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Received I7 February 200 I
The ubiquitin-proteasome pathway regulates lysosomal degradation of the
growth hormone receptor and i t s ligand
P. van Kerkhof and G. J. Strous'
Department of Cell Biology, University Medical Center Utrecht and Institute of Biomembranes, Heidelbeqlaan 100,
3584 CX Utrecht, The Netherlands
Abstract
a n t i b o d i e s against d i f f e r e n t r e c e p t o r t a i l sections
s h o w t h a t d e g r a d a t i o n of t h e GHR c y t o s o l i c
d o m a i n precedes d e g r a d a t i o n of t h e e x t r a c e l l u l a r
GH-binding d o m a i n . A p o s s i b l e r o l e for t h e
u b i q u i t i n - p r o t e a s o m e p a t h w a y in t h e d e g r a d a t i o n
of t h e r e c e p t o r a n d l i g a n d i s discussed.
T h e growth h o r m o n e (GH) r e c e p t o r (GHR) i s a
m a m m a l i a n p l a s m a m e m b r a n e p r o t e i n w h o s e int e r n a l i z a t i o n i s m e d i a t e d by t h e ubiquitinp r o t e a s o m e p a t h w a y . GH i n t e r n a l i z a t i o n a n d deg r a d a t i o n are i n h i b i t e d w h e n cells are t r e a t e d with
proteasome i n h i b i t o r s . H e r e w e s h o w t h a t a GHR
t r u n c a t e d a t r e s i d u e 369 c a n e n t e r t h e cells in t h e
presence of a proteasome inhibitor, but t h a t t h e
subsequent l y s o s o m a l d e g r a d a t i o n of GH i s
b l o c k e d . L y s o s o m a l i n h i b i t o r s prolong t h e h a l f l i f e of both r e c e p t o r a n d l i g a n d . E x p e r i m e n t s with
Introduction
D e g r a d a t i o n of c y t o k i n e receptors g e n e r a l l y
o c c u r s in lysosomes as t h e f i n a l step in s i g n a l
d o w n - r e g u l a t i o n . P r e c e d i n g events take p l a c e a t
t h e c e l l surface, i.e. d i m e r i z a t i o n of two receptors
by a h o r m o n e f o l l o w e d by i n t e r n a l i z a t i o n of t h e
c o m p l e x , a n d in t h e endosome, i.e. e i t h e r s o r t i n g
to t h e l y s o s o m e or r e c y c l i n g b a c k to t h e p l a s m a
membrane. T h e latter possibility i s unlikely a n d
g e n e r a l l y u n w a n t e d , because t h e timing a n d int e n s i t y of c y t o k i n e r e c e p t o r s i g n a l l i n g m u s t b e
Key words: degradation, EGF receptor, endocytosis, lysosome,
ubiquitination.
Abbreviations used: EGF, epidermal growth factor; GH, growth
hormone; GHR growth hormone receptor: UbE, ubiquitindependent endocytosis.
'To whom correspondence should be addressed (e-mail
[email protected]).
0 200 I Biochemical Society
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The Interface of Receptor Signalling and Trafficking
Ligand binding, internalization and
degradation
'251-labelled human G H was prepared using chloramine T [l]. For degradation studies, cells were
incubated with lZ5I-GH (8 nM) for 6 m i n at
30 "C. T h e medium was aspirated and the cells
were washed and incubated in medium without
ligand. At the indicated times, the medium was
collected and precipitated with 1 vol. of ice-cold
20% (v/v) trichloroacetic acid for 30 min on ice.
Acid-soluble radioactivity was determined in the
supernatant after centrifugation and was used as a
measurement of degraded ligand. Membraneassociated ligand was removed by acid wash
(0.15 M NaCl, 50 m M glycine, 0.1 % BSA,
p H 2.5) on ice. Internalized ligand was determined
by measuring the radioactivity after solubilization
of the acid-treated cells in 1 M NaOH using an
LKB y-radiation counter. Non-specific radioactivity was determined in the presence of excess
unlabelled ligand and subtracted.
precisely regulated. Therefore steps in downregulation are tuned such that receptors are used
just once.
