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-Chain and a Lymphoid Cell Environment1
Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33136
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (c), a subunit of the IL-2R, is essential
for high affinity ligand binding and signal transduction due to Jak3
association to c. Another consequence of IL-2/IL-2R interaction is
rapid receptor-mediated endocytosis of the receptor-ligand complex. In
the present study, we establish that this rapid endocytosis of IL-2 in
a T cell tumor line is dependent upon the cytoplasmic tail of c.
Deletion mutants of the cytoplasmic tail mapped this activity to 9 aa
of c, 45–54 aa distal to the transmembrane region. In contrast,
ligand-independent constitutive endocytosis of c occurred more
slowly and was dependent upon a PEST sequence in a more
membrane-proximal region of the cytoplasmic tail of c. Thus, this
receptor subunit may use distinct sorting signals for its constitutive
regulation and ligand-induced endocytosis. Rapid endocytosis of IL-2
was inhibited by the tyrosine kinase inhibitor genistein, implicating a
role for a signal transduction pathway in IL-2 internalization.
However, one T cell line bearing a mutant c exhibited impaired
endocytosis of IL-2, despite normal IL-2-induced Jak/STAT activation.
Furthermore, inefficient endocytosis of IL-2 was noted after
transfection of the COS7 epithelial cell line with the IL-2R, and
further reconstitution of these cells with Jak/STAT proteins did not
enhance this internalization. Collectively, these latter findings
indicate that rapid endocytosis of IL-2 is dependent upon cellular
signaling in lymphoid cell environment that is not solely a consequence
of the presence of the Jak/STAT pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (p55); the
ß-chain (p90), which is also a subunit of the IL-15R; and the common
-chain (c;3
p75), which is also a subunit of the IL-4R, IL-7R, IL-9R, and IL-15R
(reviewed in Ref. 1). These three subunits are expressed
on the cell surface largely independently of each other in the absence
of IL-2. The -chain binds IL-2 at a relatively low affinity
(Kd of 10-8 M)
with fast on/off-rates (
30 s). The - and ß-chains cooperate to
increase IL-2-binding affinity by 100-fold, but this partial receptor
still exhibits fast on/off-rates. The functional high affinity
(Kd of 10-11 M)
IL-2R, consisting of , ß, and c subunits, exhibits fast IL-2
on-rates characteristic of the -chain, but slow off-rates, which, in
practical terms, represent essentially irreversible IL-2 binding. The
sole role of the -chain is in ligand binding, whereas the ß-chain
and c contribute to ligand binding and signal transduction.
Signal transduction ensues by IL-2-induced trimerization of the ,
ß, and c subunits, which brings the cytoplasmic tails of the ß
and c subunits in close proximity for an extended period of time,
allowing receptor phosphorylation by associated tyrosine kinases
(2, 3). The Janus kinase Jak3, the only known tyrosine
kinase associated with c, importantly contributes to the initial
phosphorylation of the ß-chain (4). This initiates the
recruitment of a number of signal-transducing molecules to the
cytoplasmic tail of the ß-chain, including Jak1, STAT5, and STAT3,
the Shc-adaptor protein, Syk, and p56lck
(reviewed in Ref. 5). Thus, at a minimum, IL-2 signaling
results in the activation of the Jak/STAT, phosphatidylinositol
3-kinase, and the ras/raf/mitogen-activated
protein kinase pathways.
Besides signal transduction, another early consequence of the
IL-2/IL-2R interaction is receptor-mediated endocytosis of the
receptor-ligand complex. This process is often utilized to remove the
receptor-ligand complex from the cell surface. For the IL-2/IL-2R
complex, receptor-mediated endocytosis functions to limit IL-2 signal
transduction, and hence, the biological response to this cytokine. In T
lymphocytes, IL-2 is rapidly internalized
(t1/2 of 10–20 min), ultimately
leading to lysosomal degradation of IL-2 (6, 7, 8). After
internalization, the ß- and c-chains are sorted to late endosomal
compartment, presumably for degradation (9). The
-chain, on the other hand, was detected only in early endosomes,
colocalizing with the transferrin receptor, suggesting that this
subunit may recycle back to the plasma membrane (9). The
route of entry of IL-2/IL-2R complex into the cell has not been
established, but may be independent of clathrin-coated pit endocytosis
(10, 11).
