From the Unité de Biologie des Interactions Cellulaires, URA CNRS 1960, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
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ABSTRACT |
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The common cytokine receptor
c, shared by interleukin 2, 4, 7, 9, and 15 receptors, has a major role in lymphocyte proliferation and
differentiation, leading, when mutated, to a genetic disease, X-linked
severe combined immunodeficiency. In this study, we report that
c is internalized and degraded in lymphoid cells. To
identify
c regions involved in sorting along the
endocytic pathway, we have studied a chimeric protein composed of the
extracellular part of interleukin 2-receptor
and transmembrane and
intracellular part of
c,
wt. When
transfected in Jurkat T cells,
wt is as
efficiently internalized and degraded as
c,
demonstrating that the transmembrane and cytosolic tail of
c carry sequences involved in this process. To identify
these motifs, we have analyzed the trafficking of chimeric proteins
with serial truncations in their cytosolic tail. Internalization
studies showed that the cytosolic tail of
c contains
three regions located between cytosolic amino acids 1-35, 35-40, and
40-65 involved in
c endocytosis. Successive deletions
of these motifs result in reduced endocytosis. One region containing
the 5 cytosolic amino acids 36-40 is essential to direct
c to the degradation pathway. These sorting sequences, by participating in the fine tuning of cell surface
c
expression, might somewhat regulate the cell responsiveness to
interleukins whose receptors share this component.
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INTRODUCTION |
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Communication between cells in the immune and hematopoietic
systems is mediated by soluble factors, cytokines, which exert their
biological activities through specific cell surface receptors. The
molecular structure of a growing number of cytokine receptors has been
determined and led to the definition of a new family of receptors (1,
2). The hematopoietic cytokine receptor superfamily is defined by
structural homology in the extracellular, ligand binding domain and
limited similarity in the membrane proximal cytosolic regions (3). This
family of receptors includes receptors to many interleukins
(IL),1 such as IL2, IL3, IL4,
IL5, IL6, IL7, IL9, IL12, and IL15, receptors for hormones such as
growth hormone and prolactin, and receptors for growth factors. Another
characteristic of this family is that many receptors subfamily members
share at least one component, explaining in part the redundant action
of different cytokines. One member of this family of receptors was
named the common chain (
c): first described as the
third component of the IL2 receptor (IL2R), it participates in the
formation of high-affinity forms of IL2, IL4, IL7, IL9, and IL15
receptors (4-9). Because it is shared by these interleukin receptors,
c has a critical role in lymphocyte differentiation and
proliferation. Indeed, patients suffering from X-linked severe combined
immunodeficiency have mutations in the gene encoding
c (10, 11).
One of the early events that follows ligand binding to receptors on the
cell surface is the internalization of the ligand-receptor complex.
After internalization from the plasma membrane in early endosomes,
receptors can either recycle to the cell surface or be degraded within
intracellular compartments. Endocytosis and degradation are essential
for the rapid down-modulation of surface receptors after ligand
binding. Down-modulation results in a decrease in the number of
receptors on the cell surface and, together with biosynthesis, controls
the cell responsiveness. For several receptors, internalization is
driven by the presence of specific sequences in their cytosolic part,
recognized by the cellular machinery. These internalization signals
have been classified into two groups, one is characterized by a
tyrosine-based motif and the other by a di-leucine-based motif (12,
13). They allow the recruitment of receptors into clathrin-coated pits
and promote high-efficiency endocytosis. Whereas the molecular
mechanisms that direct internalization from the plasma membrane to
early endosomes are well documented for receptors, much less
information is available concerning the sorting from early endosomes to
the other intracellular compartments. For receptors to exit from the
recycling pathway and to move to other intracellular destinations
(e.g. in most cases toward the late endosomes and lysosomes)
or to new domains on the cell surface (as in transcytosis) requires
selective routing and depends upon the possession of additional,
specific sorting signals. Indeed, tyrosine or di-leucine families of
signals described for clathrin-coated pit endocytosis are also involved
in lysosomal targeting of lysosomal membrane glycoproteins (reviewed in
Ref. 12). Sequences necessary for the degradation of nonlysosomal
membrane proteins have been reported for two proteins, the P-selectin
and the IL2R chain (14, 15). In both cases a short sequence is
sufficient for degradation after endocytosis. These two degradation
signals are different and do not match the trafficking signals
described so far.