For the growth hormone (GH) receptor
(GHR) we have described a mechanism in which
the ubiquitin-proteasome system is involved in its
endocytosis. Since this system regulates many
basic mechanisms, such as cell cycle progression,
apoptosis and transcription control, it is tempting
to speculate about its role in G H R function, and
thus in metabolic processes. Until now, our data
showed that a 10-amino-acid motif within the
G H R cytosolic tail, the UbE (ubiquitin-dependent endocytosis) motif, is involved in endocytosis. In addition, we found that the proteasome
is involved in endocytosis of the full-length receptor, but not of receptors truncated beyond
amino acid residue 369. Such truncated receptors
can enter the cells in the presence of proteasome
inhibitors, but ligand and receptor are not degraded. This may imply that the degradation of
the G H R cytosolic tail starts early, i.e. at the cell
surface or in the endosomes. At the same time, it
confirms earlier observations that most degradation of the GH-GHR complex occurs within
lysosomes. Here we compare the effects of proteasomal and lysosomal inhibitors on endocytosis
and degradation to show that both systems are
required and act together in degradation of the
receptor. T h e data also show that partial degradation of the cytosolic tail precedes complete
lysosomal degradation of the GHR.
Results and discussion
Lysosomal degradationof GH
G H binds to the G H R at the cell surface and
induces receptor dimerization [4]. T h e GH-GHR
complex is internalized via clathrin-coated pits
[5,6], and it has been shown that the G H is
transported to the lysosomes and degraded [7].
Based on the observation that dissociation of the
GH-GHR complex does not occur at the endosoma1 p H of 5.5, it was suggested that, in cultured
rat adipocytes, approx. 75 yo of internalized G H R
is targeted to lysosomes to be degraded [8]. T h e
G H R was initially found to be ubiquitinated upon
amino acid sequencing of the receptor from rabbit
liver [9]. Binding of G H stimulates ubiquitination,
internalization and degradation of the receptor [l].
Ubiquitin plays an essential role as a signal for
protein degradation in eukaryotes [lo]. Multiple
ubiquitin moieties are covalently conjugated to
substrate proteins in the form of a branched
polymeric chain, resulting in the targeting of these
substrates to 26 S proteasomes. In addition to
recognition by the proteasome, ubiquitination is
also involved in the endocytosis and down-regulation of membrane receptors, transporters and
channels [11,121.
Internalization of the G H R is mediated by
the ubiquitin-proteasome pathway via a 10amino-acid motif within the cytosolic tail (UbE
motif) [1,13]. G H R ubiquitination coincides with
Materials and methods
Plasmids, cell culture and transfection
Full-length rabbit G H R cDNA in pCB6, truncated G H R cDNA GHR(369) and mutant GHR(F327A) in pcDNA 3.1 were described previously
[l-31. T h e Chinese hamster cell line ts20, bearing
a thermolabile ubiquitin-activating enzyme E l ,
was used [l]. cDNA constructs were transfected
into ts20 cells using the calcium phosphate
transfection procedure. For all constructs, stably
expressing clonal cell lines were obtained. T h e
ts20 cells were grown at 30 "C in minimal essential
medium tl supplemented with 10 yo (v/v) fetal calf
serum, 4.5 g/l glucose, 100 units/ml penicillin,
100 ,ug/ml streptomycin and 0.45 mg/ml geneticin. For experiments, cells were grown in the
absence of geneticin to a confluence of approx.
75 Yo. Sodium butyrate was added overnight to
increase G H R expression [11.
489
0 2001 Biochemical Society
Biochemical Society Transactions (200 I) Volume 29, part 4
acid-insoluble (intact) lZ5I-GHinto the medium
was consistently less than 5 yo (results not shown).