For most hormone receptors, ligand-induced receptor-mediated
endocytosis is dependent upon the cytoplasmic tail of the receptor,
often through a tyrosine-based or di-leucine-based motif
(12). The structural basis for IL-2/IL-2R internalization
has not been extensively investigated. Transfection of wild-type (WT)
(3) and mutant IL-2R subunits points to an important role
for the c subunit for internalization of IL-2 (13). In
fact, rapid IL-2 internalization has been noted for T cells expressing
cytoplasmic tailless IL-2R or IL-2Rß (14, 15),
suggesting no essential role for these subunits in this process other
than ligand binding. The present study, therefore, was undertaken to
more precisely define the contribution of the cytoplasmic tail of the
c subunit in internalization of IL-2. We establish that efficient
internalization of IL-2 depends on 9 aa within the cytoplasmic tail of
c and at least one other IL-2R-independent lymphocyte-specific
component.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CXß is a variant of the mouse EL4 thymoma that expresses mouse
IL-2R, ß, and c subunits, the former two after transfection
with the respective cDNA (14). 1F1 is a mutant variant of
CXß that lacks cell surface mouse c (16). These cells
were cultured in RPMI 1640 medium supplemented with 5% FCS,
L-glutamine (300 µg/ml), penicillin (100 U/ml),
streptomycin (100 µg/ml), and 2-ME (5 x
10-5 M) (complete medium). To maintain
expression of the transfected cDNAs, the cells were periodically (every
10–14 days) passed in complete medium supplemented with mycophenolic
acid (2 µg/ml), xanthine (25 µg/ml), hypoxanthine (15 µg/ml), and
G418 (1 mg/ml; Life Technologies, Grand Island, NY).
Mouse IL-2R constructs
Preparation of the cytoplasmic tailless c284 mutant
(c284Arg
stop codon; in which 1 represents the initial methionine
residue of the leader peptide), which contains only 1 aa of the
predicted cytoplasmic tail, was previously described (17)
and cloned into the pSI expression vector (Promega, Madison, WI). The
cytoplasmic truncations c295 (c295Glustop codon) and c328
(328Cysstop) were prepared by site-directed mutagenesis using the
Chameleon site-directed mutagenesis kit (Stratagene, La Jolla, CA),
according to the manufacturer’s instructions, using the full-length
mouse c cDNA in the pSI vector as the target DNA. The mutagenic
oligonucleotides were 5'-CCCCCCATCAAGAATCTATAGGATCTGGTTACTGAATACC
for c295 and 5'-CTACAGTGAACGGTTCTGACACGTCAGCGAGATTCCCCCC for
c328. The c337 (c337Lysstop) and the cPEST
(c317GluVal; c318SerGly; c321ProLeu;
c322AspAla) were prepared using the Quick Change site-directed
mutagenesis kit (Stratagene), according to the manufacturer’s
instructions. The forward mutagenic primers were
5'-GCGAGATTCCCCCTTAAGGAGGGGCCCTAGG for c337 and
5'-CTAAAGGGCTGACTGTGGGTCTGCAGCTAGCCTACAGTGAACGG for cPEST.
Mutations were confirmed by DNA sequence analysis. The mouse IL-2R
and IL-2Rß cDNAs were cloned into the pSI expression vector. To
prepare pSI-IL-2Rß, the expression cassette from pSI-IL-2Rß was
excised and subcloned adjacent to the expression cassette in
pSI-IL-2R.
Transfection
1F1 cells (8 x 106) were stably
cotransfected with either pSI-c284, pSI-c295, or pSI-c328
(38.5 µg/ml) and BMG-His (18) (kindly provided by E.