The c chain is a part of IL2R, IL4R, IL7R, IL9R, and
IL15R. Its role in endocytosis of these cytokines and their receptors is not known. We have previously shown that
c is
internalized and degraded when part of the IL2·IL2R complex (16) and
that the cytosolic part of
c is involved in IL2
endocytosis and in the down-regulation of the IL2R
chain (17). These
results suggest that
c may carry the necessary
information for endocytosis and degradation. In the present study we
show that the cytosolic tail of
c contains sequences
involved in receptor endocytosis and degradation.
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EXPERIMENTAL PROCEDURES |
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Cells, Monoclonal Antibodies, and Reagents
YT 12881, a subclone from the NK cell line YT, was obtained from
Dr. K. Smith (Dartmouth Medical School, Hanover, NH). The Jurkat
77.31.13 T cell line was a kind gift of Dr. A. Alcover (Institut
Pasteur, Paris, France) and will be referred to as J77 cells from now
on (18). All cells were grown in suspension in RPMI 1640, 10%
decomplemented fetal calf serum, 10 mM HEPES, pH 7.2, supplemented with 2 mM L-glutamine. Stably
transfected Jurkat cells were grown in the same medium supplemented
with 400 µg/ml hygromycin (Boehringer Mannheim, Mannheim, Germany).
Monoclonal antibodies (mAb) 2A3A1H (mouse IgG1) or 7G7B6 (mouse IgG2a),
directed against the chain of the IL2 receptor, were obtained from
the American Tissue Culture Collection (Manassas, VA). TUGh4 (rat IgG2b), directed against the
chain of the IL2 receptor, was obtained from PharMingen (San Diego, CA). Monoclonal antibodies FG 1/6
(mouse IgG1), directed against the human transferrin receptor, were a
kind gift from Dr. B. Alarcon (Centro de Biologia Molecular Severo
Ochoa, Madrid, Spain). The second antibodies used with mAb TUGh4 were
FITC-conjugated anti-rat IgG antibodies (Southern Biotechnology
Associates, Birmingham, AL), with 2A3A1H F(ab)'2 phycoerythrin-conjugated goat anti-mouse IgG (Immunotech, Marseilles, France) and with 7G7B6 F(ab)'2 FITC-conjugated rabbit
anti-mouse Ig (Dako, Glostrup, Denmark). Cycloheximide and saponin were
obtained from Sigma.
Plasmids
The constructs were subcloned in the NT expression vector,
containing the SR promoter, a kind gift of Dr. C. Bonnerot (Institut Curie, Paris, France). The plasmid pRCH
, coding for the
c chain, and the plasmid pREP4, were kindly provided by
Dr. J. Di Santo (Hôpital Necker, Paris, France). The plasmid
T-XO, coding for the IL2R
chain, has already been described (15).
The plasmid pME18S
/
/
was kindly provided by Dr. W. Leonard
(National Institutes of Health, Bethesda, Md).
pREP was constructed by subcloning IL2R
from TXO into the
KpnI/NotI site of the plasmid pREP4. It contains
a BglII restriction enzyme site just upstream the
transmembrane part of IL2R
. The transmembrane and cytosolic regions
of human
c were derived by polymerase chain reactions
(PCR) using pRCH
and primers with terminal BglII and
XhoI restriction enzyme sites. pREP
wt
was obtained by ligating the transmembrane and cytosolic regions of
c to the pREP
digested with BglII and
XhoI.