T o establish that degradation of G H is lysosomal,
cells were incubated in the presence of the lysosoma1 protease inhibitors leupeptin and pepstatin
(Figure 1B). T h e uptake rate was comparable in
treated and control cells, indicating that the
inhibitors did not affect G H internalization. However, in the presence of the inhibitors, the intracellular radioactivity did not decrease after a
prolonged incubation time, which resulted in less
acid-soluble radioactivity in the medium. This
strongly indicates that degradation of GH is
lysosomal.
Next, GHR(F327A) mutant cells or the proteasome inhibitor MG-132 were used to examine
the role of the ubiquitin-proteasome pathway in
the degradation of G H . As shown previously,
mutation of Phe-327 of the G H R UbE motif to
alanine abolished both G H R ubiquitination and
internalization [2]. lZ5I-GHwas initially detected
in the acid-labile cell surface pool of the G H R (F327A) cells (Figure 1C). After prolonged incubation, G H remained predominantly present at
the cell surface, with almost no detectable
degradation of lZ5I-GH, and little intracellular
radioactivity. Incubation of wild-type G H R transfected cells with lZ5I-GHin the presence of
MG-132 resulted in the prolonged presence of the
ligand at the cell surface, and consequently in a
lack of degraded G H (Figure 1D). These observations point to an indirect role for the ubiquitin-proteasome pathway in the degradation of the
GH-GHR complex, via regulation of the transport pathway to the lysosome at the level of
internalization.
Recently, we have shown that a truncated
G H R could enter cells in the presence of proteasome inhibitors, but was not degraded [15].
Here we used the same truncation [GHR(369)] to
monitor the internalization and degradation of
lZ5I-GHin the presence of MG-132 (Figure 1E).
After the initial 6 min of incubation with lZ5I-GH
( t = 0 min of chase), more than 40% of the
radioactivity was acid-resistant, increasing to 75 yo
after 15 min of incubation. Although internalization was not inhibited by the proteasome
inhibitor, no acid-soluble radioactivity was detected in the medium, indicating that MG-132
completely blocked the degradation of GH.
Therefore the ubiquitin-proteasome pathway is
involved in degradation of the GH-GHR complex
not only at the level of internalization, but also in
endosome-to-lysosome sorting.
Figure I
Endocytosis and degradation of '251-GH
Cells were incubated for 6 min at 30 "C with 8 nM 1251-GH.
The
ligand was removed and the cells were incubated forthe indicated
times at 30 "C. At each time pointthe amounts ofcell surface
internalized ( 0 )and degraded (A)ligand were determined as
described in the Materials and methods section. The amount of
1251-GH
is plotted as a percentage of total radioactivity. (A) Wildtype GHR; (B) wild-type GHR with l00pM leupeptin and
l00pM pepstatin: (C) GHR(F327A); (D) wild-type GHR with
20 p M MG- 132; (E) GHR(369) with 20 p M MG- 132.
(m),
@
gJ75
75
c
88 50
50
25
25
30
0
60 90
0
n
m
0
30
60
90
0
30 60 90
time (min)
" %0 % - z d
time (min)
the recruitment of the receptor to clathrin-coated
membrane areas [14]. Here we studied the role of
the ubiquitin-proteasome pathway in the degradation of G H and G H R . First we examined the
fate of lZ5I-GH after binding to the wild-type
receptor at the cell surface of transfected cells. As
seen in Figure 1(A), upon prolonged incubation,
lZ5I-GHdisappeared rapidly from the acid-labile
cell surface pool. Concomitantly, the pool of
intracellular, acid-resistant radioactivity increased, indicating internalization of the GHG H R complex. After 30min the amount of intracellular radioactivity decreased and acid-soluble radioactivity appeared in the culture medium,
indicating degradation of lZ5I-GH.T h e release of
0 2001 Biochemical Society
490