Podack, University of Miami) (12.5 µg/ml) in 0.4 ml of RPMI 1640 by
electroporation using a BRL cell porator (Life Technologies) set at
1180 µF and 200 V. The electroporated cells were placed on ice for 10
min; resuspended in complete medium containing mycophenolic acid,
xanthine, hypoxanthine, and G418; and cultured (4 x
104/well) in 96-well flat-bottom culture plates
at 37°C in a 7% CO2 incubator. Twenty-four
hours later, histidinol (0.5 mM) was added to the cultures. 1F1 was
similarly transfected with the cWT, c337, or cPEST cDNA, but
using only the pSI-cWT vector (50 µg/ml) containing the CMV
ZeoCassette (Invitrogen, Carlsbad, CA). Cells were selected by addition
of Zeocin (150 µg/ml; Invitrogen) 24 h after transfection.
COS7 cells were harvested by treatment with trypsin-EDTA and washed,
and 5–20 x 106 cells were transiently
transfected, as described above, at 330 µF and 215 V. These cells
were transfected with either pSI-IL-2Rß (15 µg/ml), pSI-cWT
(1–5 µg/ml), and pME18S-mJak3 (15 µg/ml) (19) (kindly
provided by J. O’Shea, National Institutes of Health), as indicated,
or pSI-IL-2Rß, pSI-cWT, pME18S-Jak3, pcDNA-mSTAT5a
(20) (kindly provided by W. Leonard, National Institutes
of Health), and Prk5-mJak1 (21) (kindly provided by J.
Ihle, St. Jude Children’s Research Hospital, Memphis, TN) at 5 µg/ml
for each vector, as indicated. In these latter transfections, empty pSI
vector was added, as required, to maintain a constant final
concentration of DNA (30 µg/ml). We noted similar expression of high
affinity IL-2R after both types of transfection conditions. After
transfection, cells were cultured in complete medium at 1 x
106 cells/100 mm2 tissue
culture plates for 3 days. Cells were then harvested for experimental
assays by first washing the plates with PBS and then harvesting the
adherent cells by incubation with prewarmed (37°C) PBS containing 5
mM EDTA for 5 min.
Abs and other reagents
mAbs to mouse IL-2R (3C7) (22), IL-2Rß (5H4)
(16), and c (4G3 and 3E12) (17) were
previously described. PE-conjugated anti-c (4G3) and
FITC-conjugated goat anti-rat Ig were obtained from PharMingen (San
Diego, CA). Rabbit antisera to mouse c, Jak1, Jak3, and STAT5 were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and to
phospho-STAT5 from Upstate Biotechnology (Lake Placid, NY).
HRP-conjugated donkey anti-rabbit Ig was obtained from Amersham
Pharmacia Biotech (Piscataway, NJ). Emetine was obtained from Sigma
(St. Louis, MO), and genistein from Calbiochem (San Diego,
CA).
FACS analysis
FACS analysis was performed as previously described (23) using a Becton Dickinson FACScan and CellQuest software. Typically, 10,000 cells/sample were analyzed. Dead cells were excluded from analysis by forward vs side scatter gating. Depending upon the primary Ab, control stained cells were incubated with either PE-streptavidin or FITC goat anti-rat Ig.
Internalization assays
CXß and the 1F1 transfectants (10 x 106/ml) or the transiently transfected COS7 cells (4 x 106/ml) were incubated at 4°C for 30 min with 50,000 cpm/ml human 125I-labeled IL-2 (125I-IL-2). The IL-2 was radiolabeled with Na125I using IODO-GEN-precoated tubes (Pierce, Rockford, IL), according to the manufacturer’s instruction, to a sp. act. of approximately 20–40 µCi/µg. The cells were washed three times with cold HBSS and resuspended in complete medium at 4 x 106/ml (CXß and 1F1 transfectants) or 2 x 106/ml (COS7) and shifted to 37°C. At the indicated times, 1 ml was removed from culture and cells were pelleted by centrifugation in a microfuge at 14,000 x g for 15 s in 1.5-ml tubes. Protein released in the supernatant was precipitated by addition of 1/4 vol of 50% TCA. The TCA-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 then centrifuged for 15 s at 14,000 x g in a microfuge. The radioactivity that remained associated with the cells after this low pH buffer wash represented the internalized (I) IL-2, while 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 80–85% of the cpm was cell surface associated. The I:S ratio was calculated at the indicated time points. The pH 2-resistant material at t0 was considered nonspecific material and was subtracted from I.