The truncated forms of n were generated by PCR by
insertion of a stop codon after the nth amino
acid of the cytosolic part of the protein (assuming that Glutamine is
the first cytosolic amino acid) and were cloned into the
NotI/XbaI sites of NT by standard techniques.
They were used for stable transfections in J77 cells. The plasmid
TXO
25-65 and TXO
35-65 were constructed
by inserting amino acids 25-65 or 35-65 from the cytosolic tail of
c derived by PCR into the HindIII/XBaI
cloning cassette, localized at the 3' end of IL2R
cDNA (15).
These plasmids were stably transfected in J77 cells. Sequences were
confirmed whenever synthetic oligonucleotides or PCR were involved.
Cell Transfection
All endocytosis and half-life analyses described here have been
performed in stably transfected J77 cells. To generate stable transfectants, 20 × 106 J77 cells were washed once in
culture medium and resuspended in 800 µl of the same medium, with 20 µg of the plasmid of interest. Electroporation was performed using
the Easyject electroporator (Eurogentec, Serain, Belgium) with a simple
pulse, 260 V, 900 microfarads. Selection with 400 µg/ml hygromycin
was initiated 2 days after transfection, and the cells were cloned in
96-well dishes. Hygromycin-resistant clones were assayed for expression by flow cytometry using anti- (2A3A1H) antibodies. The expression levels of recombinant proteins in all clones tested were the same or
less than their normal level in activated lymphocytes. J77 cells stably
transfected with the
n construct will be called
J77
n from now on.
Endocytosis of Antibodies
Endocytosis of antibodies directed against the chimeric
molecules was quantitated by flow cytometry essentially as described previously (19). Briefly, 5 × 105
J77n cells were incubated at 4 °C for 60 min with the anti-IL2R
2A3A1H mAb (1/2000 ascitic fluid). The cells were washed once in phosphate-buffered saline (PBS) at 4 °C, and after incubation at 37 °C for the indicated times, the cells were rapidly cooled to 4 °C and washed twice in PBS, 2% fetal calf serum. The cells were incubated at 4 °C for 1 h with the
IgF(ab)'2 phycoerythrin-conjugated goat anti-IgG secondary
antibody, washed once at 4 °C, and the level of expression of the
constructs on the cell surface was assessed by flow cytometry on a
FACScan (Becton Dickinson, San Jose, CA) immediately after labeling.
Base line cell fluorescence intensity was determined with cells
incubated only with the secondary antibody. For each time point
t, the percentage of internalized antibodies was calculated
as follows: 100 × (1
fluorescence intensity at time
t/fluorescence intensity at time 0). All experiments were performed at
least four times.
Cell Surface Half-life Measurement
To measure the half-life at the cell surface of c
and of the different
n constructs, cells were
incubated with cycloheximide to prevent the synthesis of new receptors.
After different times of incubation at 37 °C in culture medium with 50 µM cycloheximide, the cells were cooled to 4 °C,
washed, and cell surface expression of the constructs was assayed by
flow cytometry as described (20). Time 0 on the graph corresponds to a
30-min incubation in cycloheximide, which is the time required for a
newly synthesized IL2 receptor to reach the cell surface (21). All
experiments were performed at least four times.
Immunofluorescence and Confocal Microscopy
Endocytosis of Anti-c mAb--
YT 12881 cells
were incubated for the indicated times at 37 °C with TUGh4 mAb,
before being washed in PBS at 4 °C and fixed in 3.7%
paraformaldehyde and 0.03 M sucrose for 60 min at 4 °C. Subsequent steps were performed at room temperature. The cells were
washed once in PBS, quenched for 10 min in 50 mM
NH4Cl in PBS, and washed once in PBS supplemented with 1 mg/ml bovine serum albumin. After two washes in the permeabilizing
buffer (PBS with 1 mg/ml bovine serum albumin and 0.05% saponin), the
presence of antibodies was revealed by incubating the cells for 60 min in permeabilizing buffer containing FITC-conjugated anti-rat IgG antibody (1/50).
