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INTRODUCTION |
The high affinity interleukin-2 receptor
(IL-2R)1 is comprised of
three subunits; an
-chain, which functions solely in IL-2 binding,
and
- and common
(
c)-chains that function in both ligand
binding and signal transduction (1). IL-2 promotes oligomerization of
these subunits and activation of a number of receptor-associated signaling molecules, including Janus kinase (Jak)-1/3, signal transducer and activator of transcription (STAT) 5/3, p56lck, and
phosphatidylinositol-3-kinase that is recruited by the Shc-adapter protein (2). Another immediate consequence of IL-2·IL-2R
interaction is ligand-induced receptor-mediated endocytosis of the
receptor-ligand complex. In T lymphocytes, 50% of the receptor-ligand
complex is internalized within 10-15 min (3, 4). Ligand-induced endocytosis of the IL-2R is primarily dependent upon the cytoplasmic tail of
c (5, 6). Studies that have followed the fate of these
proteins indicate that IL-2R
recycles to the plasma membrane from an
early endosomal compartment, whereas the
- and
c-subunits as well
as IL-2 traffic to the lysosome, where they are degraded (7).
Within the immune system, the proteasome is essential for production of
peptides for MHC class I antigen presentation. The proteasome also has
a well described role in the degradation of cytosolic and nuclear
proteins, including the elimination of misfolded proteins as well as
controlling the levels of certain transcription factors or cell cycle
regulatory proteins (8). Most proteins destined for degradation are
targeted to the proteasome by ubiquitination (9). More recent studies
have implicated ubiquitination in the internalization of a growing list
of cells surface receptors (10, 11), suggesting a possible role for the
proteasome in regulating the levels of cell surface receptors. In the
present study we have used specific inhibitors to explore the potential role of the proteasome for endocytosis and degradation of the IL-2·IL-2R complex.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
1F1
cWT (6), 1F1
c284 (12), and
CX
t (13) have been described previously. Activated T cells were
prepared by the culture of C57BL/6 spleen cells with anti-CD3 for
48 h as described previously (14). E-64, MG132, PSI, and antipain
were obtained from Peptide International, Inc. (Louisville, KY),
lactacystin from Calbiochem-Novabiochem Corp. (San Diego, CA), and
chloroquine from Sigma (St. Louis, MO)
Internalization Assay--
Cells (10 × 106/ml)
were incubated in RPMI 1640 containing 5% fetal calf serum (14)
(complete medium) at 4 °C for 30 min in the presence of 50,000 cpm/ml human IL-2 (specific activity ~20-40 µCi/µg) that was
radiolabeled with Na125I using Iodo-gen precoated tubes
(Pierce Chemical Co., Rockford, IL) according to the manufacturer's
instruction. The cells were then washed three times with cold Hank's
balanced salt solution. Unless otherwise indicated, all inhibitors were
added at 50 µM final concentration to the cells just
after these washes. In the case of lactacystin, the cells were also
cultured with this proteasome inhibitor for 2 h in complete medium
and cooled on ice for 15 min prior to treatment with
125I-labeled IL-2. Dimethyl sulfoxide (Me2SO),
which was the solvent for all the proteasome inhibitors, was added to
the control cells at a concentration of
1%. The IL-2-pulsed cells
(4 × 106/ml) were then cultured in complete medium at
37 °C using a water bath in 1 ml of medium/tube using 1.5-ml
microcentrifuge tubes. At the indicated times, the cells were pelleted
by centrifugation in a microcentrifuge at 14,000 × g
for 15 s. Protein released in the supernatant was precipitated by
addition of one-fourth volume of 50% trichloroacetic acid. The
trichloroacetic acid soluble counts represented internalized IL-2 that
was degraded. The cell pellets were then resuspended in 0.5 ml of 0.01 M sodium citrate, 0.14 M NaCl, pH 2 buffer for
2 min at room temperature and centrifuged for 15 s at 14,000 × g in a microcentrifuge. The radioactivity that remained
associated with the cells after this low pH buffer wash represented the
internalized (I) IL-2 whereas the portion of radioactivity
in the pH 2 buffer supernatant represented cell surface-associated
(S) IL-2. Before shifting the cells to 37 °C, usually
75-85% of the cpm was cell-surface associated. The
I/S ratio was calculated at the indicated time
points. The pH 2-resistant material at time 0 (t0) was considered nonspecific material and was
subtracted from I.