Internalization of transferrin was similarly evaluated, except that initially CXß and the 1F1 transfectants (40 x 106/ml) or the transiently transfected COS7 cells (12 x 106/ml) were incubated at 4°C for 30 min with 250,000 cpm/ml of 125I-labeled transferrin (1 µCi/µg; NEN, Boston, MA).
Western blot analysis
Cells were extracted in buffer containing 0.5% Nonidet P-40, as previously described (24). The indicated Ab was first bound to protein G-Sepharose (Amersham Pharmacia Biotech) by incubation for 30 min at 25°C, washed three times with extraction buffer, and then used for immunoprecipitations by incubation of the Ab-coated beads with the Nonidet P-40 extracts at 4°C overnight. The immunoprecipitates were washed three times with extraction buffer containing 0.5% Nonidet P-40, and the bound material was eluted with sample buffer containing 2% SDS. Samples were resolved by 10% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and blocked by incubation with 5% nonfat dried milk in PBS (5% milk) for 1 h. The nitrocellulose was subsequently incubated with the indicated antisera in 5% milk for 90 min, washed three times with PBS containing 0.1% Nonidet P-40, and then incubated with HRP-conjugated donkey anti-rabbit Ig for 60 min, followed by washing twice with PBS containing 0.1% Nonidet P-40 and once with PBS. 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c
The CXß cell line is a transfected mutant variant
(14) of the mouse EL4 thymoma that constitutively
expresses IL-2R, ß, and c (Fig. 1
A). CXß was further
mutagenized to yield another variant, designated 1F1, containing a
deletion encompassing the transmembrane region of c, which
essentially lacks cells surface c, as assessed by FACS (Fig. 1B) and biochemical analysis (16).
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c in
internalization of IL-2 (13). To test whether the
cytoplasmic tail of c was essential, 1F1 was transfected with c
cDNA that contained the entire extracellular and transmembrane regions
of c, but only 1 aa of the predicted cytoplasmic tail. The resulting
1F1c284 cell line expressed high levels of c as well as IL-2R
- and ß-chains (Fig. 1
C). Internalization of IL-2 by
these cells was compared with the parental CXß cell line by first
pretreating the cells with radiolabeled IL-2 in the cold and then
shifting the cells to 37°C to assess the fate of the cell-bound IL-2.
It is evident that the rate at which IL-2 is internalized (Fig. 2A), is lost from the cell
surface (Fig. 2B), and is metabolized (Fig. 2C)
occurred more slowly for 1F1284 than CXß.
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D). Under these conditions, the time at which the I:S is 1
represents the t1/2 for internalization and
is directly related to the rate of internalization (25, 26). The t1/2 for CXß is 15 min,
while the t1/2 for 1F1284 is 36 min.
Therefore, in the absence of cytoplasmic tail of c, the IL-2R
internalized 125I-IL-2 approximately 2-fold more
slowly. This trend held in all our subsequent analysis, although
sometimes the absolute level and the time to reach plateau levels of
intracellular IL-2 varied.
Mapping the cytoplasmic tail of c for internalization signals
The cytoplasmic tails of many cell surface receptors have been
shown to express specific, relatively short, amino acid sequences that
function for rapid ligand-induced receptor-mediated endocytosis
(12). To determine whether c contained a
membrane-proximal internalization motif, several additional mutant c
cDNAs were expressed in 1F1 that contained progressively longer
cytoplasmic tails. 1F1c295, 1F1c328, and 1F1c337 expressed
c molecules containing 12, 45, and 54 aa, respectively, of an
85-aa-long cytoplasmic tail (Fig. 3A). 1F1 was also transfected
with the WT c cDNA to ensure that the slow internalization of
1F1c284 was not due to a secondary defect in 1F1 cell line.