Direct Observation of c, IL2R
, or
wt Inside the Cells--
Either HeLa cells, grown
on coverslips and transfected 2 days before with the plasmid of
interest, or YT 12881 cells were used. Exponentially growing cells,
incubated for 45 min with 50 µM cycloheximide, were
washed twice with PBS and permeabilized as described above. The cells
were then incubated in permeabilizing buffer for 1 h with TUGh4
mAb (1/50) for YT 12881 cells and with 7G7B6 mAb (1/800 ascites) for
HeLa cells. After two washes in permeabilization buffer, cells were
labeled with the second FITC-labeled antibody for 1 h in the same
buffer. After washes and sample mounting, cells were examined by
confocal microscopy (for the YT 12881 cells) or by epifluorescence
microscopy (for HeLa cells). Washes, sample mounting, and confocal
microscopy were performed as described (16). No immunofluorescence
staining was ever observed when second antibodies were used without the
first antibody or with an irrelevant first antibody.
Cell Iodination
YT cells (2 × 107) washed in PBS and
resuspended in PBS, pH 7.3, 1 mM Ca2+, 1 mM Mg2+ were surface-labeled using the
lactoperoxidase method with 1.5 mCi of Na125I (22). After
iodination, the cells were washed twice in culture medium and then kept
in a 37 °C incubator. After a 10-min incubation, considered as time
0, cells were harvested at 30-min intervals, washed in PBS, pelleted by
centrifugation, and kept frozen at 20 °C before analysis by
immunoprecipitation.
Immunoprecipitation and Gel Analysis
Cells were lysed for 30 min at 4 °C in lysis buffer (0.5%
Nonidet P-40, 150 mM NaCl, 10 mM Tris-HCl, pH
8.0) complemented with 1 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml leupeptin, 20 µg/ml aprotinin, and 2 mM phenylmethylsulfonyl fluoride).
Insoluble material was pelleted at 15,000 × g for 15 min, and the supernatant was then precleared for 120 min at 4 °C
with protein A-Sepharose CL-4B (Amersham Pharmacia Biotech, Les Ulis,
France) before being immunoprecipitated overnight at 4 °C with
relevant first antibodies and protein A-Sepharose coated with anti-rat
antibody (Southern Biotechnology Associates). The first antibodies were
anti-c mAb TUGh4 (1 µg/ml) and anti-transferrin
receptor mAb FG1/6 (1/200 ascitic fluid) for the measurement of
c and transferrin receptor half-life after iodination.
The Sepharose beads were then washed three times in 0.5% Nonidet P-40,
0.5 M NaCl, 10 mM Tris-HCl, pH 8.0, and once in
10 mM Tris-HCl, pH 8.0. Bound proteins were eluted into
electrophoresis sample buffer (60 mM Tris-HCl, pH 6.8, 2%
SDS, 10% glycerol, 5%
-mercaptoethanol) before analysis by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. The gels were fixed
and dried, and radioactivity in the gel bands was quantitated using a
PhosphorImager and ImageQuaNT software (Molecular Dynamics, Inc.,
Sunnyvale, CA). Gels were subsequently exposed to Hyperfilm-MP
(Amersham, Pharmacia Biotech, Les Ulis, France) at
80 °C.
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RESULTS |
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The c Chain Is Constitutively Internalized in the NK
Cell Line YT12881--
When receptors are constitutively internalized,
some of them are found in endocytic intracellular compartments at any
time. To study the endocytic behavior of
c, we observed
its intracellular localization by immunofluorescence. First, the
cellular location of
c inside the cells was studied in
the absence of any ligand. To rule out a potential
c
staining from the secretory pathway, the cells were treated with
cycloheximide to prevent protein synthesis. In the NK cell line
YT12881,
c staining appears as bright intracellular vesicles dispersed in the cytoplasm (Fig.
