adaptor complex AP-2 binds directly to the
tyrosine-based motif and nucleates assembly of clathrin triskelions
onto the plasma membrane (for review, see Ref. 7). Invagination of the
plasma membrane results in the formation of constricted coated pits,
followed by the dynamin-dependent detachment of coated
vesicles from the plasma membrane. More complex situations exist when
plasma membrane proteins enter cells on stimuli such as hormone binding
or specific signal transduction events. In this case the
internalization signal is only recognized on ligand binding, or the
stimulus induces the addition of a signal, which results in the
subsequent recruitment of the receptor into the coated pit. The
agonist-induced phosphorylation of the
2-adrenergic receptor
resulting in the binding of
-arrestin, a specialized adaptor that
binds to clathrin, is an example of a protein modification that
regulates internalization (8). Recently, it was shown that the
attachment of ubiquitin moieties is involved in the internalization of
several plasma membrane proteins (for review, see Refs. 9, 10). In
mammalian cells, the ubiquitin conjugation system regulates the
endocytosis of the epithelial sodium channel (11) and the growth
hormone receptor (GHR1; Ref.
12).
The GHR was initially found to be ubiquitinated on amino acid
sequencing of the receptor from rabbit liver (13). The ubiquitin conjugation system is involved in GHR internalization and degradation (12, 14). In particular, a 10-amino acid motif within the GHR cytosolic
tail (the ubiquitin-dependent endocytosis motif, DSWVEFIELD) is involved in both receptor ubiquitination and endocytosis (15). We have recently shown that the proteasome is also involved in
GHR internalization (16). Ligand-induced internalization of the GHR is
blocked in the presence of specific proteasomal inhibitors such as
carbobenzoxy-L-leucyl-L-leucyl-L-leucinal
and
-lactone, the more membrane-permeable analogue of lactacystin. Disruption of clathrin-mediated endocytosis by cellular potassium depletion (17), hypertonic medium treatment (18), or cellular cytosol
acidification (19) inhibits internalization and ubiquitination of the
GHR (14). Although ubiquitination of the GHR itself is not required for
endocytosis (15), GHR internalization requires the activity of the
ubiquitin conjugation system, which acts together with the endocytic
machinery in targeting the receptor into the coated pit.
In this study the question was addressed of at which stage the
ubiquitin conjugation system acts in the process of GHR endocytosis. GHR ubiquitination was taken as a biochemical marker for the location of the activity of the ubiquitin conjugation system. Conditions in
which coated pit formation was prevented were compared with conditions
that allowed coated pit formation but prevented coated vesicle formation.
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EXPERIMENTAL PROCEDURES |
Materials and Antibodies--
Antibody mAb5 recognizing the
lumenal part of the GHR was from AGEN Inc. (Parsippany, NJ). Polyclonal
antibodies against amino acid residues 271-318 of the cytosolic tail
of the GHR (Anti-T; Ref. 16) and against human growth hormone (GH) were
raised in rabbits. Antiserum specific for protein-ubiquitin conjugates
was a generous gift from Dr. A. Ciechanover (Technion-Israel
Institute of Technology, Haifa, Israel). Antibody 4G10
(anti-pY), recognizing phosphotyrosine residues, was obtained
from Upstate Biotechnologies Inc. (Lake Placid, NY). Anti-biotin was
from Rockland (Gilbertsville, PA); monoclonal anti-clathrin was from
Transduction Laboratories (Lexington, NY); anti-mouse IgG was
from Nordic Immunological Laboratories (Tilburg, The Netherlands); and
monoclonal anti-hemagglutinin (HA) antibody 12CA5 was from Babco
(Richmond, CA). Human GH was a kind gift from Lilly;
methyl-
-cyclodextrin (M
CD) was from Sigma; LipofectAMINE was from
Life Technologies, Inc.; and
carbobenzoxy-L-leucyl-L-leucyl-L-leucinal was from Calbiochem.
Plasmids, Cell Culture, and Transfection--
Wild-type rabbit
GHR cDNA was cloned into the cytomegalovirus-Neo expression plasmid
pcDNA3.1 (Invitrogen BV/Novex) and used for transient
transfections. The internalization-deficient mutant GHR(F327A) was
constructed by site-directed mutagenesis and cloned into pcDNA3.1
as described (20). The Chinese hamster cell line ts20, stably
transfected with a pCB6 construct containing the rabbit GHR cDNA
sequence, was used in this study (12). Cells were grown at 30 °C in
MEM
supplemented with 10% fetal calf serum, 4.5 g/l glucose, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.45 mg/ml
geneticin. For experiments cells were grown on 35- or 60-mm dishes in
the absence of geneticin to ~75% confluence, and 10 mM
sodium butyrate was added overnight to increase GHR expression
(12).
tTA-HeLa cell lines stably transfected with the HA-tagged
temperature-sensitive mutant of dynamin (dynTS) or
wild-type dynamin (wtdyn) were kindly provided by Dr. S. Schmid (The
Scripps Research Institute, La Jolla, CA). DynTS carries a
mutation of the glycine at position 273 to aspartic acid. Cells were
cultured at 37 °C in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.4 mg/ml geneticin, 2 µg/ml tetracycline, and
200 ng/ml puromycin. For transfection experiments subconfluent cultures
of tTA-HeLa cells were washed with phosphate-buffered saline (PBS),
detached with trypsin-EDTA, plated on 60-mm dishes, and incubated at
the permissive temperature of 32 °C in the absence of tetracycline
for 24 h. Cells were 30-40% confluent when transfected with 1 µg cDNA/dish, using LipofectAMINE according to the
manufacturer's protocol. After 24 h, cells were washed with
medium free of tetracycline. Cells were used for experiments 48 h
after transfection and 72 h after removal of tetracycline.