The Interface of Receptor Signalling and Trafficking
Degradation of the GHR
T h e results obtained so far suggested that the
ubiquitin-proteasome pathway and the endocytic
lysosomal sorting system co-operate in G H R
degradation. Therefore we examined the possibility that, while the cytosolic tail of the G H R is
degraded by the proteasome, the lumenal part
is degraded within the lysosome. In order to investigate the formation of intermediate degradation products of the G H R after ligand-induced
endocytosis, cells were incubated for 6 h with G H
in the absence or presence of the lysosomal
protease inhibitor leupeptin (Figure 2). Cell lysates were analysed with antibodies against the
extracellular domain of the G H R (Mab5) and
against a membrane-proximal domain of the cytosolic tail (anti-T). In the presence of G H , lowmolecular-mass species of the receptor were detected with Mab5, indicating partial degradation of
the wild-type GHR. When the internalization
mutant GHR(F327A) was used, no such intermediates were present. Leupeptin increased the
amount of intermediate degradation products detectable with the Mab5 antibody, indicating the
involvement of lysosomes in degradation of
the luminal part of the receptor. Surprisingly, the
majority of the degradation products reactive with
Mab5 migrated on SDS/polyacrylamide gels as
proteins of 50-60 kDa, similar in size to the
circulating GH-binding protein (50-60 kDa). In
most species, the 60 kDa GH-binding proteins
originate from proteolysis of the extracellular part
of the G H R [16]. No signal was detected when an
antiserum against the C-terminal portion of the
cytosolic tail was used (results not shown). T h e
degradation products that accumulated in the
presence of G H , and were detected with Mab5,
were not detected with anti-T. Given their sizes
and their reactivity towards the different antibodies, our data indicate that most of the cytosolic
tail is removed from the 50-60 kDa degradation
products. One extra band (designated ‘t’) as
compared with the Mab5 detection could be
observed in the anti-T Western blot. This band
was formed whether or not the cells were incubated with G H in both the wild-type G H R and
the UbE mutant GHR(F327A) cell lines. Most
probably it represents the remaining cytosolic tail
of the receptor after the proteolytic release of G H binding protein, indicating that the shedding
process is not regulated by the cytosolic UbE
motif.
Although the data show that the lysosome is
involved in the degradation of the extracellular
domain of the GHR, they do not exclude the
involvement of the proteasome in degradation of
the cytosolic tail. Detection of proteasomal degradation intermediates may be difficult due to the
presence of degradation intermediates of various
lengths, which are not easily detectable by immunoblotting. Indeed, with both Mab5 and anti-T, a
smear of immunoreactive proteins was detected
upon G H incubation, primarily in lanes containing the wild-type receptor. When using an antiserum against the C-terminal part of the G H R tail,
no epitopes were detectable in this smear (results
not shown). This indicates a gradual degradation
of the receptor cytosolic tail after its endocytosis.
Recently we detected a small amount of fulllength receptor endocytosed and bound to G H ,
indicating that degradation of the cytosolic tail
begins shortly after its endocytosis [17].
Figure 2
Degradation products of the GHR
Cells were incubated for 6 h with GH in the presence or absence
of I00 p M leupeptin as indicated. Western blots of cell lysates
were detected with antibodies against the extracellular (Mab5) or
cytosolic (anti-T) domains ofthe GHR. rn, mature GHR ( I 30 kDa):
p, precursor GHR ( I 10 kDa): t, cytosolic tail (70 kDa); wt, wild
type. Positions of molecular mass standards (kDa) are shown at
the left.