1a). Cell surface receptors are too sparse to be detectable. Second, mAbs were used as ligands to
study the capacity of
c to be internalized in YT12881
cells. The cells were incubated with anti-
c mAb TUGh4
for 30 min at 37 °C and after permeabilization stained with a
FITC-labeled secondary antibody. Anti-
c antibodies are
found in intracellular endocytic vesicles (Fig. 1b). These
two methods show that
c is endocytosed in the YT 12881 cell line.
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The c Chain Is Degraded after Endocytosis--
We
have shown previously that the measure of the half-life of a receptor
on the cell surface allows for the analysis of both internalization and
degradation of the receptor (15, 16, 20). At steady state, the total
number of cell surface receptors results from balance between receptor
biosynthesis and endocytosis. After endocytosis, most membrane
molecules are recycled back to the cell surface or degraded. The level
of expression at the cell surface of a protein that is entirely
recycled after internalization remains the same, even when protein
synthesis is inhibited. On the other hand, the level of expression at
the cell surface of a protein that is not recycled after
internalization decreases with time when protein synthesis is
inhibited.
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The Transmembrane and Cytosolic Region of c Chain
Are Sufficient for Endocytosis and Degradation--
The
c chain is a component of at least five interleukin
receptors. The regulation of its surface expression by endocytosis and
degradation may affect the response to the corresponding cytokines. IL2R
, an another IL2R component, also expressed in the YT12881 cells, carries its own endocytic and degradation signals (20). To rule
out a potential role of IL2R
in the endocytic behavior of
c, even though these two proteins are not associated in
the absence of ligand (23), and to study the intrinsic molecular mechanisms involved in
c internalization and
degradation, we have transfected and studied the intracellular traffic
of a chimeric molecule composed of the extracellular region of IL2R
and the transmembrane and intracellular regions of
c,
called
wt, in the Jurkat cell line J77. We have
used this T cell line because it does not express any of the IL2
receptor components on its cell surface, as assessed by cytofluorimetry
(not shown). IL2R
does not carry any signals for its endocytosis and
degradation (15, 16) and has been used previously to prepare other
chimeric membrane proteins, because good antibodies against its
extracellular domain are available (15, 24, 25). It was therefore a
good tool to study the potential role of the transmembrane and
cytosolic domain of
c in endocytosis and degradation.
The endocytosis of IL2R
and
wt was quantitated
by flow cytometry in stably transfected J77 cells as described under
"Experimental Procedures." The chimeric molecule
wt was internalized very efficiently in contrast
to IL2R
, which was very poorly internalized (Fig.
3A). Similar results were
obtained by studying the endocytosis of both proteins with radiolabeled
antibodies (data not shown). Also, HeLa cells were transiently
transfected with IL2R
or the
wt construct, and 2 days after transfection, the cells were processed for
immunofluorescence using anti-IL2R
mAb. Staining of
IL2R
-transfected cells showed a strong surface labeling (Fig.
3B, left), whereas in
wt-transfected cells,
wt was
found in intracellular compartments (Fig. 3B, right).
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The Cytosolic Tail of c Contains Different Regions
Involved in Its Internalization--
To further characterize the
region(s) involved in the internalization of
c, we
constructed truncated forms of the chimeric molecule
wt, with 1, 25, 35, 40, and 65 cytosolic amino
acids (
1,
25,
35,
40,
65, respectively) represented schematically in
Fig. 4A. These constructs were
stably transfected in J77 cells, and their endocytosis was measured by
flow cytometry as described above. Four patterns of internalization
kinetics were obtained (Fig. 5). The
construct
65 was internalized as fast as
wt, showing that the last 21 carboxyl amino acids
of
c are not necessary for endocytosis. Internalization
of the chimera
1 was slow and inefficient, with
only 10-15% of the molecules being internalized at 20 min. Thus, most
of the internalization signals are located between the first and the
65th cytosolic amino acid. The third pattern of
internalization, observed for
25 and
35, and the fourth one, observed for
40, show two intermediate profiles of
internalization. The
25 and
35 constructs were endocytosed in a similar
fashion, with
25% of internalization at 20 min, whereas
40 was more efficiently internalized (
40% of
endocytosis at 20 min) but not as fast as the
wt
and the
65 constructs.