GH Binding and Internalization--
125I-Human GH
was prepared using chloramine T (12). For internalization studies,
cells were grown in 12-well cluster plates, washed with MEM
supplemented with 20 mM Hepes, pH 7.4, and 0.1% bovine
serum albumin (BSA), and incubated in a water bath. 125I-GH
(8 nM) was bound on ice for 2 h, and the cells were
washed free of unbound GH and incubated for 0-30 min at 30 °C.
Membrane-associated GH was removed by acid wash (0.15 M
NaCl, 50 mM glycine, 0.1% BSA, pH 2.5) on ice.
Internalized GH was determined by measuring the radioactivity after
solubilization of the acid-treated cells in 1 M NaOH using
an LKB gamma counter.
Metabolic Labeling--
Cells were grown in 60-mm dishes and
incubated in methionine- and cysteine-free MEM. Then 3.7 MBq/ml
[35S]methionine (Tran35S Label,,
40 TBq/mmol; ICN, Costa Mesa, CA) was added, and the incubation was
continued at 30 °C in a CO2 incubator. The radioactivity was replaced with medium containing 100 µM unlabeled
methionine, 0.1% BSA, and 8 nM GH and chased for 0-60
min. Cells were lysed, and samples were immunoprecipitated (see below).
Radioactivity was determined using a Storm imaging system (Molecular
Dynamics, Sunnyvale, CA) and quantified with Molecular Dynamics Image
Quant software, version 4.2a.
Cell Lysis, Immunoprecipitation, and Western
Blotting--
Immunoprecipitations were performed as described
previously (12). For GHR immunoprecipitations, cells were lysed on ice in 0.3 ml of lysis mix containing 1% Triton X-100, 1 mM
EDTA, 50 mM NaF, 1 mM
Na3VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, 2 µM
carbobenzoxy-L-leucyl-L-leucyl-L-leucinal,
and 100 mM phenylmethylsulfonyl fluoride in PBS. In
ubiquitin immunoprecipitation experiments the cells were lysed in 0.3 ml boiling lysis buffer containing 1% SDS, 1 mM EDTA, 50 mM NaF, and 1 mM
Na3VO4. The lysate was heated for 5 min at
100 °C, after which the DNA was sheared using a 25-G needle.
Immunoprecipitation of the supernatant was carried out in 1% Triton
X-100, 0.5% SDS, 0.25% sodium deoxycholate, and 0.5% BSA in PBS plus
various inhibitors. For anti-GH immunoprecipitations of GH-GHR
complexes, the cells were lysed on ice in a lysis mix containing 1%
Triton X-100, 150 mM NaCl, 10% glycerol, 50 mM
Tris-HCl, pH 8.0, 10 mM N-ethylmaleimide, and
various inhibitors. The immunoprecipitation was carried out in the same
buffer. The lysates were incubated with the indicated antibodies for
2 h on ice, and immune complexes were isolated using protein
A-agarose beads (Repligen Co., Cambridge, MA). The immunoprecipitates
were washed twice with the same buffer and twice with 10-fold diluted
PBS. Immune complexes were subjected to SDS-polyacrylamide gel
electrophoresis and immunoblotting as described (14). For detection the
enhanced chemiluminescence system (Roche Molecular Biochemicals) was
used. To reprobe blots, the membranes were incubated for 1 h at
room temperature in 0.15 M NaCl, 50 mM glycine,
pH 2.5, buffer. The efficiency of the stripping procedure was checked
and was found to remove >95% of the signal.
Potassium Depletion--
Cells were subjected to potassium
depletion as described previously (14, 18). All incubation steps were
performed at 30 °C. Cells were washed twice with isotonic,
potassium-free buffer A, (0.14 M NaCl, 20 mM
Hepes, 1 mM CaCl2, 1 mM
MgCl2, 1 g/l glucose, and 0.1% BSA, pH 7.4) and subjected
to a hypotonic shock for 5 min in buffer A diluted 1:1 with
H2O. After incubation in buffer A for 30 min, GH was added
for an additional 30 min. Parallel cultures, which had also been
hypotonically shocked, were incubated in buffer A supplemented with 10 mM KCl.
Light Microscopy--
Fluorescently labeled GH (Cy3-GH)
was prepared as described before (16). Cells grown on coverslips were
incubated in MEM
supplemented with 20 mM Hepes, pH 7.4, and 0.1% BSA. Cy3-GH (0, 8 µg/ml) was added, and the incubation was
continued. Cells were washed with PBS to remove unbound label and fixed
for 2 h in 3% paraformaldehyde in PBS. After fixation, the cells
were embedded in Mowiol, and confocal laser scanning microscopy was
performed using a Leica TCS 4D system.
Immunogold Electron Microscopy--
Cells were incubated in
MEM
plus 0.1% BSA and 8 nM biotinylated GH (21) in a
CO2 incubator. Before fixation and processing for
immunoelectron microscopy, cells were washed three times with MEM
and 0.1% BSA. The cells were fixed in 2% paraformaldehyde and 0.2%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min on ice followed by 3 h at room temperature. Further processing for ultrathin cryosectioning and labeling according to the protein A-gold method was done as described previously (22). To pick up
ultrathin cryosections, a 1:1 mixture of 2.3 M sucrose and 1.8% methylcellulose was used (23). A rabbit polyclonal antibody against biotin was directly visualized by protein A-gold. A mouse monoclonal antibody against clathrin was first incubated with rabbit
anti-mouse IgG, to provide binding sides for protein A, which binds
poorly to mouse antibodies.