Western Blot Analysis--
1F1
cWT cells were extracted in
buffer containing 0.5% Nonidet P-40, phenylmethylsulfonyl fluoride (1 mM), leupeptin (10 µg/ml), aprotinin (10 µg/ml),
iodoacetamide (10 mM), sodium orthovanadate (1 mM), and sodium fluoride (1 mM) as described
previously (15) and precipitated with anti-STAT5 antibodies (Santa Cruz
Biotechnology, Inc., Santa Cruz, CA) associated to protein G-Sepharose.
The precipitated material was resolved by 10% SDS-polyacrylamide
electrophoresis under reducing conditions, transferred to
nitrocellulose, and blocked by incubation with 5% nonfat dried milk in
PBS, pH 7.2, (5% milk) for 1 h. The nitrocellulose was
subsequently incubated with either anti-phospho-STAT5 (Upstate
Biotechnology, Lake Placid, NY) or anti-STAT5 antisera in 5% milk for
90 min, washed three times with PBS containing 0.1% Nonidet P-40, and
then incubated with horseradish peroxidase-conjugated donkey
anti-rabbit Ig for 60 min followed by washing twice with PBS containing
0.1% Nonidet P-40 and once with PBS alone. Bands were visualized by
chemiluminescence using ECL Western blotting detection reagents
(Amersham Pharmacia Biotech) according to the manufacturer's instructions.
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RESULTS |
Effect of Proteasome Inhibitors on the Internalization and
Degradation of IL-2--
After IL-2 binds to the high affinity IL-2R,
~50% is detected intracellularly (I/S = 1) within 10-15 min (3, 4), and a similar rate of internalization has
been noted for IL-2R-bearing EL4, including 1F1
cWT (Fig.
1 and Refs. 6 and 13). By 45 min, ~70%
of the 125I-labeled IL-2 was detected intracellularly
without appreciable degradation (Fig. 1, B and C;
Refs. 6 and 13). To test whether proteasome function was required for
receptor-mediated endocytosis of IL-2, 1F1
cWT cells were treated
with 125I-labeled IL-2 in the cold, and internalization of
the surface-associated IL-2 was initiated by culturing the cells at
37 °C in the absence or presence of the proteasome inhibitor MG132
(Fig. 1). During the first 10-15 min, the MG132 minimally affected the
initial internalization of IL-2 as measured by the initial loss of cell surface IL-2 (Fig. 1A) or the accumulation of intracellular
IL-2 (Fig. 1B). However, an altered
I/S ratio was evident for 1F1
cWT 45 min after
initiation of endocytosis when the cells were cultured with either
MG132 or the highly proteasome specific inhibitor lactacystin (Fig.
2A). This effect was because
of an increased level of surface IL-2 that was evident for the
MG132-treated cell by 30 min (Fig. 1A). The altered
endocytosis by MG132 and lactacystin was specific as the
I/S ratio was not affected when 1F1
cWT were treated with the calpain inhibitor antipain or the cysteine protease inhibitor E-64 (Fig. 2A).

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Fig. 1.
Effect of a proteasome inhibitor on
receptor-mediated endocytosis of IL-2. 1F1 cWT cells were pulsed
with 125I-labeled IL-2, treated with MG132 (50 µM), and cultured for 3 h at 37 °C. At the
indicated time, the fraction of IL-2 that was on the cell surface
(A), intracellular (B), or released into the
medium (C) was determined. Data are the mean ± S.D. of
three determinations.
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Fig. 2.