Mab5
anti-T
blot
wGHR F327A wGHR F327A
-
184
11684
62
51
38
--
The ubiquitin-proteasome pathway in
endosomal sorting
Ubiquitination is a key mechanism for targeting
membrane proteins to be internalized (Figure 3,
step 1). T h e attachment of a single ubiquitin
moiety or short Lys-63-linked ubiquitin oligomers
appears to be sufficient to mediate their endocytosis [18-201. Recent studies have also implicated the ubiquitin-proteasome pathway in the
regulation of protein sorting at the endosome
-m
-P
-t
-
25
-
- + + - + + - + + - ++GH
- - + - - + - - + - - +leupeptin
49 I
0 200 I Biochemical Society
Biochemical Society Transactions (200 I) Volume 29, part 4
(Figure 3, step 2) (reviewed in [21]). Some plasma
membrane proteins are known to be ubiquitinated; however, in most cases it is not clear whether
the ubiquitinated proteins are degraded through
proteasomes or lysosomes, or both. Internalization
of the epithelial sodium channel depends on the
ubiquitination of its y and a subunits by the
ubiquitin ligase Nedd4, the mammalian homologue of yeast Rsp5p. T h e rapid turnover of the
epithelial sodium channel is affected by inhibitors
of both the proteasome and the lysosome, indicating that ubiquitination and lysosomal degradation are somehow linked [22]. It was shown that
a functional proteasome is required for optimal
endocytosis of the interleukin-2 receptor-ligand
complex and is essential for the subsequent lysosoma1 degradation of interleukin-2 [23]. Studies
with proteasome inhibitors have indicated that the
ubiquitin-proteasome pathway is also involved in
the degradation of the platelet-derived growth
factor receptor [24] and the Met receptor [25].
T h e tyrosine kinase adaptor protein c-Cbl was
shown to mediate ubiquitination of the epidermal
growth factor (EGF) receptor [ 2 6 ] , the plateletderived growth factor receptor [27], the colonystimulating factor-1 receptor [28] and the Neu
oncogene (ErbB2) [29]. Acting as a ubiquitin
ligase, c-Cbl recognizes tyrosine-phosphorylated
substrates through its SH2 (Src homology 2)
domain and recruits/activates an E2 ubiquitinconjugating enzyme through its R I N G domain in
trans [30]. At the endosome, c-Cbl may act to
facilitate sorting of the E G F receptor into multivesicular bodies, thereby attenuating kinase signalling [26]. Recent data show that ubiquitination
of the E G F receptor can also occur at the cell
surface, but the role of ubiquitination in its
endocytosis remains to be established [311. For the
colony-stimulating factor-1 receptor, it was shown
that c-Cbl stimulates multi-ubiquitination and
endocytosis, thereby attenuating macrophage proliferation [28].
Amerik and co-workers [32] provided more
evidence for a role for ubiquitination in the
endocytic pathway, when they showed that the
yeast deubiquitinating enzyme, Doa4, acts at the
late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane
proteins en route to the vacuole. T h e mammalian
tumour-susceptibility gene product TSGlOl regulates the lysosomal degradation of cell surface
proteins. Endocytosed E G F receptors were re-
Figure 3
Model for sorting in the endocytic pathway
The ubiquitin-proteasome pathway is involved in selecting membrane proteins for internalization at the plasma membrane (step I ) and in sorting membrane proteins t o the lysosome
(step 2). Proteasome inhibitorscan inhibit the internalization of membrane proteins (GHR), but
also the degradation of internalized ligand and receptor. Proteins of the ubiquitin-proteasome
pathway that play a role in the different sorting steps are indicated.
0 2001 Biochemical Society
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The Interface of Receptor Signalling and Trafficking
cycled rapidly back to the plasma membrane in
tsglOl mutant cells [33]. The N-terminus of
TSGlOl shares homology with the catalytic domain of ubiquitin-conjugating E2 enzymes, but
lacks the active-site cysteine and is, therefore,
unlikely to catalyse ubiquitination. In yeast, the
F-box protein Rcylp is involved in endocytic
membrane traffic and recycling out of an early
endosome. Members of the F-box family of proteins have been shown to mediate ubiquitination
of substrate proteins as components of SKPl/
cullin/F-box (SCF) ubiquitin ligase complexes.
Degradation of the a-factor receptor and uracil
permease is inhibited at a post-internalization step
in RcyA mutant cells [34]. The presence of the
F-box domain in a protein that affects vesicular
traffic could point to a role for ubiquitination in
regulating the sorting machinery itself.
In conclusion, it is clear that the ubiquitinproteasome pathway regulates hormone receptor
degradation at the endosomal level. How this is
accomplished for such a variety of receptors is a
challenging question.
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