The effect of M
CD on clathrin-coated pit morphology was quantified
by determining the number of clathrin-coated structures at the plasma
membrane. M
CD-treated and control cells were labeled for clathrin,
and the plasma membrane of 50 cell profiles with visible nucleus was
screened for clathrin-coated structures, which were subdivided into
four categories: 1) flat or slightly invaginated coated pits, 2)
invaginated coated pits with a wide opening, 3) invaginated coated pits
with a constricted neck, and 4) vesicles in close vicinity to the
plasma membrane, which were not connected to the plasma membrane in the
plane of the section. The frequency of each category was expressed as a
percentage of the total number of clathrin-coated structures at the
plasma membrane.
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RESULTS |
M
CD Inhibits the Endocytosis of the GHR--
The GHR enters the
cell via clathrin-coated pits (14, 24, 25). Previously we used methods
that deplete the cytosol of free clathrin triskelions and interfere
with the early stages of coated pit formation (18, 26). These methods
inhibited GHR internalization and abolished GHR ubiquitination (14),
suggesting that GHR ubiquitination occurs during or after coated pit
formation. Acute cholesterol depletion of the plasma membrane using
M
CD inhibits clathrin-coated pit budding. This method reduces the internalization of the transferrin receptor by >85% (27). Cholesterol depletion results in the formation of shallow coated pits, indicating that cholesterol is essential for clathrin coated vesicle formation (28). To monitor GH uptake, GHR-expressing ts20 cells were incubated for 30 min in the presence of Cy3-labeled GH, which resulted in the
presence of Cy3-GH in endosomal and lysosomal compartments (Fig.
1A, Control). As expected, the
Cy3-GH was protected against acid treatment, confirming that the label
is in intracellular structures (Fig. 1A, Acid Wash). When
cells were preincubated in the presence of M
CD, virtually no Cy3-GH
entered the cells (Fig. 1A, M
CD), and the
majority of label could be removed on acid treatment (Fig. 1A,
Acid Wash). Neither uptake nor binding was observed when excess
unlabeled ligand was added together with Cy3-GH or when untransfected
cells were used (data not shown).

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Fig. 1.
Effect of M CD on GH
internalization. A, GHR-expressing ts20 cells were
incubated for 30 min at 30 °C in the absence (Control) or
presence of 10 mM M CD and incubated with Cy3-GH for 30 min. Cells were fixed before (30 min, 30 °C) or after acid wash
(Acid Wash). Cy3-GH was visualized by confocal microscopy.
B, GHR-expressing ts20 cells were incubated for 30 min at
30 °C in the absence (control) or presence of 10 mM
M CD and put on ice for 2 h with 125I-GH. Unbound
label was removed, and the cells were incubated at 30 °C in the
absence or presence of M CD as indicated. Background label was
determined in the presence of excess unlabeled GH and subtracted. The
amounts of 125I-GH internalized are plotted as a percentage
of the cell-associated radioactivity at the start of the incubation.
Each point represents the mean value of two experiments
performed in duplicate ± S.D. , control; , M CD.
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To confirm and quantify the effect of M
CD on endocytosis, uptake of
125I-GH was measured in a time course experiment. As seen
in Fig. 1B, M
CD inhibited the internalization of GH
efficiently. There was no effect of M
CD on the total binding of
125I-GH to the cells (data not shown). Two control
experiments were performed to ascertain that M
CD treatment did not
affect other relevant cellular processes. The effect of M
CD on GHR
biosynthesis was measured using pulse-chase labeling with
[35S]methionine (Fig.
2A). The receptor was
synthesized as a 110-kDa glycoprotein precursor (double band; Fig.
2A, p) and on "complex glycosylation"
in the Golgi complex converted to a 130-kDa mature species (Fig.
2A, m). Quantification of the radioactivity showed that the
GHR signal in the M
CD cells is ~85-90% of the control cells,
indicating a slight inhibition of protein synthesis. Conversion of
precursor to mature receptor was detectable after 30 min of chase both
in control and in M
CD-treated cells, indicating that transport to
the Golgi compartment was not affected by the cholesterol depletion. To
examine the effect of M
CD on GHR phosphorylation, a second control
experiment was performed. Allevato and colleagues (29) showed that a
mutated GHR, deficient in internalization, was capable of stimulating
transcription of the serine protease inhibitor 2.1 promoter, and we
showed that the GHR cytosolic tail is tyrosine-phosphorylated, while
internalization is inhibited (14, 20). From these studies it was
concluded that GHR phosphorylation is independent of GHR endocytosis.
M
CD-pretreated cells were incubated for various periods with GH. As
seen in Fig. 2B, phosphorylation became detectable after 5 min of incubation, reaching a maximum after 15 min for both control and
M
CD-treated cells. The blot was reprobed with anti-GHR to show that
comparable amounts of receptor were loaded in each lane. From these
results we conclude that M
CD treatment has no effect on GH-induced
phosphorylation of the GHR, indicating that activation of the tyrosine
kinase (Janus kinase 2) and receptor dimerization can take place
in the presence of M
CD.

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Fig. 2.
Effect of M CD on the
biosynthesis and the tyrosine-phosphorylation of the GHR.