Comparison of proteasome and protease
inhibitors on the internalization and degradation of IL-2. Effect
on internalization (A) or degradation (B) of
IL-2. C, dose-response of proteasome inhibitors. 1F1 cWT
cells were pulsed with 125I-labeled IL-2, treated with 50 µM indicated inhibitors as described under
"Experimental Procedures," and cultured for 45 min (A)
or 180 min (B and C) at 37 °C. In
A, the fraction of cell-associated IL-2 that was
intracellular (I) versus cell surface
(S) was determined and plotted as the
I/S ratio where 1 represents 50% of the
cell-associated IL-2 within an intracellular compartment. Data in
A are the and range of two independent determinations,
except for lactacystin. In B, degraded IL-2 was assessed by
the fraction of trichloroacetic acid-soluble cpm in the culture medium
and is the mean ± S.D. of 3-6 determinations, except for
antipain
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After 60 min in culture, an obvious fraction of the IL-2 that was
initially associated with the 1F1
cWT cells was detected in the
culture supernatant, and at this and all subsequent time points, >90%
of this released IL-2 was soluble in trichloroacetic acid, consistent
with its degradation to small peptides. The small amount of IL-2
released during the first 30 min in culture was precipitated by
trichloroacetic acid, which most likely reflected the release of IL-2
from the IL-2R prior to its endocytosis. Surprisingly, MG132
substantially inhibited the degradation of IL-2 (Fig. 1C). Like MG132, the related peptide aldehyde proteasome inhibitor PSI, but
not antipain or E-64, prevented the degradation of IL-2 (Fig.
2B). This finding suggests that the inhibition by these two
peptide aldehydes is caused by effects on the proteasome rather than
effects on lysosomal cysteine proteases or calpains. Again, the highly
proteasome-specific inhibitors lactacystin and clasto-lactacystin
-lactone (not shown) also inhibited the degradation of IL-2 (Fig. 2B). Dose-response studies indicated that 2-5
µM MG132, PSI, or lactacystin were required for
inhibition of 50% degradation of IL-2 (Fig. 2C). This
concentration of inhibitors is consistent with those required to
inhibit the proteasome (16-18). Collectively, these data indicate that
proteasome function is necessary for normal receptor-mediated
endocytosis and subsequent degradation of IL-2.
Relationship Between the Proteasome and Lysosome for
Internalization and Degradation of IL-2--
Previous studies have
demonstrated that upon internalization of the IL-2·IL-2R complex,
IL-2 traffics to the lysosome and is degraded (3, 4, 7). We confirmed
that degradation of IL-2 by 1F1
cWT occurred in the lysosome as its
degradation was inhibited by 50 µM chloroquine (Fig.
3A). The peptide aldehyde proteasome inhibitor MG132 was even a somewhat more potent inhibitor of
degradation of IL-2.

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Fig. 3.
Comparison of proteasome and lysosome
inhibitors on the internalization and degradation of IL-2. Effect
on degradation (A) and internalization (B) of
IL-2. These experiments were performed as described in the legend to
Fig. 2 and assayed after 2-3 h (A) or the indicated time
(B). Data in A represent the mean ± S.D. of
three experiments and B is representative of three
experiments.
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During the first 15 min of culture, time-course studies revealed that
MG132 only minimally blocked internalization of IL-2 (Figs.
1A and 3B). However, after a 20-30 min culture
in the presence of MG132, internalization of IL-2 reached a maximal
level, and after that time the fraction of surface and intracellular
IL-2 remained constant. By contrast, chloroquine, which directly
inhibits lysosomal function, showed much lower capacity to affect the
internalization of IL-2 with consistently a higher
I/S ratio than MG132-treated cells (Fig.
3B). This finding in conjunction with the complete inhibition of degradation of IL-2 by proteasome and lysosome inhibitors suggests that impairment of IL-2 internalization by the proteasome inhibitors may be secondary because of the blockade in trafficking of
IL-2 to the lysosome, preventing the degradation of IL-2.
