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J Biol Chem, Vol. 274, Issue 38, 26978-26984, September 17, 1999
-Chain mRNA Results in a Shortened Form with a Distinct
Pattern of Expression*
,
From the Groupe de Recherche Cytokines et Récepteurs, Unité INSERM 463, Institut de Biologie, 9 Quai Moncousu, 44035 Nantes Cedex 01, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We report the existence of eight different
interleukin-15 receptor Interleukin
(IL)1-15 is a cytokine that
was discovered through its capacity to replace IL-2 in supporting the
growth of the murine IL-2-dependent CTLL cell line (1, 2).
Both cytokines belong to the short IL-15R Three isoforms of IL-15R Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Analysis
Total RNA was extracted from various human cell lines and
tissues using guanidinium thiocyanate/phenol as described (21). Total
RNAs from human liver, brain, and small intestine were purchased from
CLONTECH (Basingstoke, United Kingdom). Reverse
transcription and PCR amplifications were performed as described
previously (22). For PCR, the cycling conditions were as follows:
denaturation for 1 min at 94 °C; annealing for 1 min at 66 °C
(E1/E7), 68 °C (E1/E7'), 52 °C (E4/p3BGH), 55 °C
( Molecular Constructs
The four products resulting from RT-PCR using primers E1 and E7
were ligated into the pNoTA/T7 plasmid, leading to pNo15R, pNo15R Cell Culture and Transfections
The U937 human histiocytic lymphoma cell line, the SAOS-2 human
osteogenic sarcoma cell line, and COS-7 monkey kidney epithelial cells
were purchased from the American Type Culture Collection (Manassas, VA)
and cultured in RPMI 1640 medium containing 10% fetal calf serum and 2 mM glutamine (U937 and SAOS-2 cells) or Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and 2 mM glutamine (COS-7 cells). The Kit 225 human T lymphoma cell line (obtained from Dr. Doreen Cantrell, Imperial Cancer Research
Fund, London, UK) was cultured in RPMI 1640 medium containing 6% fetal
calf serum, 5 ng/ml IL-2, and 2 mM glutamine. COS-7 cells (1.5 × 106 cells/plate) were transfected using the
DEAE-dextran/chloroquine method (23) with 10 µg of each
pcDNA3.1/Myc-His construct. The same plasmids (30 µg) were used
to transfect Kit 225 cells by electroporation (220 V, 960 microfarads),
and transfectants were selected in culture medium containing 750 µg/ml Geneticin and 5 ng/ml IL-2.
Antibodies
Rabbit polyclonal anti-histidine antibody and monoclonal
antibody to the c-Myc epitope were from Santa Cruz Biotechnology (Santa
Cruz, CA). Anti-p300 monoclonal antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). Biotin-conjugated affinipure goat anti-mouse antibody was from Immunotech (Marseille, France). Cy5-conjugated streptavidin was from Jackson ImmunoResearch
Laboratories, Inc. (West Grove, PA). Fluorescein
isothiocyanate-conjugated affinity-purified anti-rabbit F(ab')2
fragments were from BioAtlantic (Nantes, France).
Confocal Immunofluorescence Microscopy
Confluent COS-7 cells collected 48 h after transfection
were fixed with 50% methanol and 50% acetone at 4 °C for 10 min
and incubated with anti-p300 monoclonal antibody (10 µg/ml) overnight at 4 °C, with biotin-conjugated goat anti-mouse antibody (1:100 dilution) for 1 h, and with Cy5-conjugated streptavidin for 30 min
at room temperature. Cells were further incubated for 1 h at room
temperature with anti-histidine antibody (1:200 dilution) and with
fluorescein isothiocyanate-conjugated anti-rabbit F(ab')2 for 30 min
and analyzed by scanning confocal microscopy (Leica, Rueil-Malmaison, France).
Subcellular Fractionation and Biochemical Analysis
Enzymatic Treatments--
COS-7 cells were lysed in radioimmune
precipitation assay buffer (50 mM Tris buffer (pH 7.5)
containing 100 mM NaCl, 0.25% sodium deoxycholate, 0.1%
Nonidet P-40, 1 mM sodium vanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml
aprotinin). After 30 min on ice, cell lysates were clarified by
centrifugation (12,000 rpm, 20 min, 4 °C). Samples (30 µg of protein) were treated either with 250 milliunits of endoglycosidase F
(Roche Molecular Biochemicals, Meylan, France) according to the
manufacturer's instructions or with 3 mM neuraminidase
(Roche Molecular Biochemicals) plus 2 milliunits of
O-glycosidase (Roche Molecular Biochemicals) for 18 h
at 37 °C in 50 mM sodium acetate buffer (pH 6)
containing 9 mM CaCl2 and 150 mM NaCl.