A, GHR-expressing ts20 cells were incubated for 30 min at
30 °C in methionine-free medium in the absence (Control)
or presence of 10 mM M CD. [35S]Methionine
was added, and the incubation was continued for 10 min. Cells were
chased in MEM supplemented with 0.1% BSA, 100 µM
methionine, and 8 nM GH in the absence or presence of
M CD for the periods indicated. Upper panel, GHR was
immunoprecipitated using anti-T; p, precursor GHR (110 kDa.); m, mature GHR (130 kDa.). Lower panel,
radioactivity was quantified and expressed as a percentage of the
radioactivity incorporated in the precursor GHR. , precursor GHR;
, mature GHR. B, GHR-expressing ts20 cells were incubated
at 30 °C in the absence (control) or presence of 10 mM
M CD. All dishes were incubated for 60 min in total; GH was present
during the last 5, 15, or 30 min. Upper panel, cells were
lysed, and the GHR was immunoprecipitated with anti-T and immunoblotted
using anti-pY. Lower panel, the same blot was
reprobed using anti-GHR (mAb5). p, precursor GHR (110 kDa.);
m, mature GHR (130 kDa.).
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M
CD Does Not Affect Accessibility of Clathrin-coated Pits for
GHR--
Previously, it was shown that M
CD does not interfere with
the association of clathrin to the plasma membrane but has an
inhibitory effect on the invagination and fission of clathrin-coated
pits (28). For the present study it was important to determine whether the GHR enters these clathrin-coated areas at the plasma membrane of
M
CD-treated cells. Immunogold labeling of clathrin in GHR-expressing ts20 cells revealed that the majority of plasma membrane-associated clathrin was present on deeply invaginated pits and coated vesicles in
close vicinity to the plasma membrane (Fig.
3A and Table
I). GH was regularly found in the deeply
invaginated clathrin-coated pits and vesicles (Fig. 3, C and
D), as well as in later compartments of the endocytic
pathway (data not shown). In M
CD-treated cells, the total number of
clathrin-coated structures at the plasma membrane was increased twice
compared with that in control cells. In contrast to control cells,
>85% of the clathrin-containing structures were flattened coated pits
(Fig. 3B). Deeply invaginated coated pits and coated
vesicles were rarely seen (Table I). GH accumulated at the plasma
membrane, where it regularly but not exclusively occurred in the
flattened clathrin-coated pits (Fig. 3E). These findings
suggest that the GH-GHR complex in M
CD-treated cells indeed enters
the clathrin-coated areas of the plasma membrane.

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Fig. 3.
M CD does not effect
accessibility of clathrin-coated pits for GHR. Ultrathin
cryosections show plasma membrane (pm) regions of
GHR-expressing ts-20 cells incubated 30 min without (A, C,
and D) or with (B and E) 10 mM M CD before addition of biotinylated GH. Sections were
labeled for clathrin (A and B) or biotinylated GH
(C-E). A, in control cells, clathrin (10-nm
gold) was associated with invaginated clathrin-coated pits. The dense
cytosolic coating is characteristic of the presence of clathrin.
B, in M CD-treated cells, clathrin (10-nm gold) assembled
in longer stretches along the plasma membrane, and only flattened
invaginations were formed. C and D, biotinylated
GH (10-nm gold) had access to deeply invaginated clathrin-coated pits
in control cells, as well as to the flattened clathrin-coated pits in
M CD-treated cells (E). n, nucleus. Scale
bars, 200 nm.
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Effect of M
CD on GHR Ubiquitination--
To address the
question of whether the GHR is ubiquitinated after M
CD treatment,
cells were incubated with or without GH, and ubiquitinated proteins
were immunoprecipitated and analyzed by Western blotting, as indicated
in Fig. 4. Ubiquitinated GHR appeared as
high molecular weight species in the top part of the gel (Fig.
4A). Control cells (lanes 3 and 4)
showed increased GHR ubiquitination on ligand binding. Both in
unstimulated and stimulated cells the level of GHR ubiquitination
increased when M
CD was present (Fig. 4A, compare
lane 3 with lane 9 and lane 4 with
lane 10). The use of untransfected cells resulted, as
expected, in the absence of signal for ubiquitinated GHR (ts20; Fig.
4A, lanes 1 and 2). When endocytosis was
inhibited by potassium depletion, GHR ubiquitination was almost
completely abolished (lanes 5 and 6).
Ubiquitination was restored to control values by adding 10 mM KCl (lanes 7 and 8). Reprobing the
blot from Fig. 4A with anti-ubiquitin showed that the amount
of immunoprecipitated, ubiquitinated protein in each lane was
comparable (Fig. 4B). These results show that the GHR is
ubiquitinated at the cell surface before constriction of the coated pit
occurs and suggest that assembly of the clathrin coat is a requirement
for GHR ubiquitination.

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Fig. 4.
Effect of potassium depletion and
M CD on GHR ubiquitination. GHR-expressing
ts20 cells (lanes 3-10) or untransfected cells (ts20;
lanes 1 and 2) were incubated with medium alone
(lanes 1-4), in the absence
( K+; lanes 5 and 6) or
presence (+K+; lanes 7 and
8) of potassium after hypotonic shock, or with 10 mM M CD (lanes 9 and 10) for 30 min
at 30 °C. The incubation was continued for 30 min with (lanes
2, 4, 6, 8, and 10, +GH) or without GH.
A, cells were lysed with boiling lysis buffer; equal amounts
of cell lysates were immunoprecipitated with anti-ubiquitin and
analyzed by immunoblotting with anti-GHR (mAb5). B, the same
blot was reprobed with anti-ubiquitin (Ubi). Relative
molecular weight standards (Mr × 10 3) are shown at the left.