Biochemical analysis confirmed that the transfected cells expressed the
appropriate c molecules of increasing
Mr (Fig. 3B). The material
detected in band 1 (B1) represented the heavily glycosylated mature
cell surface c, while that in band 2 (B2) represented an
endo-H-sensitive intracellular c intermediate (16)
(data not shown). FACS analysis with anti-c mAb to the
extracytoplasmic region revealed relatively high expression of these
c molecules on the surface of all four transfectants, with the
highest expression on 1F1c295 (Fig. 3C).
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).
This analysis indicates that the internalization of IL-2 by 1F1c284
(not shown), 1F1c295, and 1F1c328 are all similarly impaired when
compared with CXß (not shown) and 1F1cWT. These data directly
demonstrate that the 1F1 cell line is competent to support normal
internalization of IL-2, provided that these cells express WT c.
These data also indicate that rapid internalization of IL-2 required
the proximal 54 aa of the cytoplasmic tail of c and maps to a 9-aa
region between residues 328 and 337.
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c for constitutive turnover
The proximal 44 aa of the cytoplasmic tail of c have been
implicated in ligand-independent endocytosis (27) and
contain a PEST sequence that may function to regulate c surface
levels (28). To assess whether this region of c
similarly functioned in our EL4-transfected variants, the constitutive
turnover of c by CXß and the 1F1c transfectants was compared.
These cells were treated with emetine, a protein synthesis inhibitor,
and the loss of cell surface c was assessed by cell surface FACS
analysis using an anti-c mAb (Fig. 5). This same approach has been
previously used to study the turnover of human c in the YT cell
line, and the decrease in surface c as measured by FACS was
essentially identical to the decrease in total c protein as measured
by Western blot analysis (27). Cells that expressed a
severely truncated c cytoplasmic tail, i.e., 1F1284 (not shown)
and 1F1c295, exhibited a relatively slow and linear decrease in
their c molecules with a t1/2 of
approximately 290 min. By contrast, the c molecules of CXß (not
shown), 1F1cWT, and 1F1c328 each exhibited a similar more rapid
and biphasic decrease, with a t1/2 of
approximately 150 min, which is two times faster than seen for
1F1284 and 1F1c295. The turnover
(t1/2) of human WT c in YT cells
has been reported to be 120 min (27), similar to what we
have observed for the decrease of mouse WT c by this FACS analysis.
Collectively, our results indicate that the constitutive turnover of
c is dependent upon 33 aa between positions 295 and 328 of the
cytoplasmic tail. Furthermore, this finding demonstrates that the
region of the cytoplasmic tail responsible for the constitutive
endocytosis of c is distinct from that required for IL-2-induced
receptor-mediated endocytosis.
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c contains a species-conserved
calpain-sensitive PEST sequence
(GLTESLQPDYSE) (28)
that may function to regulate the level of this protein. To determine
whether the PEST sequence in mouse c contributed to
ligand-independent endocytosis, 3 aa between residues 317 and 321 were
mutated (GLTVGLQLAYSE) (Fig. 3A), as
previously done for human c (28). The transfected
1F1cPEST cells expressed c, as assessed by biochemical (Fig. 3B) and FACS (Fig. 3C) analysis. The somewhat
lower levels of the mature form of c from 1F1cPEST (B1, Fig. 3B) most likely reflected some variability that we have
detected in the overall expression of c transfected in 1F1 cells,
when analyzed at different times. Importantly, the rate of
internalization of c in 1F1cPEST was comparable with that seen
for 1F1c295 (Fig. 5). Thus, the integrity of the PEST sequence is
required for normal ligand-independent endocytosis of c. Tissue specificity for IL-2-induced endocytosis
We developed a transient assay with the aim to rapidly
characterize internalization of variants of the IL-2R. The approach was
to cotransfect COS7 cells with IL-2R, ß, and c cDNAs and assess
IL-2 internalization 72 h later by binding IL-2 to the cell
surface. Surprisingly, when compared with CXß, the transfected COS7
cells inefficiently internalized 125I-IL-2 (Fig. 6A). By contrast, the
internalization of transferrin, as assessed by the release of
apo-transferrin into the culture media, was identical for CXß-,
1F1c284-, and IL-2R-transfected COS7 (Fig. 6B).