Non-nuclear Membrane Fractions--
They were prepared as
described previously (22). After depletion of nuclei, the supernatant
was centrifuged at 100,000 × g for 45 min at 4 °C
to separate the cytosolic fraction (supernatant) from the membrane
fraction (pellet).
Nuclear Membrane Fractions--
Nuclei were purified according
to a previously described procedure (24). The nuclear pellet was lysed
for 20 min at 4 °C in 20 mM Hepes buffer (pH 7.9)
containing 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation
(12,000 rpm, 30 min, 4 °C), the pellet containing nuclear membranes
was resuspended in 50 µl of the same buffer. The protein content of
each subcellular fraction was determined by the BCA method (Pierce).
For immunoblot analysis, fraction samples (30 µg) were resolved by
SDS-polyacrylamide gel electrophoresis under reducing conditions,
transferred to polyvinylidene difluoride membranes (Millipore Corp.,
Bedford, MA), and probed with anti-histidine antibody (1:1000
dilution). Detection of the antibody-antigen complexes was achieved by
enhanced chemiluminescence (ECL kit, Roche Molecular Biochemicals) and
exposure to X-Omat films (Eastman Kodak Co.).
Cell-surface Labeling and Immunoprecipitation
COS-7 cells were iodinated with 100 µg of IODO-GEN (Pierce)
and 0.4 mCi of [125I]iodine for 20 min at room
temperature. Cell lysates were made in radioimmune precipitation assay
buffer, cleared by centrifugation, and immunoprecipitated overnight
with anti-histidine antibody (10 µg) and protein A-coupled Sepharose
beads (Amersham Pharmacia Biotech, Orsay, France). Protein A-bound
material was eluted with 1× Laemmli sample buffer (Bio-Rad, Ivry sur
Seine, France) at 95 °C for 5 min and resolved by SDS-polyacrylamide
gel electrophoresis under reducing conditions. Iodinated proteins were
detected by autoradiography (PhosphorImager, Molecular Dynamics, Inc.,
Sunnyvale, CA).
IL-15 Binding Assays
Human recombinant IL-15 (Peprotech, Rocky Hill, NJ) was
iodinated as described (25) with a specific radioactivity of ~2000 cpm/fmol. Binding experiments were carried out for 75 min at 4 °C in
phosphate-buffered saline/bovine serum albumin as described previously
(26) using 106 cells/well and increasing concentrations of
labeled IL-15 in a final volume of 50 µl. Nonspecific binding was
determined in the presence of a 100-fold excess of unlabeled cytokine.
Regression analysis of the binding data was accomplished using a
one-site equilibrium binding equation (Grafit, Erithacus Software,
Staines, UK).
IL-15R Proliferation Assays
Transfected Kit 225 cells were washed twice to remove IL-2 or
IL-15 and starved for 1 h in RPMI 1640 medium with 6% fetal calf
serum and 2 mM glutamine. Cells were then plated (5 × 104 cells/well) and cultured for 48 h in culture
medium containing 750 µg/ml Geneticin and supplemented with
increasing concentrations of IL-2 or IL-15. They were pulsed for 6 h with 4 µCi of [3H]thymidine and harvested onto
Whatman filters. Cell-associated [3H]thymidine was
measured using a Microbeta counter (Wallack, Turku, Finland).
Characterization of New IL-15R
These eight isoforms were detected in most of the cell lines and
tissues examined (Fig. 2), except fetal bone marrow and the choriocarcinoma JAR cell line. The respective expression levels of the
different isoforms varied from one cell/tissue type to another.
Whereas, in most cases, the full-length and Biochemical Analysis of IL-15R
In the case of Subcellular Localization of the IL-15R
To support these observations, Western blot analyses were carried out
on subcellular fractions prepared by biochemical means (Fig.
3B). In the membrane fraction prepared from isolated nuclei, IL-15R
Confocal microscopy did not reveal detectable expression of IL-15R Deletion of Exon 2 Results in a Loss of IL-15 Binding--
To
evaluate the function of the sushi domain encoded by exon 2, we first
analyzed the binding of radioiodinated IL-15 to transfected COS-7 cells
(Fig. 5A). Mock-transfected
COS-7 cells did not bind IL-15, whereas cells transfected with
IL-15R Recently, three different IL-15R Expression of human IL-15R IL-15R IL-15R Of major interest with respect to these findings is the recent
demonstration that a newly identified isoform of IL-15 that uses a
short (21 amino acids) signal
peptide (SSP-IL-15) is not directed to the secretory
pathway, but rather is stored intracellularly, appearing in the
cytoplasm and nucleus (31). The other IL-15 isoform containing a
long (48 amino acids) signal
peptide (LSP-IL-15), in contrast to SSP-IL-15, has no
nuclear localization and is directed to the secretory pathway. The two
isoforms have also distinct tissue distribution and are likely to be
generated by the usage of alternate promoters rather than by
alternative splicing, suggesting that they might serve different roles.