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Effect of a Temperature-sensitive Dynamin Mutation on GHR
Ubiquitination--
To examine the effect of inhibition of
clathrin-mediated endocytosis on GHR ubiquitination by an independent
method, we used a HeLa cell line expressing a temperature-sensitive
mutant of human dynamin, dynTS. DynTS carries a
point mutation corresponding to the Drosophila
shibirets1 allele (30). For this dynamin mutant it has
been shown that transferrin internalization is inhibited at the
nonpermissive temperature, that the phenotype is rapid and reversible,
and that invaginated but not constricted coated pits accumulate on the cytoplasmic surface of the plasma membrane. HeLa cells were transiently transfected with wild-type GHR cDNA and incubated in the presence of GH; afterward the GH-GHR complex was immunoprecipitated with anti-GH. Using this approach, only mature GHR species from the cell
surface were immunoprecipitated. At the permissive temperature, ubiquitinated protein was detected in both the wild-type and mutant dynamin cells (Fig. 5A,
anti-Ubi). After shifting the cells to the nonpermissive
temperature, an increase in the amount of ubiquitinated GHR was
detected in dynTS and wild-type dynamin cells (Fig.
5A, anti-Ubi). Ubiquitination of proteins is dynamic with
rapid addition and removal of ubiquitin (31). The increase in
ubiquitination in the wild-type dynamin cells might reflect the higher
endocytotic activity of the cells caused by the elevated temperature or
a temperature-dependent shift in balance between
ubiquitinating and deubiquitinating enzymes. The increase in amount of
ubiquitinated GHR in the dynTS cells at the nonpermissive
temperature was consistently severalfold higher compared with wild-type
dynamin cells. The data indicate that GHR ubiquitination precedes GHR
endocytosis, which is in agreement with the experiments in which M
CD
was used. Reprobing the blot with anti-GHR showed that the amounts of
mature receptor expressed in dynTS and wild-type dynamin
cells were comparable (Fig. 5A, anti-GHR). The amount of
ubiquitinated GHR is only a small fraction of the total amount of GHR
bound to GH (see below). The expression of HA-tagged dynamin in the
HeLa cells was controlled on a Western blot of cell lysate using
anti-HA (Fig. 5A, anti-HA). Both dynTS and
wild-type dynamin cells expressed the HA-tagged dynamin, albeit in
different amounts, whereas cells cultured in the presence of
tetracycline showed no detectable HA signal (results not shown). To
ascertain that indeed GH uptake was inhibited, we measured internalization of 125I-GH. At the permissive temperature
the percentage of GH uptake was comparable in the two cell lines,
whereas at the nonpermissive temperature GH internalization was
strongly inhibited in dynTS cells compared with wild-type
dynamin cells (Fig. 5B). The results demonstrate that
overexpression of a dominant negative mutant of dynamin-1 inhibits the
clathrin-mediated endocytosis of the GHR. Because we analyzed a complex
of proteins immunoprecipitated with anti-GH to monitor ubiquitination
of the GHR, the possibility exists that other ubiquitinated proteins
coimmunoprecipitate in this complex. To show that the ubiquitination of
this complex depends on the GHR, we transfected the
internalization-deficient GHR(F327A) mutant in dynTS cells.
Previously, we have shown that this mutant is not ubiquitinated because
of a defective ubiquitin-dependent endocytosis motif (15). After immunoprecipitation of the GH-GHR(F327A) complex, almost no
ubiquitinated protein was isolated either at the permissive or the
nonpermissive temperature (Fig. 5C, left panel). This result shows that the ubiquitination of the GH-GHR complex is dependent on an
intact ubiquitin-dependent endocytosis motif and that most likely the receptor itself is ubiquitinated, perhaps in complex with
other ubiquitinated proteins. Control experiments with mock-transfected cells and incubations without GH showed no signal on the
ubiquitin blots (results not shown). As seen in the Fig. 5C,
right panel, mature GHR and GHR(F327A) were detected in the
complex, and virtually no GHR signal is present at the top of the
lanes, indicating that only a small percentage of total GHR is
ubiquitinated. Most likely, the GHR is ubiquitinated during a very
short period, presumably the resident time in the coated pit.

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Fig. 5.
Effect of overexpression of the
dynTS mutant on GHR ubiquitination and
internalization. A, Wild-type (wtDyn) and
mutant (DynTS) dynamin cells were transfected with
GHR cDNA. Cells were incubated at the permissive or nonpermissive
temperature for 30 min; afterward, GH was added to all dishes, and the
incubation was continued for 30 min. Left panel, cells were
lysed on ice; equal amount of cell lysates were immunoprecipitated with
anti-GH; and the immunoprecipitates were analyzed by immunoblotting
with anti-ubiquitin (anti-Ubi). Middle panel, the
anti-ubiquitin blot was reprobed with anti-GHR (mAb5). Right
panel, equal amounts of cell lysates were loaded on a gel and
immunoblotted with anti-HA. Relative molecular weight standards
(Mr × 10 3) are shown
at the left. B, wild-type (wtDyn) and
mutant (DynTS) dynamin cells were transfected with
GHR cDNA. Cells were incubated at the permissive or nonpermissive
temperature for 30 min; afterward, 125I-GH was added to all
dishes, and the incubation was continued for 30 min. Background signal
was measured in the presence of excess unlabeled GH and subtracted.