Proteasome Function Is Not Required for Initial Internalization of
IL-2--
From the preceding data, only the initial endocytosis of
IL-2 was not influenced by MG132. However, this apparently normal internalization might simply be related to a short delay for MG132 to
effectively block the proteasome. To directly test whether the entry of
IL-2 into the cells was unaffected by proteasome inhibitors, 1F1
cWT
cells were pretreated with proteasome inhibitors prior to initiation of
receptor-mediated endocytosis (Table I). When analyzing the continuous internalization of IL-2 for 45 min (Table
I, Experiment 1), the fraction of surface and intracellular IL-2 was comparable for cells pretreated with either the
Me2SO vehicle or proteasome inhibitors. In the latter two
experiments (Table I, Experiments 2 and 3), the
cellular distribution of IL-2 was assessed 3 h after initiation of
endocytosis of IL-2-pulsed cells. In this scenario, most of the
cell-associated IL-2 was found in an intracellular fraction, a result
that was essentially identical to experiments where the proteasome
inhibitor was added simultaneous to initiation of internalization.
Because lactacystin is an irreversible proteasome inhibitor, the
accumulation of intracellular IL-2 in the presence of this agent (Table
I, Experiment 3) strongly supports the notion that initial
endocytosis of IL-2 was unaffected by blockade of the proteasome.
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Table I
Pretreatment of 1F1 cWT with proteasome inhibitors does not impair
initial internalization of IL-2
In Experiment 1, 1F1 cWT cells were pretreated with MG132 (50 µM)
for 60 min at 37 °C prior to adding 125I-labeled IL-2
without washing. The cells were further cultured at 37 °C for 45 min, and cell surface- and intracellular-associated cpm were
determined. In experiments 2 and 3, 1F1 cWT cells were pretreated
with PSI (50 µM) or lactacystin for 120 min at 37 °C and then
cooled on ice for 15 min. The cells were then pulsed with
125I-labeled IL-2 at 4 °C for 30 min. After washing, the
cells were cultured at 37 °C for 180 min with further addition of
PSI or lactacystin. 70-85% of the cpm were cell surface-associated
prior to initiation of endocytosis as determined by cells maintained at
4 °C (Experiment 1) or by assay before shifting to 37 °C
(Experiments 2 and 3).
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After a 30-min culture of IL-2-treated 1F1
cWT, usually 65-75% of
the cell-associated IL-2 was detected as an intracellular fraction of
ligand (see Figs. 1B and 2B and Ref. 6). The
addition of MG132 at this time resulted in inhibition of degradation of IL-2 to a level that was comparable when MG132 was added at the initiation of the culture (Fig.
4A). Substantially less
inhibition was noted when MG132 was added 60 min after initiation of
endocytosis of IL-2, a time when degradation of IL-2 is beginning to be
detected in the control cells. Furthermore, when compared with
Me2SO vehicle-treated cells, an accumulation of
intracellular IL-2 occurred when MG132 was added at culture initiation
(t0) or 30 min later. The increased fraction of
intracellular IL-2 was most prominent when the proteasome inhibitor was
delayed for 30 min, as normal levels of IL-2 were allowed to first be
internalized before blocking the proteasome. Delayed addition of MG132
similarly affected the degradation of 125I-labeled IL-2 by
C57BL/6 T blasts (Fig. 4B), demonstrating that proteasome
function for IL-2 degradation was not unique to only IL-2R-bearing EL4
cells, but was also required for a normal population of T cells.
Collectively, these data suggest that blockade of the proteasome
inhibits a relatively early step in the intracellular traffic of the
IL-2·IL-2R complex, but a step later than required for the initial
endocytosis of the receptor-ligand complex.

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Fig. 4.
Effect of delayed addition of MG132 on the
degradation and internalization of IL-2. 1F1 cWT (A)
or C57BL/6 T blasts (B) were pulsed with
125I-labeled IL-2 and cultured at 37 °C for 3 h.
MG132 was added at various times after the initiation of
internalization by culture at 37 °C. The cpm associated with the
cell surface, an intracellular pool, or released as IL-2
trichloroacetic acid-soluble cpm in the supernatant were determined.
Data are the mean and range of two independent determinations.