Our observations raise the interesting possibility that IL-15R Exon 2-truncated IL-15R
-chain (IL-15R) transcripts resulting
from exon-splicing mechanisms within the IL-15R gene. Two main
classes of transcripts can be distinguished that do or do not (
2
isoforms) contain the exon 2-coding sequence. Both classes were
expressed in numerous cell lines and tissues (including peripheral
blood lymphocytes) at comparable levels and could be transcribed in
COS-7 cells, and the proteins were expressed at the cell surface. Both
receptor forms displayed numerous glycosylation states, reflecting
differential usage of a single N-glycosylation site as well
as extensive O-glycosylations. Whereas IL-15R bound
IL-15 with high affinity, 2IL-15R was unable to bind IL-15, thus
revealing the indispensable role of the exon 2-encoded domain in
cytokine binding. A large proportion of IL-15R was expressed at the
nuclear membrane with some intranuclear localization, supporting a
potential direct action of the IL-15·IL-15R complex at the nuclear
level. In sharp contrast, 2IL-15R was found only in the
non-nuclear membrane compartments, indicating that the exon 2-encoded
domain (which is shown to contain a potential nuclear localization
signal) plays an important role in receptor post-translational routing.
Together, our data indicate that exon 2 splicing of human IL-15R is
a natural process that might play regulatory roles at different levels.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical cytokine family (3).
Unlike IL-2 mRNA, whose expression is restricted to activated T
cells, IL-15 mRNA is expressed by a variety of tissues and cell
types, including monocyte/macrophages, kidney epithelial cells,
keratinocytes, fibroblasts, nerve cells, placenta, skeletal muscle, and
heart (1, 4, 5). IL-15 can replace IL-2 in most of its activities in
the lymphoid system, including T cell chemotaxis (6), induction of
proliferation and cytotoxicity of activated T cells (1) and natural
killer cells (2, 7), and co-stimulation of B cell proliferation and
immunoglobulin synthesis (8). This redundancy was shown to be due in
part to the sharing, within their high affinity receptors, of common
transducing subunits, the IL-2 receptor (IL-2R) 
- and 
-chains
(9). Cytokine specificity is conferred by short receptor chains,
IL-2R and IL-15R (10-13). Unlike IL-2R and IL-2R, these
two -chains are not members of the hematopoietin receptor family,
but instead contain, in their extracellular parts, structural domains
previously defined as protein-binding motifs (called the "sushi
domain"; also known as the glycoprotein-I motif or short consensus
repeat) present in some complement and adhesion molecules (14).
IL-2R contains two such domains, whereas IL-15R contains only
one. In contrast to IL-2R, which alone binds IL-2 with low affinity
(Kd = 10 nM) (15), IL-15R on its own
binds IL-15 with high affinity (Kd = 10 pM) (13). IL-2R and IL-2R build up a functional
receptor of intermediate affinity (Kd = 1 nM) competitively shared by both cytokines, and association
of the -chains leads to the formation of cytokine-specific functional high affinity ternary () receptor complexes
(Kd = 10 pM) (16, 17).
mRNA are expressed in T cells, B cells, and macrophages
as well as thymic and bone marrow stromal cell lines. Like IL-15
mRNA, IL-15R messages are also expressed in various nonimmune cell types and tissues, including liver, skeletal muscle, heart, and
lung (12, 13), suggesting that the IL-15 system may operate at multiple
levels within and beyond the immune system. Accordingly, IL-15 has
already been described as an anabolic agent on skeletal muscle (18) and
to stimulate intestinal cell proliferation (19). IL-15 also stimulates
the proliferation of murine mast cell lines, but this effect is
mediated through a novel, as yet unidentified, receptor not involving
IL-15R or IL-2R and IL-2R (20).
mRNA have been described that result
from alternative splicing of exon 3 and/or alternate usage of exon 7 or
7' (12). These isoforms are equally capable of binding IL-15. In this
paper, we show the existence of a novel type of IL-15R mRNA
lacking exon 2. The IL-15-binding capacities, intracellular routing,
and biochemical features of the IL-15R proteins encoded by these
different transcripts in COS-7 cells are compared.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5'/3'), or 60 °C (5E4.1/E7.2); and elongation for 1 min at
72 °C (30 cycles). The following primers were used: E1,
5'-AGTCCAGCGGTGTCCTGTGG; E7, 5'-TCATAGGTGGTGAGAGCAGT; E7',
5'-TCAACAGACGCTTCCCACTG; E4, 5'-GAACTCACAGCATCCGCC; p3BGH, 5'-TAGAAGGCACAGTCGAGG; 5' (sense), 5'-CGTGCTGCTGACCGAGGCC; 3' (antisense), 5'-TTCGTGGATGCCACAGGAC; 5E4.1, 5'-GCAGCTTCATCTCCCAG; and
E7.2, 5'-TAGGTGGTGAGAGC.