Internalization in the dynTS mutant was compared with the
internalization in the wtdyn; specifically internalized
125I-GH was calculated as a percentage of total
cell-associated label and set to 100% for the wtdyn cells. The amount
of cell surface expression of GHR was comparable in the two cell lines;
mock-transfected cells showed no detectable specific binding of
125I-GH (data not shown). The values are the mean ± S.D. of two experiments performed in duplicate. C, mutant
(dynTS) dynamin cells were transfected with wild-type GHR
(wt) or GHR(F327A) cDNA (F327A). Experimental
conditions and immunoprecipitations were as described in A. Left panel, the Western blot was detected with
anti-ubiquitin (anti-Ubi). Right panel, the same
blot was reprobed with anti-GHR (mAb5).
|
|
 |
DISCUSSION |
In this study two independent methods were used to inhibit
clathrin-mediated endocytosis at the level of clathrin-coated vesicle formation. Both methods inhibited the endocytosis of the GHR, resulting
in an accumulation of ubiquitinated receptors at the cell surface. A
morphological approach detected the GHR in deeply invaginated coated
pits in control cells and in flattened clathrin-coated pits in
M
CD-treated cells. Disruption of clathrin-mediated endocytosis by
hypertonic medium treatment, cytosol acidification, or potassium depletion resulted in the accumulation of nonubiquitinated receptors at
the cell surface (Ref. 14 and Fig. 4A). Why is the GHR not ubiquitinated under these conditions? The ubiquitination state of a
protein is the result of a dynamic process of ubiquitination and
deubiquitination. Changing the intracellular milieu by depleting potassium or modifying the pH could alter the balance between those two
processes. Do the used methods interfere with the ubiquitination machinery itself and cause a complete inhibition of cellular
ubiquitination? Analysis of cell lysates from potassium-depleted or
hypertonic medium-treated cells showed no reduction in total cellular
ubiquitin conjugates but rather an increase in high molecular mass
ubiquitinated proteins (Ref. 14 and Fig. 4B). The amount of
free ubiquitin under these circumstances as measured with Western
blotting was reduced (data not shown). However, cellular cytosol
acidification showed increased free and less conjugated ubiquitin (data
not shown). Cytosol acidification causes the same precipitation of small clathrin microcages as seen after hypertonicity and potassium depletion (32). Recently, it was shown that after hypertonic treatment
or cytoplasmic acidification, free clathrin triskelions within the
cytosol are depleted, and all of the clathrin becomes associated with
membranes (26). Because the presence of free clathrin triskelions is
required for the stabilization of AP-2 coated pit nucleation sites,
depletion of clathrin interferes with coated pit formation. Because the
methods used have a varying effect on the ratio of free
versus conjugated ubiquitin, it is most likely that the
inhibition of GHR ubiquitination is the result of the interference with
the coated pit formation rather than with ubiquitin conjugation itself.
The observation that GHR ubiquitination coincides with the recruitment
of the GHR to clathrin-coated membrane areas suggests that the
ubiquitin conjugation system and the endocytosis machinery act together
in the endocytosis of the GHR. The earlier observation that
ubiquitination of the receptor itself is not important for endocytosis
suggests that ancillary proteins might be ubiquitinated or that factors
of the ubiquitin conjugation system itself might act as adaptors for
the endocytosis machinery. Ubiquitin-protein ligases have been
implicated in endocytosis. For the epithelial sodium channel, it was
shown that the ubiquitin-protein ligase Nedd4 mediates the
down-regulation of the Na+ channel activity by
ubiquitinating the channel, which leads to its endocytosis and
degradation (33). The yeast homologue of Nedd4, Npi1/Rsp5, participates
via its C2 domain in the endocytosis of Gap1 permease. A truncated Npi1
protein lacking the C2 domain can still promote ubiquitination but not
the endocytosis of Gap1 permease, which is consistent with direct
participation of Npi1 in endocytosis of the permease (34). Whether an
E2/E3 ubiquitin ligase directly serves as an endocytic adaptor for GHR,
analogous to the role of arrestin for the
2-adrenergic receptor (8) or binding of the ubiquitin conjugation system, results in the interaction with an endocytic adaptor (e.g. AP-2), remains
to be established. Recently, GH-dependent association of
AP-2 with the chicken GHR was reported (35). Also, a possible role for the ubiquitin polypeptide itself, conjugated to a GHR-associated protein, cannot be excluded, as has been described for Ste2p (36) and
Ste3p (37). Ligand-induced ubiquitination was shown for Eps15, a
clathrin-coated pit associated protein that is ubiquitinated on
epidermal growth factor (EGF) receptor activation (38). Eps15 is
required for clathrin-mediated endocytosis, and perturbation of Eps15
function inhibits receptor-mediated endocytosis of transferrin (39).
The biological significance of Eps15 monoubiquitination is not known.
Recently, it was shown that polyubiquitination of the EGF receptor
occurs at the plasma membrane on ligand-induced activation (40).
Inhibition of endocytosis caused by overexpression of mutant dynamin
resulted in a transient polyubiquitination of the EGF receptor. The
mechanisms for GHR and EGF receptor ubiquitination are probably
different. Ubiquitination of the EGF receptor is mediated by Cbl
adaptor proteins, and both EGF receptor and Cbl must undergo
phosphorylation on specific sites for productive ubiquitination (41),
whereas GHR ubiquitination occurs in the absence of GHR tyrosine
phosphorylation and is dependent on the ubiquitin-dependent
endocytosis motif (20).