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The Cytosolic Amino Acids 36-40 of c Are Necessary
for Degradation--
We have shown above and elsewhere (16, 17) that a
receptor that is degraded after its internalization has a short
half-life on the cell surface, whereas the half-life of an internalized receptor that recycles to the plasma membrane is long. Therefore, to
study the fate of the different chimeric constructs after
internalization, and to determine the regions involved in sorting
toward degradation, we have measured their half-life on the cell
surface in stably transfected cells. The
40 and
65 constructs have a short half-life of about 120 min, similar to that of
wt (Fig.
6). These results suggest that the three
constructs are efficiently degraded after their internalization, in the
same fashion as
c. Thus, the first 40 cytosolic amino
acids of
c contain sequences sufficient to promote the
degradation of the receptor. In contrast, the half-life of the
25 and
35 constructs was very
long (Fig. 6). Thus, it appears that these two chimeric proteins are not degraded. Taken together with the results presented in Fig. 5,
these experiments show that the deletion of the last 5 amino acids of
40 leads to a complete lost of degradation and
only a partial decrease of internalization. Therefore, these amino acids may belong to a strong degradation and a weak endocytic signal.
The
65 and
wt constructs,
which are internalized faster than
40, are not
degraded faster, suggesting that the last 46 cytosolic amino acids of
c do not contain another degradation signal.
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The Cytosolic Amino Acids 25-65 of c Contain
Transferable Internalization and Degradation Signals--
To
demonstrate positively that the distal region of
c
contains sequences directing internalization and degradation, we
constructed chimeric molecules,
25-65 and
35-65, between the entire IL2R
chain and amino
acids 25-65 or 35-65 from the cytosolic part of the
c
chain (Figs. 7C and
8C).
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DISCUSSION |
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Endocytosis and degradation of the cytokine receptors participate
in the fine tuning of their expression on the cell surface and control
the cell responsiveness to some extent. In this report we show that
c, common to at least five interleukin receptors, is
constitutively internalized and degraded in lymphoid cells. We have
replaced the extracellular region of
c by the
extracellular part of IL2R
. This allowed us to follow the derived
chimeric constructs with available anti-
antibodies. Internalization
studies show that three regions in the cytosolic tail of
c are involved in endocytosis. These regions are located
between amino acids 1-35, 35-40, and 40-65 (Fig. 5). Interestingly,
successive deletions of these regions lead to a decrease in
endocytosis, suggesting that normal endocytosis involves several motifs
along the cytosolic tail and not only one specific short sequence as
described for other receptors (12). The role of various cytosolic
regions in endocytosis has been described previously for receptors such as the P-selectin (26), the prolactin receptor (27), and is also found
in the IL2R
chain (28). The cytosolic domains of many membrane
proteins have short sequences, usually including a tyrosine or a
di-leucine motif, which mediate their rapid internalization through
clathrin-coated pits. The cytosolic tail of
c contains two Tyr and two Leu-Val sequences between the first and the
65th amino acid (Fig. 4B). Further studies are
needed to determine whether these amino acids are part of the
internalization motifs of IL2R
. The
1
construct, although poorly internalized, is endocytosed more
efficiently than IL2R
, suggesting that the transmembrane part of
c may also participate in the endocytic process.