3,
pNo15R2, and pNo15R23. PCR amplification of pNo15R with
primers 5E4.1 (located in exon 4) and E7.2 enabled the elimination of
the receptor stop codon. The resulting PCR product was purified and
inserted into pNoTA/T7, leading to pNo15R*. SmaI fragments from pNo15R, pNo315R, pNo215R, and pNo2315R were inserted into SmaI-digested and dephosphorylated pNo15R*. The
XbaI fragments from each construct were then inserted into
the pcDNA3.1/Myc-His vector (pcDNA3mh; Invitrogen, Groningen,
Netherlands), yielding pcDNA-15Rmh, pcDNA-315Rmh,
pcDNA-215Rmh, and pcDNA-2315Rmh, respectively.
and 2IL-15R solubilized from transfected COS-7 cells as
described above were purified by nickel-nitrilotriacetic acid (QIAGEN
Inc., Courtaboeuf, France) affinity chromatography (27) according to
the manufacturer's instructions. The IL-15R content of each
preparation was analyzed by a specific enzyme-linked immunosorbent
assay as described (26), in which anti-Myc antibody (0.5 µg/well) was
used as coating antibody and the combination of anti-histidine antibody
(0.5 µg/well) plus peroxidase-labeled anti-rabbit IgG (1:5000
dilution; BioAtlantic) was used as indicator. Purified receptors were
incubated for 2 h at 4 °C in phosphate-buffered saline/bovine
serum albumin with labeled IL-15 (2 nM) and 8 µg/ml anti-histidine antibody in a final volume of 25 µl. Protein
A-Sepharose beads were then added (25 µl) for 1 h at 4 °C.
Centrifugation through a layer of dibutyl phthalate (87.5%) and
paraffin oil (12.5%) allowed separation of protein A-bound
immunocomplexes (pellet) from unbound reactants (supernatant), and the
radioactivity associated which each fraction was determined.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Transcripts Lacking Exon
2--
Different IL-15R isoforms using alternate C-terminal exons
(exons 7 and 7') and containing or not exon 3 have already been described (Fig. 1) (12). Using
oligonucleotide primers corresponding to the N-terminal end of exon 1 (primer E1) and the C-terminal end of exon 7 (primer E7) or 7' (primer
E7'), RT-PCR amplifications were carried out on mRNAs prepared from
different cell lines and tissues (Fig.
2). For each couple of primers (E1/E7 or
E1/E7'), four amplification products were detected. The products from
the E1/E7 amplification carried out on human peripheral blood
mononuclear cells were cloned and sequenced. The two upper bands (834 and 735 bp) corresponded to full-length IL-15R and IL-15R lacking exon 3 (3IL-15R), respectively. The two lower bands corresponded to two new mRNA species, one lacking exon 2 (641 bp; 2IL-15R) and one lacking both exons 2 and 3 (542 bp; 23IL-15R).
Sequence analysis showed that exon 2 and/or exon 3 deletions did not
change the reading frame (data not shown). The four products obtained from E1/E7' amplification had sizes that were ~100 bp lower than those obtained from the E1/E7 amplification, suggesting that they corresponded to similar IL-15R isoforms (full-length, 3, 2, and 23), but using the alternate exon 7', which is 100 bp shorter than exon 7 (12). The structures of the coding sequences of the eight
mRNA isoforms are shown in Fig. 1.
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Fig. 1.
Schematic diagram of the human
IL-15R gene, transcription products, and IL-15
receptor subunits. A, the positions of oligonucleotide
primers (E1, E7, and E7') used for RT-PCR amplifications are shown by
arrows. Asterisks indicate the three isoforms
already described by Anderson et al. (12). B, the
T cell high affinity IL-15 receptor includes IL-15R and the
IL-2R/IL-2R transducing subunits. IL-2R and IL-2R each
contain one hematopoietic receptor domain with two conserved disulfide
bonds and a consensus WSXWS motif. In IL-15R is shown the
exon 2-encoded sushi domain characterized by two interwoven disulfide
bonds (C-1-C-3 and C-2-C-4). This domain is absent in
2IL-15R.
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Fig. 2.
Detection of eight distinct
IL-15R transcripts. RT-PCR analysis of
IL-15R was performed on different human tissues (A) and
cell lines (B) as indicated using either primer pair E1/E7
(lanes 1) or primer pair E1/E7' (lanes 2).
Lane M shows the 100-bp DNA ladder. In C are
shown the sizes (in base pairs) of the E1/E7 amplification products
obtained from SAOS-2 cells. PCR amplification of -actin served as
internal control for the amount of RNA analyzed. FBM, fetal
bone marrow; PBMC, peripheral blood mononuclear cells.