The amount of ubiquitinated GHR is very low compared with the total
amount of cell surface GHR. The fact that only a small percentage of
total GHR is ubiquitinated indicates a coated pit restricted function
of the ubiquitin conjugation system. Whether the ubiquitinated GHR is
(partially) degraded soon after endocytosis or perhaps rapidly
deubiquitinated is not clear at present. A role for deubiquitinating
enzymes in regulating endocytosis cannot be excluded, because
deubiquitination has been recognized as an important regulatory step
(31, 42). Recently, genetic data were presented that support a model
whereby the Drosophila fat facets deubiquitinating enzyme
removes ubiquitin from the product of the liquid facets
gene. The liquid facets locus encodes epsin, a protein
involved in clathrin-mediated endocytosis (43). Thus the ubiquitin
system may add another layer of complexity to the membrane-sorting
machinery at the plasma membrane and regulate, together with the
classical endocytosis machinery, the time span of the GHR at the cell surface.
 |
ACKNOWLEDGEMENTS |
We thank Rene Scriwanek for the excellent
preparation of EM photographs, Dr. Guojun Bu for carefully reading the
manuscript, Jürgen Gent, Julia Schantl, Cristina Alves dos
Santos, and Toine ten Broeke for stimulating discussions, Dr. William
Wood (Genentech) for providing the GHR cDNA, Dr. A. Ciechanover for
the kind gift of anti-ubiquitin antibody, and Dr. S. Schmid for
providing the HeLa cells.
 |
FOOTNOTES |
*
This work was supported by Netherlands Organization for
Scientific Research Grant NWO-902-23-lg2) and European Union Network Grant ERBFMRXCT96-0026.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cell Biology,
University Medical Center Utrecht and Institute of Biomembranes, Heidelberglaan 100, AZU-G02.525, 3584CX Utrecht, The Netherlands. Tel.:
31-30-250-6476; Fax: 31-30-254-1797; E-mail: strous@med.uu.nl.
Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M007326200
 |
ABBREVIATIONS |
The abbreviations used are:
GHR, growth hormone
receptor;
GH, growth hormone;
M
CD, methyl-
-cyclodextrin;
mAb, monoclonal antibody;
HA, hemagglutinin;
MEM, minimal essential medium;
dynTS, temperature-sensitive mutant of dynamin;
wtdyn, wild-type dynamin;
PBS, phosphate-buffered saline;
BSA, bovine serum
albumin;
EGF, epidermal growth factor.
 |
REFERENCES |
1.
|
Mellman, I.
(1996)
Annu. Rev. Cell Dev. Biol.
12,
575-625[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Schmid, S. L.
(1997)
Annu. Rev. Biochem.
66,
511-548[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Chen, W.-J.,
Goldstein, J. L.,
and Brown, M. S.
(1990)
J. Biol. Chem.
265,
3116-3123[Abstract/Free Full Text]
|
4.
|
Collawn, J. F.,
Stangel, M.,
Kuhn, L. A.,
Esekogwu, V.,
Jing, S. Q.,
Trowbridge, I. S.,
and Tainer, J. A.
(1990)
Cell
63,
1061-1072[Medline]
[Order article via Infotrieve]
|
5.
|
Haft, C. R.,
Klausner, R. D.,
and Taylor, S. I.
(1994)
J. Biol. Chem.
269,
26286-26294[Abstract/Free Full Text]
|
6.
|
Gabilondo, A. M.,
Hegler, J.,
Krasel, C.,
Boivinjahns, V.,
Hein, L.,
and Lohse, M. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
12285-12290[Abstract/Free Full Text]
|
7.
|
Kirchhausen, T.
(1999)
Annu. Rev. Cell Dev. Biol.
15,
705-732[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Goodman, O. B.,
Krupnick, J. G.,
Santini, F.,
Gurevich, V. V.,
Penn, R. B.,
Gagnon, A. W.,
Keen, J.,
and Benovic, J. L.
(1996)
Nature
383,
447-450[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Strous, G. J.,
and Govers, R.
(1999)
J. Cell Sci.
112,
1417-1423[Abstract/Free Full Text]
|
10.
|
Hicke, L.
(1999)
Trends Cell Biol.
9,
107-112[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Staub, O.,
Gautschi, I.,
Ishikawa, T.,
Breitschopf, K.,
Ciechanover, A.,
Schild, L.,
and Rotin, D.
(1997)
EMBO J.
16,
6325-6336[Abstract/Free Full Text]
|
12.
|
Strous, G. J.,
van Kerkhof, P.,
Govers, R.,
Ciechanover, A.,
and Schwartz, A. L.
(1996)
EMBO J.
15,
3806-3812[Abstract]
|
13.
|
Leung, D. W.,
Spencer, S. A.,
Cachianes, G.,
Hammonds, R. G.,
Collins, C.,
Henzel, W. J.,
and Wood, W. I.
(1987)
Nature
330,
537-544[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Govers, R.,
van Kerkhof, P.,
Schwartz, A. L.,
and Strous, G. J.
(1997)
EMBO J.
16,
4851-4858[Abstract/Free Full Text]
|
15.
|
Govers, R.,
ten Broeke, T.,
van Kerkhof, P.,
Schwartz, A. L.,
and Strous, G. J.
(1999)
EMBO J.
18,
28-36[Abstract/Free Full Text]
|
16.
|
van Kerkhof, P.,
Govers, R.,
Alves Dos Santos, C. M.,
and Strous, G. J.
(2000)
J. Biol. Chem.
275,
1575-1580[Abstract/Free Full Text]
|
17.
|
Larkin, J. M.,
Brown, M. S.,
Goldstein, J. L.,
and Anderson, R. G. W.