Besides the clathrin-mediated endocytic pathway, receptors can also be
internalized via an alternative pathway. Indeed, receptor-mediated endocytosis pathways through non-clathrin-coated pits have been reported for ricin (29, 30), epidermal growth factor (31), and cholera
and tetanus toxins (32, 33). Interestingly, this pathway has also been
described for IL2 (34) whose receptors contain c. The
three regions found in the cytosolic tail of
c may carry
signals recognized by the cellular components of this alternative
pathway. In this case, the other cytokines whose receptors share the
c chain might also be similarly internalized.
In the absence of clathrin-coated-pit structures, IL2 endocytosis is
2-fold less efficient (34), suggesting that some IL2Rs may also be
internalized via the clathrin-coated pit pathway. In this case, the
three cytosolic regions of c may contain sequences recognized with low affinity by the adaptor complexes. Addition of
three weak signals may constitute an efficient signal. We have already
prepared a chimeric molecule in which the internalization signal of the
transferrin receptor YTRF was inserted in the cytosolic region of
IL2R
(15). When transfected in J77 cells, this chimeric molecule is
internalized more efficiently than
wt,2
indicating that the YTRF motif forms a stronger internalization signal
than the addition of the three regions described above.
The cytosolic domain of c associates constitutively with
the tyrosine kinase Jak3 (8, 35). Jak3 has a key role in
c-mediated signal transduction. It is phosphorylated and
activated by cytokines such as IL2 and IL4, whose receptors contain
c (36, 37), and its activation correlates with
lymphocyte proliferation (37, 38). The cytosolic region of
c necessary for binding and activating Jak3 is located
between residues 40 and 52 (38). Here we show that the first 40 cytosolic amino acids of
c are sufficient for internalization and degradation, suggesting that Jak3 is dispensable in
endocytic process. The region between residues 40 and 65, which contains the association site with Jak3, increases endocytosis, which
might suggest that activation of Jak3 might participate in efficient
endocytosis, as do other kinases in endocytosis of epidermal growth
factor and insulin receptors (39-41). However, in the case of
IL2-dependent Jak3 stimulation, the serine-rich region of
the IL2R
chain is required to obtain the phosphorylation of Jak3
(37), indicating that stimulation via
c alone does not
allow the activation of Jak3. In our cells, the chimeric constructs were studied independently of other components of cytokine receptors and should not be able to activate Jak3. Therefore, it is unlikely that
Jak3 plays a role in the process of endocytosis.
The half-lives of the different chimeric constructs show that a region
containing the 36-40 cytosolic amino acids is involved in the
degradation of c. Indeed, whereas the deletion of the last 46 carboxyl amino acids does not modify degradation, deletion of
amino acids 36-40 leads to a complete loss of degradation. Although
35 is internalized less efficiently than
40 (Fig. 5), this reduced efficiency of
endocytosis is unlikely to account for the absence of degradation of
35. In this respect, it is worth noting that
40 is degraded as fast as
65
and
wt, although its internalization is less
efficient (Fig. 5). Thus, the 36-40-amino acid ESLQP sequence may
belong to a strong degradation signal. Interestingly, these amino acids
are a potential target for a protease, calpain, found to associate to
c in a double hybrid assay and to degrade it in
vitro (42). Although many internalization signals have now been
described, less is known about the mechanisms by which internalized
receptors are directed toward degradation. Sequences necessary for
internalization and degradation of plasma membrane proteins have been
reported (24, 43-46). However, in these cases, it has not been shown
that a short sequence is sufficient to serve as degradation signal. Two
cases of a membrane molecule with a short sequence sufficient for
lysosomal degradation after endocytosis have been reported for the cell adhesion molecule P-selectin (14) and for the IL2R
chain (15). In
both cases, the degradation signal does not match with any of the
signals described so far.
Altogether, our results suggest that endocytosis of c is
mediated by discrete internalization and degradation motifs. This is
confirmed by the fact that addition of amino acids 25-65 of
c confers internalization and degradation to a membrane
protein, which is by itself neither internalized nor degraded
(IL2R
). In conclusion the 25-65-amino acid cytosolic sequence of
c contains both internalization and degradation signals.