3 isoforms appeared to
predominate, the mRNAs corresponding to the 2 and 23 forms
were as abundant as the others in a number of cell types (Kit 225 lymphoma cells and SAOS-2 osteocarcinoma cells) and even more in normal
human peripheral blood mononuclear cells. The eight isoforms were also
expressed by normal human T cell clones, either CD4+ or
CD8+ (data not shown).
and 2IL-15R Expressed in
COS-7 Cells--
The different IL-15R Myc/polyhistidine-tagged
cDNA isoforms were transfected in COS-7 cells, and the expression
products were analyzed by Western blotting on whole cell extracts (Fig. 3A). With the IL-15R
cDNA (E7 isoform), two doublets of bands were revealed (best seen
in panel c) at 59-62 and 39-41 kDa. Treatment with
N-glycosidase induced a disappearance of the 62- and 41-kDa bands, leaving the 59- and 39-kDa bands unaffected, whereas treatment with O-glycosidase induced a disappearance of the 59- and
62-kDa bands, but not of the 39- and 41-kDa bands. Together, these
results suggest that the 62- and 41-kDa bands are
N-glycosylated forms of the 59- and 39-kDa bands,
respectively, and that the 59-62-kDa bands are highly
O-glycosylated forms of the 39-41-kDa bands.
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Fig. 3.
Biochemical analysis and subcellular
localization of IL-15R and 2IL-15R in COS-7
cells. A, Western blots with anti-histidine antibody on
whole cell extracts. Panels a and b, IL-15R
and 2IL-15R, respectively, before (
) and after (+) treatment
with endoglycosidase F; panels c and d, IL-15R
and 2IL-15R, respectively, before () and after (+) treatment
with neuraminidase and O-glycosidase. Molecular mass
standards are indicated in kilodaltons. B, Western blotting
performed on nuclear and non-nuclear membrane fractions prepared from
mock-transfected (vector) or IL-15R- or
2IL-15R-transfected cells. C, anti-histidine antibody-
and protein A-driven immunoprecipitation of cell surface-iodinated
IL-15R and 2IL-15R. M, molecular mass
standards.
2IL-15R, similar results were observed. The lower
band at 32.5 kDa corresponds to an unglycosylated form of the receptor.
Its size is ~7 kDa lower than the corresponding 39-kDa band of
IL-15R, in agreement with the deletion of the exon 2-encoded domain
(12). The band at 35 kDa represents 2IL-15R with a carbohydrate
linked to the single N-glycosylation site present in
IL-15R and retained in 2IL-15R, whereas bands at 54-57 kDa
correspond to O-glycosylated forms of the 32.5-35-kDa bands. Some material at ~40 kDa was also observed with variable intensities and might represent intermediate states of glycosylation.
Isoforms in COS-7
Cells--
The localization of the receptor was first analyzed by
confocal microscopy. As shown in Fig. 4,
IL-15R (in green) was mainly found associated with the
nuclear membrane, giving a ring-like pattern. By comparison,
localization of the nuclear protein p300 (in red) was purely
intranuclear. There was also some co-localization of IL-15R and p300
(in yellow), suggesting that part of the receptor was
localized at the inner side of the nuclear membrane and/or in the
intranuclear space. 2IL-15R behaved very differently, with an
expression pattern suggesting localization within the endoplasmic
reticulum, Golgi, and cytoplasmic vesicles, but not with the nuclear
membrane or intranuclear space. Accordingly, it did not co-localize
with p300. Other experiments (data not shown) indicated that exon 3 deletion had no influence on the localization of the receptor:
3IL-15R and 23IL-15R showed patterns similar to those of
IL-15R and 2IL-15R, respectively.
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Fig. 4.
Confocal microscopic analysis of
intracellular IL-15R and 2IL-15R localization in
COS-7 cells. Transfected cells were labeled with anti-p300
antibody (Cy5; red) and anti-histidine antibody (fluorescein
isothiocyanate; green). Red and green
fluorescence signals are shown separately or together as indicated.
Yellow (upper right) indicates co-localization of
the two antibodies.
was expressed at a much higher level than the 2 isoform. Conversely, 2IL-15R was predominant in the membrane fraction prepared after depletion of nuclei (non-nuclear membrane fraction), and
in comparison, IL-15R was expressed at a much lower level in that
fraction. Both unglycosylated and glycosylated forms of the receptors
were observed. Examination of the cytosolic extracts did not show
detectable expression of either receptor form (data not shown).
or 2IL-15R at the plasma membrane. However, this could be due to
low cell-surface receptor density as often observed with cytokine
receptors. To analyze this point further, cells were externally labeled
with [125I]iodine before lysis and immunoprecipitation
with anti-histidine antibody (Fig. 3C). Specific bands at 62 and 53 kDa were precipitated from IL-15R-transfected cells and a
specific band at 48 kDa from 2IL-15R-transfected cells,
indicating that both receptor isoforms are routed to the plasma
membrane and exposed at the cell surface as glycosylated proteins.
expressed ~1200 high affinity receptors
(Kd = 60 pM). COS-7 cells transfected with the 2 isoform did not show any detectable specific IL-15 binding. As far as both receptors were expressed at the cell surface (Fig. 3C), these results suggested a loss of IL-15-binding
capacity as a result of exon 2 deletion. To rule out the possibility
that a low expression level of 2IL-15R as compared with that of
IL-15R at the cell surface could account for the absence of
detectable cytokine binding, IL-15R and 2IL-15R were
solubilized from transfected COS-7 cells and purified by nickel
affinity chromatography, and their IL-15-binding capacities were
assessed. An enzyme-linked immunosorbent assay using anti-Myc antibody
as coating antibody and anti-histidine antibody as tracer antibody
showed similar receptor contents for the two preparations (Fig.