Untransfected COS7 cells failed to bind and internalize IL-2 (not
shown), demonstrating specificity for the transfected cells. The
t1/2 for internalization of IL-2 by
IL-2Rßc-transfected COS7 cells was 39 min, more than 2-fold
slower than detected for CXß and slightly slower than observed for
1F1284. These data suggest that a lymphoid-specific component
independent of the IL-2R is also required for rapid endocytosis of
IL-2.
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One potential lymphoid-specific component that associates with the
cytoplasmic tail of c is the tyrosine kinase Jak3. Initially, the
effect of the tyrosine kinase inhibitor genistein was tested on IL-2
internalization. When internalization was allowed to proceed for 45
min, genistein substantially inhibited IL-2 internalization by CXß
and 1F1c284 (Fig. 7). Interestingly,
in the presence of genistein, the proportion of internalized IL-2 by
CXß was still greater than detected for 1F1c284. This result
suggests that internalization may depend upon two components, the
cytoplasmic tail of c and a tyrosine kinase activity that at least
in part is independent of the cytoplasmic tail of c. At 45 min, the
IL-2Rß-transfected COS7 cells showed even greater impairment in
internalization than 1F1c284, and this internalization was minimally
sensitive to the effects of genistein. This finding suggests that the
tyrosine kinase-dependent component for IL-2 internalization may be
lymphoid specific.
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Jak3 is a lymphoid-specific tyrosine kinase that associates with
c. Therefore, we tested whether functional Jak3 activity might be
required for IL-2-induced receptor-mediated endocytosis. For these
experiments, we compared the ability of IL-2 to induce STAT5
phosphorylation in 1F1 cells transfected with WT or truncated mutant
c (Fig. 8). As expected, three
independently derived transfected 1F1c295 cell lines failed to
phosphorylate STAT5, demonstrating the dependence on the cytoplasmic
tail of c for this phosphorylation. By contrast, STAT5 was
phosphorylated in all the 1F1c328 and 1F1c337 cells, albeit at
somewhat different levels. This activation of STAT5 was strictly
dependent upon the addition of IL-2 to each type of transfectant (not
shown). Importantly, STAT5 was phosphorylated in 1F1c328, which,
like 1F1c295, inefficiently internalized IL-2 (see Fig. 4). This
finding demonstrates that c-dependent Jak3 functional activity is
not the sole c signal required for rapid ligand-dependent
receptor-mediated endocytosis.
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ßc-transfected COS7 cells were also cotransfected with
Jak3 or Jak1, Jak3, and STAT5. The expression of these multiple genes
in COS7 for the cytokine receptor subunits was confirmed by
irreversible binding of IL-2 and FACS analysis with anti-IL-2R
subunit-specific mAbs (not shown) and for Jak/STAT molecules by Western
blot analysis of untransfected and transfected COS7 cells (Fig. 9A). Essentially, no Jak3 and
minimal STAT5a were present in the untransfected cells, while these
proteins were readily detected after transfection. As reported by
others (20), we detected a low amount of Jak1 in the
untransfected COS7, which was also substantially increased after
cotransfection of this cDNA.
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ßc-transfected COS7 cells
in the absence or presence of Jak3 or Jak1, Jak3, and STAT5a was
largely comparable and substantially lower than seen for CXß T cells
when examined at both 15 min and 30 min after placing the
125I-IL-2-treated cells at 37°C (Fig. 9B). These data indicate that reconstitution of Jak3 and
STAT5a in COS7 is not sufficient to increase IL-2 internalization by
this nonlymphoid cell. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c in lymphoid cells in general
and T lymphocytes in particular have been hampered because virtually
all lymphoid cells constitutively express c, making such cell lines
unsuitable as recipients for transfected mutant c constructs.