Interestingly, when only amino acids 35-65 of
c were
transferred instead of amino acids 25-65, the chimera was similarly
internalized, but it was not degraded. This confirms that the
internalization and degradation signals are not the same. The positive
data shown thus far map the degradation motif to amino acids 25-40
(conferred by the 25-65-amino acid chimera, retained by the truncation
mutant). Further experiments will be necessary to precisely define the
minimal transferable degradation signal.
The presence of endocytic and degradation signals in c
suggests that it participates in endocytosis and degradation of
cytokines whose receptors share the
c chain. The role of
c in cytokine receptors internalization has been
essentially reported for IL2Rs. High-affinity IL2Rs are composed of
three chains (IL2R
, IL2R
, and
c) that associate
noncovalently on the cell surface after ligand binding. The first
report concerning the role of
c in IL2Rs endocytosis
showed that IL2 is internalized only if
c is present
(47). However, this does not clarify the molecular mechanisms and the
respective roles of IL2R
and
c in IL2 endocytosis, as the IL2 binding also depends on the composition of the receptor. We
have shown previously that in a B-cell line derived from a patient
suffering from a X-linked severe combined immunodeficiency (ST B-cell
line), whose IL2Rs are composed of normal IL2R
and IL2R
chains,
and truncated
c (containing only the 6 first residues in
its cytosolic tail), IL2 is internalized 2-fold less efficiently than
in the normal control B-cell lines (17). This difference between the
B-cell lines was not because of a modification in the ability of IL2 to
bind to IL2Rs. It is rather likely because of the loss of the
c endocytic signals that we have characterized in this
report. This indicates that in normal IL2Rs, the cytosolic regions of
c can account for 50% of the ligand internalization. In
contrast to IL2R
, which does not carry any internalization sequences, IL2R
carries also endocytic and degradation signals (15,
20). Therefore, from these and other previously reported data (15-17,
20, 21, 48), it appears that IL2R endocytosis takes place as follows:
both IL2R
and
c carry their own endocytic signals.
Upon heterodimerization of both chains by IL2, these signals are used.
Therefore, the rate of entry of high-affinity receptors is higher than
that of the separate chains, leading to a decrease in surface receptor
expression. The role of
c in receptor endocytosis, also
reported for IL15 (49), is likely to concern the other receptors
containing
c. Its behavior after endocytosis induced by
one cytokine may allow for a fine balance to be achieved between the
expression of the other cytokine receptors containing
c
on the same cells. For instance, in NK cells, where both IL2Rs and
IL15Rs are expressed on the cell surface, IL2-induced down-modulation
of
c (50) decreases the number of
c on
the cell surface available for the formation of IL15 receptors and is
likely to control the response of both cytokines. It will be worthwhile
to determine whether such regulation, previously reported for IL6R
(51), can apply to the other
c-containing cytokine receptors and to members of the expanding family of cytokine receptors sharing one component.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. J. Di Santo and W. Leonard for providing plasmids pRCH and pME18S
/
/
,
respectively; to Véronique Colin for excellent technical help;
and to Drs. F. Niedergang, D. Ojcius, A. Rocca, and A. Subtil for
critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the CNRS "Action Biologie Cellulaire," by the Human Science Frontier Program, and by the Ligue Nationale contre le Cancer, Comité de Paris (for support to purchase a charge-coupled device camera).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.
Supported by an Assistance Publique-CNRS program.
§ To whom all correspondence should be addressed: Unité de Biologie des Interactions Cellulaires, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. Tel.: 33-1-45-68-85-74; Fax: 33-1-40-61-32-38; E-mail: adautry{at}pasteur.fr.
The abbreviations used are: IL, interleukin; ILR, interleukin receptor; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; mAb(s), monoclonal antibody(ies); FITC, fluorescein isothiocyanate.
2 E. Morelon, unpublished results.
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REFERENCES |
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