5B). Immunoprecipitation of the iodinated IL-15·receptor
complex with anti-histidine antibody and protein A (Fig. 5C)
revealed dose-dependent binding of IL-15 to IL-15R,
whereas no IL-15 binding was found when using 2IL-15R. IL-15·IL-15R complex formation was competed out with unlabeled IL-15 (Fig. 5D). The half-maximal effect was obtained with
an IL-15 concentration of ~100 pM, indicating that
solubilized IL-15R retained a high affinity similar to that measured
on intact cells.
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Fig. 5.
IL-15-binding capacities of
IL-15R and 2IL-15R isoforms.
A, transfected cells (as indicated) were equilibrated with
increasing amounts of 125I-labeled IL-15, and cell-bound
and unbound fractions were determined. Nonspecific binding
(dashed line) was measured in the presence of a 100-fold
excess of unlabeled IL-15. B, the purified receptor
fractions were compared at various dilutions as indicated in an
enzyme-linked immunosorbent assay using anti-Myc antibody as coating
antibody and anti-histidine antibody as tracer antibody. C,
the same fractions were analyzed for their ability to bind
125I-labeled IL-15 (2 nM) using an
anti-histidine antibody/protein A precipitation assay. Nonspecific
immunoprecipitation was evaluated by including a 100-fold excess of
unlabeled cytokine. An irrelevant rabbit antibody used instead of
anti-histidine antibody served as a negative control. D,
shown is the inhibition of the binding of 125I-labeled
IL-15 (2 nM) to IL-15R by increasing concentrations of
unlabeled IL-15.
2IL-15R Transfection Does Not Affect IL-15- or IL-2-driven
Cell Proliferation--
To investigate potential regulatory effects,
IL-15R and 2IL-15R (E7 forms) cDNAs were transfected in
the human T lymphoma cell line Kit 225. RT-PCR using the E4/p3BGH
primer pair showed that the corresponding transcripts were expressed at
comparable levels in the IL-15R and 2IL-15R transfectants
(data not shown). Kit 225 cells are dependent on exogenous IL-2 for
proliferation (28). We also observed that this growth requirement could
be replaced by IL-15, in agreement with the observation that this cell
line expressed endogenous IL-15R mRNA (Fig. 2). Proliferation experiments (Fig. 6) showed that
transfection of IL-15R or 2IL-15R did not change the
proliferative response of Kit 225 cells. The three cell lines responded
with similar dose-response curves to IL-2 or IL-15 in terms of
thymidine incorporation at 48 h.
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Fig. 6.
Proliferation of transfected Kit 225 cell
lines. Kit 225 cells transfected with empty vector, IL-15R , or
2IL-15R (as indicated) were tested for their proliferative
response ([3H]thymidine incorporation) to graded doses of
IL-2 (A) or IL-15 (B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoforms have been described
that either lack exon 3 and/or use an alternate exon 7 (exon 7') (12).
Here, by RT-PCR amplification using appropriate sets of oligonucleotide
primers, we confirm the existence of these isoforms and show, in
addition, the existence of novel IL-15R transcripts corresponding to
the deletion of exon 2. This new deletion can combine with the ones
already described, leading to eight different transcripts corresponding
to all the possible combinations of exon 2 deletion, exon 3 deletion,
and alternate usage of exon 7 or 7'. In agreement with previous reports
(12, 13), IL-15R transcripts were found to be expressed by various cell lines and tissues. Each positive tissue or cell line expressed the
eight different IL-15R transcripts, although at relative levels that
varied from one cell/tissue type to another.
in COS-7 cells gave rise to several
protein bands, in agreement with a previous report suggesting several
bands for a soluble form of mouse IL-15R (13). We show in this study
that these different products are due to alternate N- and
O-glycosylations of a 39-kDa precursor. Similarly,
2IL-15R gave rise to different glycosylated bands from a 32.5-kDa
precursor. The size difference between IL-15R and 2IL-15R
precursors is in agreement with the theoretical molecular mass of the
exon 2-encoded sushi domain (7329 Da). The human IL-15R sequence
displays a single potential N-glycosylation site at
Asn107 within the exon 3-encoded domain (12). Our data show
that this site was used in COS-7 cells for both full-length and exon
2-deleted forms. Most of the size increases due to carbohydrate
addition were the results of extensive O-glycosylations,
which accounted for ~23 kDa in the highest molecular mass species.