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IL-2-induced Signaling, Degradation of IL-2, and Proteasome
Function--
Normal internalization of IL-2 is dependent upon the
cytoplasmic tail of the
c-subunit of the IL-2R (5, 6). For the 1F1
c284-transfected variant of EL4, which expresses wild-type IL-2R
- and
-chains but the cytoplasmic tailless
c-chain, the rate
of internalization of IL-2 is ~2-3-fold slower than 1F1
cWT, with
a corresponding lower fraction of degraded IL-2 after 2-3 h in culture
(Table II and Ref. 6). By
contrast, the CX
t-transfected variant of EL4, which expresses
wild-type IL-2R
and -
c, but a cytoplasmic tailless
-chain,
demonstrated essentially normal rates of internalization and
degradation of IL-2 (Ref. 13 and Table II). Importantly, MG132 very
efficiently inhibited the degradation of the fraction of IL-2 that was
internalized by the IL-2R on 1F1
c284 or CX
t (Table II). These
findings indicate that the proteasome-dependent pathway for
degradation of IL-2 is independent of the cytoplasmic tail of IL-2R
or -
c, including the respective IL-2R-associated signaling
molecules.
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Table II
Internalization and degradation of IL-2 by IL-2R lacking the
cytoplasmic tail of the - or c-subunits
The indicated cells or 1F1 cWT (not shown) were pulsed with
125I-labeled IL-2, treated with MG132 (50 µM) or
Me2SO (0.5%), and cultured at 37 °C for 3 hr. Degraded IL-2
was assessed by the fraction of trichloroacetic acid-soluble cpm in the
culture medium.
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Several studies have implicated the proteasome in the regulation of
Jak-STAT signal transduction, including IL-2-induced activation of
STAT5 (19, 20). In 1F1
cWT, IL-2 induced tyrosine phosphorylation of
STAT5 (Fig. 5A, lane
1 versus 3), and upon extended culture without IL-2,
the level of phosphorylated STAT5 was considerably reduced (Fig.
5A, lane 3 versus 5 and
7). Furthermore, as expected, MG132 stabilized IL-2-induced
activation of phosphorylated STAT5, which was especially evident after
2 h in culture (Fig. 5A, lane 7 versus 8).
To investigate whether this enhanced signaling might be related to the
proteasome-dependent blockade of the IL-2 degradation pathway, we examined the ability of delayed addition of MG132 to
potentiate STAT5 phosphorylation in 1F1
cWT (Fig. 5B).
Because the IL-2 degradation pathway was nearly completely inhibited
even when MG132 was administered 30 min after initiation of
IL-2·IL-2R endocytosis, we reasoned that the stabilized
phosphorylation of STAT5 by MG132 would also be evident after delayed
addition of MG132, if these two processes were related. However, unlike
degradation of IL-2, MG132 was required to be continuously present to
stabilize STAT5 phosphorylation (Fig. 5B, lanes 3 and 4 versus lanes 5 and 6). Thus, proteasome regulation of IL-2 signaling and
subsequent endocytosis appear to be unrelated.

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Fig. 5.
Effect of the proteasome inhibitor MG132 on
IL-2-induced STAT5 activation. 1F1 cWT cells were treated with
IL-2 (400 units/ml) at 4 °C for 30 min in complete medium, washed
three times, and cultured at 37 °C for the time indicated (time
assayed). MG132 (50 µM) or Me2SO (0.5%) was
added at the initiation (A) (t0) of
this 37 °C culture or delayed (B), as indicated. Nonidet
P-40 extracts were prepared from these cells, precipitated with
anti-STAT5 antiserum, and subjected to Western blot analysis using
antiserum specific for phosphotyrosine-STAT5 (upper panel).
The blot was stripped and reprobed with antiserum to STAT5 protein
(lower panel) to verify the levels of STAT5 in each
lane. Data are representative of three experiments.