Several laboratories have overcome this problem by expressing chimeric
receptors in T lymphocytes in which the WT or mutant cytoplasmic tail
of c is linked to extracytoplasmic domain of a distinct protein
(3, 27). The function of c is then inferred by
stimulating the T cells through the extracytoplasmic region of the
chimeric molecule. This approach by the nature of its design is not
suitable for direct analysis of IL-2-induced receptor-mediated
endocytosis. In the present study, a mutant variant of the mouse EL4
thymoma, designated 1F1 (16), which constitutively
expresses IL-2R - and ß-chains, but not cell surface c, was
exploited to begin to study the structural basis by which c controls
IL-2R-mediated endocytosis of IL-2. Two major new observations emerge
from this study. First, there are distinct cytoplasmic regions of c
that function during endocytosis, one for ligand-independent
constitutive endocytosis of c and another for c-dependent
IL-2-induced endocytosis. Second, efficient internalization of IL-2 is
dependent upon a lymphoid cell environment.
Internalization of IL-2 by T lymphocytes after binding to the high
affinity IL-2R has been reported to occur at a
t1/2 of 10–20 min (6, 7, 8). The
t1/2 for CXß and 1F1cWT was
approximately 10–15 min, a value typically expected for IL-2
internalization by a T cell. 1F1 cells transfected with cytoplasmic
tailless c (1F1c284) internalized IL-2 at a rate approximately
twice as slow as the cells expressing WT c, indicating that IL-2
internalization is dependent upon c (13) and its
cytoplasmic tail. Expression of WT IL-2R, ß, and c in COS7
monkey kidney epithelial cells resulted in endocytosis of IL-2 at a
rate slightly slower than detected for 1F1c284, demonstrating that
the cytoplasmic tail of c is necessary, but not sufficient, for IL-2
internalization. This finding suggests that efficient internalization
of IL-2 is dependent not only on c, but also upon one or more
IL-2R-independent lymphoid-specific factors.
The cytoplasmic tail of c contains a calpain-sensitive PEST sequence
that has been implicated in T cell function (28). This
sequence is found in the proximal region of the 85-aa cytoplasmic tail
of c, between aa 35 and 43. Deletion mutants of the cytoplasmic tail
of c in the context of an IL-2R/c chimeric molecule have
mapped the region between residues 35 and 40 in the cytoplasmic tail of
c as being critical for the constitutive endocytosis of this
molecule (27). Our c328 mutant, which contains the
first 45 aa of the cytoplasmic tail, exhibited rapid constitutive
endocytosis of c when transfected into the 1F1 cell line that was
comparable with WT c. The constitutive turnover of the c284 and
c295 mutants, containing 1 and 12 aa of the cytoplasmic tail, was
approximately two times slower. Importantly, site-directed mutagenesis
of this PEST site in the context of full-length c resulted in c
turnover essentially identical to that seen for cytoplasmic tailless
c. Thus, these results are consistent with the above findings
(27, 28) and directly demonstrate a role for this sequence
in regulation of the constitutive endocytosis.
Rapid c-dependent endocytosis of IL-2 clearly requires more than
these first 45 aa, including the PEST sequence, of the cytoplasmic
tail. The rate of internalization by IL-2R in the context of the
c284, c295, and c328 mutants, the latter of which includes the
PEST sequence, was approximately 2-fold slower than IL-2R containing
cWT and c337. This analysis maps IL-2-induced endocytosis to 9 aa
between residues 45 and 54 of the cytoplasmic tail. We have not defined
which of these nine residues function as an endocytic signal. This
region lacks a di-leucine motif and does not obviously contain a
tyrosine motif similar to those reported to target receptor-ligand
complexes to clathrin-coated pits during the initial phase of
internalization. The importance of coated-pit structures in IL-2/IL-2R
internalization has not been established unequivocally. If fact,
substantial internalization of IL-2 was noted even after effectively
blocking clathrin-coated pit internalization of transferrin (10, 11). Thus, the IL-2/IL-2R may utilize a novel pathway to deliver
the ligand-receptor complex to the endosome that is dependent upon c
cytoplasmic signals that do not show obvious similarity to other cell
surface receptors.