The extent of O-glycosylation of 2IL-15R was as large
as in IL-15R, indicating that few glycosylations are associated with
the exon 2-encoded domain. This is in agreement with the fact that most
(74%) of the serine and threonine residues (which are potential
targets for O-linked sugar addition) in the extracellular
part of the receptor are located in the exon 3/4-encoded domains (12).
extracted from transfected COS-7 cells was shown to bind
IL-15 with a high affinity (Kd = 60 pM).
In sharp contrast, 2IL-15R did not show any detectable
IL-15-binding capacity. These results clearly demonstrate that the
exon 2-encoded sushi domain is the essential element involved in
IL-15 binding. They fully complement earlier work showing that exon
3-encoded linker sequence as well as exon 7/7'-encoded cytoplasmic
domains were dispensable for binding and signaling (12).
was expressed at the cell surface, although at low density
(~1000 sites/cell). Unexpectedly, confocal immunofluorescence studies
and analysis of subcellular fractions showed that most of IL-15R was
associated with the nuclear membrane. A large proportion of this
nuclear receptor was heavily O-glycosylated, suggesting that
it was routed to the nuclear membrane through the Golgi. Some
co-localization with the nuclear protein p300 was also observed, indicating that part of the receptor was inside the nucleus, possibly at the inner side of the nuclear membrane. Due to the relatively large
size (~60 kDa) of the glycosylated receptor, this observation suggests that an active mechanism is involved in its nuclear
translocation, rather than passive diffusion through the nuclear pores.
In support of this, we found, within the human IL-15R sequence, the
presence of a putative nuclear localization signal (NLS). This sequence consists of two clusters of polycationic residues separated by a spacer
(RERYICNSGFKRK, amino acids 24-36)
(12). Such putative NLSs have been demonstrated in the sequence of a
number of ligands and receptors, including those that activate Stat
transcription factors (29, 30). In this study, the possible involvement of this putative NLS in the nuclear routing of IL-15R is supported by the fact that the exon 2-truncated receptor (which does not contain
this putative NLS motif located in the sushi domain) does not show
nuclear localization. More specific deletions/mutations of this
sequence are, however, required to demonstrate its role as an NLS.
,
which has a high affinity on its own for IL-15, might bind SSP-IL-15
inside the cell and, through its putative NLS, translocate the
cytokine-receptor complex in the nuclear compartment. Following that
hypothesis, IL-15R might be involved in the different roles
postulated for the two forms of IL-15, at the cell surface for
LSP-IL-15 and at the nuclear level for SSP-IL-15.
has completely lost its capacity to bind
IL-15, therefore raising the question of its biological role. Two
findings suggest that it might serve some biological function: (i) a
number of cell lines and tissues expressed 2IL-15R mRNAs at
levels comparable to full-length IL-15R mRNAs; and (ii) upon
transfection in COS-7 cells, 2IL-15R was expressed at the plasma
membrane with similar efficiency as IL-15R. It is therefore possible
that 2IL-15R might compete with IL-15R for recruitment of the
transducing subunits IL-2R and IL-2R. Similar observations have
been made in the case of IL-2R. A naturally occurring truncated form
of IL-2R has been described that lacks the exon 4-encoded domain
(second sushi domain) as a result of alternative mRNA splicing (32). This truncated IL-2R, whose function is presently unknown, was
transported to the cell surface and was unable to bind IL-2. It was,
however, expressed at low levels in human T cells. In this study,
preliminary experiments on Kit 225 cells showed that 2IL-15R
transfection did not affect IL-15- or IL-2-induced proliferation. However, a potential regulatory effect of transfected 2IL-15R in
this system might have been masked by the fact that Kit 225 cells
already express endogenous IL-15R and 2IL-15R. Additional studies addressing signal transduction, proliferation, and apoptosis are required to determine the functional role of the 2IL-15R isoforms.
| |
ACKNOWLEDGEMENT |
|---|
We thank Dr. Doreen Cantrell for kindly providing the Kit 225 cell line.
| |
FOOTNOTES |
|---|
* This work was supported in part by INSERM and CNRS.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.
Recipient of fellowships from the Ligue Nationale contre le Cancer
(Comité de Vendée) and from the Association pour la
Recherche contre le Cancer.
§ Recipient of a fellowship from the Ministère de l'Education Nationale de la Recherche et de la Technologie.
¶ Recipient of a fellowship from the European Commission.