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DISCUSSION |
The proteasome is not typically considered as a key regulator of
ligand-induced receptor-mediated endocytosis. In this study we show a
mandatory role for the proteasome for normal endocytosis of
IL-2·IL-2R and subsequent lysosomal degradation of IL-2. Blockade of
the 26 S proteasome did not affect the initial endocytosis of IL-2, but
was necessary for sustained endocytosis and was essential for lysosomal
degradation of IL-2. These findings add to increasing evidence that
rapid ligand-induced internalization of cell surface IL-2·IL-2R
complexes occurs by a novel endocytic mechanism. For example, although
IL-2R and transferrin receptors are both detected in early endosomes,
blockade of coated-pit internalization of the transferrin receptor had
minimal effect on the internalization of IL-2·IL-2R (21), suggesting
that these molecules may utilize distinct pathways to traffic to the
early endosome. Furthermore, recently a critical endocytic signal has
been identified within a 9-amino acid sequence of the cytoplasmic tail
of
c (6), and this region lacks tyrosine or dileucine motifs that
target receptor-ligand complexes to clathrin-coated pits, which are
typically necessary for entry into the early endosome compartment.
It was possible to delay addition of the proteasome inhibitor MG132 to
a point (30 min) where substantial endocytosis of IL-2·IL-2R occurred
and still potently inhibit lysosomal degradation of IL-2. This finding
raises the possibility that proteasome function may be solely required
for trafficking internalized IL-2 from an endosomal compartment to the
lysosome. In this scenario the capacity of proteasome inhibitors to
impair internalization of IL-2·IL-2R may be secondary to this
regulatory role in trafficking. We cannot, however, rule out a broader
role for proteasome function for endocytosis of this ligand-receptor complex.
The molecular target for proteasome action in IL-2·IL-2R endocytosis
has not been established. Generally, polyubiquitination of
intracellular proteins targets them for recognition and proteolytic degradation by the proteasome. As a number a studies have demonstrated ubiquitination of cell surface receptors (10), we considered the
possibility the cytoplasmic tail of IL-2R
- or
c-subunits might
be critical targets for the proteasome. Importantly, proteasome function was still required for lysosomal degradation of IL-2 after
internalization by IL-2R that contained cytoplasmic tailless
- or
c-subunits. So far, we have also been unsuccessful in directly demonstrating that these subunits are modified by ubiquitin (data not
shown). These results strongly suggest that the cytoplasmic portion of
the IL-2R or their associated signaling components are unlikely to be
targets for the proteasome. Consistent with this notion, we showed
distinct kinetic requirements for proteasome function for IL-2-mediated
endocytosis and IL-2-induced STAT5 tyrosine phosphorylation.
A number of cell surface receptors are ubiquitinated upon ligand
activation. In mammalian cells, the platelet-derived growth factor
receptor (22), growth hormone receptor (23), TCR
, (24), and c-kit
(25, 26) were among the first receptors shown to undergo ligand-induced
ubiquitination of their cytoplasmic tails. In these cases, the lysosome
appears to be the primary target for degradation of these receptors. In
Saccharomyces cerevisiae the G-protein coupled
-factor
receptor undergoes enhanced mono- or short chain ubiquitination upon
binding
-factor, which facilitates endocytosis and subsequent
degradation by the lysosome (27, 28). The use of yeast defective in
ubiquitin-conjugating enzymes established a role for this protein
modification in endocytosis (27, 28). So far, there has been little
direct evidence that proteasome function is involved in this process,
and it has been suggested that ubiquitination may serve as a targeting
sequence for entry into the endocytic pathway leading to
down-regulation of the cell surface receptor (10). One exception
appears to be endocytosis of the growth hormone receptor. Similar to
our results on the IL-2R, proteasome activity appears to be required for ligand-induced endocytosis and lysosomal degradation of growth hormone receptor that is independent of ubiquitination of the cytoplasmic tail of growth hormone receptor (29). The fate of the
receptor-associated ligand was not determined in that study. Proteasome
inhibitors have also been reported to affect the levels of
platelet-derived growth factor receptor (22) and the Met kinase
receptor (30), but these effects have not been ascribed to regulation
of the endocytic pathway. If our findings on the IL-2R are generalized,
perhaps the role for proteasome function in regulating these cell
surface receptors is to gain entry into a cellular compartment for
lysosomal/vacuolar degradation that is independent of ubiquitination of
the cytoplasmic tail of the receptor.