Nelson and colleagues (29) mapped the box 2 region of c
to aa 40–52 of the cytoplasmic tail, which, along with the upstream
box 1 region, are required for Jak3 binding to c. As c-dependent
STAT5 phosphorylation readily occurred in 1F1c328, our study refines
the region required for Jak3 binding to c to the first 45 aa of the
cytoplasmic tail. Furthermore, since 1F1c328 showed impaired
internalization of IL-2, this mutant clearly illustrates that Jak/STAT
activation is not sufficient for rapid endocytosis of IL-2 in T cells,
although this does not rule out a possible contribution to endocytosis
by these molecules. We also found that expression of Jak3, either alone
or with Jak1 and STAT5a, in IL-2R-bearing COS7 did not increase the
impaired IL-2 endocytosis by these nonlymphoid cells. These findings
further emphasize that Jak3 and the Jak/STAT pathway are insufficient
by themselves to target IL-2/IL-2R for rapid endocytosis and indicate
that Jak3 is not the key lymphoid-specific component required for
normal endocytosis of IL-2. The requirement for tyrosine kinase
activity for IL-2 endocytosis might be the result of an effect on
intracellular signaling independent of c or Jak3, perhaps on some
downstream signaling molecule. Beside Jak1, the tyrosine kinases
p56lck and Syk also associate with the
cytoplasmic tail of the IL-2R ß-chain (30, 31). It is
highly unlikely that either of these tyrosine kinases are the targets
for genistein because we have previously shown that another variant of
the EL4 thymoma normally internalizes IL-2 even though their IL-2R
consists of WT IL-2R and c, but a cytoplasmic tailless ß-chain
(14). Further studies are necessary to define the
signaling requirements for endocytosis of IL-2, including the possible
relationship to lymphocyte-specific components that control endocytosis
of this cytokine.
|
Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Malek, Department of Microbiology and Immunology, University of Miami School of Medicine, 1600 N.W. 10th Avenue, Miami, FL 33136.
3 Abbreviations used in this paper: c, common -chain; I, intracellular; 125I-IL-2, 125I-labeled IL-2; S, surface; WT, wild type.
Received for publication November 18, 1999. Accepted for publication June 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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c-dependent cytokines and receptors: regulation of T lymphocyte development and homeostasis. Crit. Rev. Immunol. 18:503.[Medline]
-chain cytoplasmic domains is required for signaling. Nature 369:330.[Medline]
chains mediate the signal for T-cell proliferation. Nature 369:333.[Medline]
and ß chains are internalized and remain associated inside the cells after interleukin 2 endocytosis. J. Biol. Chem. 267:18639., ß, and chains. J. Cell Biol. 129:55. chain of the human IL-2 receptor. Science 257:379.c subunits. J. Interferon Cytokine Res. 15:447.[Medline]
c subunit of the IL-2, IL-4, and IL-7 receptors: distinct interaction of c in the IL-4 receptor. J. Immunol. 154:1596.[Abstract]
/ß and - signal transduction. Nature 366:129.[Medline]
c chain. J. Biol. Chem. 273:22044. chain (c) by calpain. Proc. Natl. Acad. Sci. USA 94:11534.c chain sufficient for Jak kinase activation and induction of proliferation in T cells. Mol. Cell. Biol. 16:309.[Abstract]
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) were treated with 125I-IL-2 at 4°C, and
then t0 were cultured at 37°C.
A, Rate of internalization of IL-2. B,
Rate of loss of cell surface IL-2. C, Rate of
degradation of IL-2. Data in A–C are taken from one
experiment representative of six. D, Ratio of
intracellular (I) to surface (S) IL-2. Linear regression of data from
six experiments (mean ± SD).
r2 was 0.99 for CXß and 0.86 for
1F1
) were treated
with 125I-labeled ferric-transferrin and then cultured at
37°C. The release of transferrin into the culture supernatant was
determined. The ferric-transferrin/transferrin receptor complex is
rapidly internalized in early endosomes, and the receptor recycles back
to the cell surface and undegraded apo-transferrin is released by the
cell.