(1983)
Cell
33,
273-285[Medline]
[Order article via Infotrieve]
|
18.
|
Hansen, S. H.,
Sandvig, K.,
and VanDeurs, B.
(1993)
J. Cell Biol.
121,
61-72[Abstract]
|
19.
|
Sandvig, K.,
Olsnes, S.,
Petersen, O. W.,
and van Deurs, B.
(1987)
J. Cell Biol.
105,
679-689[Abstract]
|
20.
|
Strous, G. J.,
van Kerkhof, P.,
Govers, R.,
Rotwein, P.,
and Schwartz, A. L.
(1997)
J. Biol. Chem.
272,
40-43[Abstract/Free Full Text]
|
21.
|
Bentham, J.,
Aplin, R.,
and Norman, M. R.
(1994)
J. Histochem. Cytochem.
42,
103-107[Abstract/Free Full Text]
|
22.
|
Slot, J. W.,
Geuze, H. J.,
Gigengack, S.,
Lienhard, G. E.,
and James, D. E.
(1991)
J. Cell Biol.
113,
123-135[Abstract]
|
23.
|
Liou, W.,
Geuze, H. J.,
and Slot, J. W.
(1996)
Histochem. Cell Biol.
106,
41-58[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Ilondo, M. M.,
Courtoy, P. J.,
Geiger, D.,
Carpentier, J.,
Rousseau, G. G.,
and de Meyts, P.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
6460-6464[Abstract]
|
25.
|
Ilondo, M. M.,
Smal, J.,
DeMeyts, P.,
and Courtoy, P. J.
(1991)
Endocrinology
128,
1597-1602[Abstract]
|
26.
|
Brown, C. M.,
and Petersen, N. O.
(1999)
Biochem. Cell Biol.
77,
439-448[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Subtil, A.,
Gaidarov, I.,
Kobylarz, K.,
Lampson, M. A.,
Keen, J. H.,
and Mcgraw, T. E.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6775-6780[Abstract/Free Full Text]
|
28.
|
Rodal, S. K.,
Skretting, G.,
Garred, O.,
Vilhardt, F.,
Vandeurs, B.,
and Sandvig, K.
(1999)
Mol. Biol. Cell
10,
961-974[Abstract/Free Full Text]
|
29.
|
Allevato, G.,
Billestrup, N.,
Goujon, L.,
Galsgaard, E. D.,
Norstedt, G.,
and Nielsen, J. H.
(1995)
J. Biol. Chem.
270,
17210-17214[Abstract/Free Full Text]
|
30.
|
Damke, H.,
Baba, T.,
van der Bliek, A. M.,
and Schmid, S. L.
(1995)
J. Cell Biol.
131,
69-80[Abstract]
|
31.
|
Hochstrasser, M.
(1996)
Annu. Rev. Genet.
30,
405-439[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Heuser, J.
(1989)
J. Cell Biol.
108,
401-411[Abstract]
|
33.
|
Harvey, K. F.,
Dinudom, A.,
Komwatana, P.,
Jolliffe, C. N.,
Day, M. L.,
Parasivam, G.,
Cook, D. I.,
and Kumar, S.
(1999)
J. Biol. Chem.
274,
12525-12530[Abstract/Free Full Text]
|
34.
|
Springael, J. Y.,
Decraene, J. O.,
and Andre, B.
(1999)
Biochem. Biophys. Res. Commun.
257,
561-566[CrossRef][Medline]
[Order article via Infotrieve]
|
35.
|
Vleurick, L.,
Pezet, A.,
Kuhn, E. R.,
Decuypere, E.,
and Edery, M.
(1999)
Mol. Endocrinol.
13,
1823-1831[Abstract/Free Full Text]
|
36.
|
Shih, S. C.,
Sloper-Mould, K. E.,
and Hicke, L.
(2000)
EMBO J.
19,
187-198[Abstract/Free Full Text]
|
37.
|
Roth, A. F.,
and Davis, N. G.
(2000)
J. Biol. Chem.
275,
8143-8153[Abstract/Free Full Text]
|
38.
|
van Delft, S.,
Govers, R.,
Strous, G. J.,
Verkleij, A. J.,
and van Bergen en Henegouwen, P. M.
(1997)
J. Biol. Chem.
272,
14013-14016[Abstract/Free Full Text]
|
39.
|
Benmerah, A.,
Bayrou, M.,
Cerfbensussan, N.,
and Dautry-Varsat, A.
(1999)
J. Cell Sci.
112,
1303-1311[Abstract/Free Full Text]
|
40.
|
Stang, E.,
Johannessen, L. E.,
Knardal, S. L.,
and Madshus, I. H.
(2000)
J. Biol. Chem.
275,
13940-13947[Abstract/Free Full Text]
|
41.
|
Levkowitz, G.,
Waterman, H.,
Ettenberg, S. A.,
Katz, M.,
Tsygankov, A. Y.,
Alroy, I.,
Lavi, S.,
Iwai, K.,
Reiss, Y.,
Ciechanover, A.,
Lipkowitz, S.,
and Yarden, Y.
(1999)
Mol. Cell
4,
1029-1040[Medline]
[Order article via Infotrieve]
|
42.
|
Chung, C. H.,
and Baek, S. H.
(1999)
Biochem. Biophys. Res. Commun.
266,
633-640[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Cadavid, A. L.,
Ginzel, A.,
and Fischer, J. A.
(2000)
Development
127,
1727-1736[Abstract/Free Full Text]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.