To whom correspondence should be addressed. Tel.:
33-2-40-08-47-23; Fax: 33-2-40-35-66-97; E-mail:
yjacques@nantes.inserm.fr.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
IL, interleukin;
IL-2R, IL-2R, and IL-2R, interleukin-2 receptor -, -,
and -chains, respectively;
IL-15R, interleukin-15 receptor
-chain;
RT-PCR, reverse transcription-polymerase chain reaction;
bp, base pair(s);
NLS, nuclear localization signal.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Grabstein, K. H.,
Eisenmann, J.,
Shanebeck, K.,
Rauch, C.,
Srinivasan, S.,
Fung, V.,
Beers, C.,
Richardson, J.,
Schoenborn, M. A.,
Ahdieh, M.,
Johnson, L.,
Alderson, M. R.,
Watson, J. D.,
Anderson, D. M.,
and Giri, J. G.
(1994)
Science
264,
965-968 |
| 2. |
Burton, J. D.,
Bamford, R. N.,
Peters, C.,
Grant, A. J.,
Kurys, G.,
Goldman, C.,
Brennan, J.,
Roessler, E.,
and Waldmann, T. A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
4935-4939 |
| 3. | Sprang, S. R., and Bazan, J. F. (1993) Curr. Opin. Struct. Biol. 3, 815-827[CrossRef] |
| 4. | Sorel, M., Cherel, M., Dreno, B., Bouyge, I., Guilbert, J., Dubois, S., Lebeau, B., Raher, S., Minvielle, S., and Jacques, Y. (1996) Eur. J. Dermatol. 6, 209-212 |
| 5. | Satoh, J., Kurohara, K., Yukitake, M., and Kuroda, Y. (1998) J. Neurol. Sci. 155, 170-177[CrossRef][Medline] [Order article via Infotrieve] |
| 6. |
Wilkinson, P. C.,
and Y., L. F.
(1995)
J. Exp. Med.
181,
1255-1259 |
| 7. |
Carson, W. E.,
Giri, J. G.,
Lindemann, M. J.,
Linett, M. L.,
Ahdieh, M.,
Paxton, R.,
Anderson, D.,
Eisenmann, J.,
Grabstein, K.,
and Caliguri, M. A.
(1994)
J. Exp. Med.
180,
1395-1403 |
| 8. | Armitage, R. J., Macduff, B. M., Eisenmann, J., Paxton, R., and Grabstein, K. H. (1995) J. Immunol. 154, 483-490[Abstract] |
| 9. | Giri, J. G., Ahdieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L. S., Cosman, D., and Anderson, D. (1994) EMBO J. 13, 2822-2830[Medline] [Order article via Infotrieve] |
| 10. | Leonard, W. J., Depper, J. M., Crabtree, G. R., Rudikoff, S., Pumphrey, J., Robb, R. J., Kronke, M., Svetlik, P. B., Peffer, N. J., Waldmann, T. A., and Greene, W. C. (1984) Nature 311, 626-631[CrossRef][Medline] [Order article via Infotrieve] |
| 11. | Nikaido, T., Shimizu, A., Ishida, N., Sabe, H., Teshigawara, K., Maeda, M., Uchiyama, T., Yodoi, J., and Honjo, T. (1984) Nature 311, 631-635[CrossRef][Medline] [Order article via Infotrieve] |
| 12. |
Anderson, D. M.,
Kumaki, S.,
Ahdieh, M.,
Bertles, J.,
Tometsko, M.,
Loomis, A.,
Giri, J.,
Copeland, N. G.,
Gilbert, D. J.,
Jenkins, N. A.,
Valentine, V.,
Shapiro, D. N.,
Morris, S. W.,
Park, L. S.,
and Cosman, D.
(1995)
J. Biol. Chem.
270,
29862-29869 |
| 13. | Giri, J. G., Kumaki, S., Ahdieh, M., Friend, D. J., Loomis, A., Shanebeck, K., Dubose, R., Cosman, D., Park, L., and Anderson, D. M. (1995) EMBO J. 14, 3654-3663[Medline] [Order article via Infotrieve] |
| 14. | Norman, D. G., Barlow, P. N., Baron, M., Day, A. J., Sim, R. B., and Campbell, I. D. (1991) J. Mol. Biol. 219, 717-725[CrossRef][Medline] [Order article via Infotrieve] |
| 15. |
Greene, W. C.,
Robb, R. J.,
Svetlik, P. B.,
Rusk, C. M.,
Depper, J. M.,
and Leonard, W. J.
(1985)
J. Exp. Med.
162,
363-368 |
| 16. | Minami, Y., Kono, T., Miyazaki, T., and Taniguchi, T. (1993) Annu. Rev. Immunol. 11, 245-268[CrossRef][Medline] [Order article via Infotrieve] |
| 17. | Giri, J. G., Anderson, D. M., Kumaki, S., Park, L. S., Grabstein, K. H., and Cosman, D. (1995) J. Leukocyte Biol. 57, 763-766[Abstract] |
| 18. | Quinn, L. S., Haugk, K. L., and Grabstein, K. H. (1995) Endocrinology 136, 3669-3672[Abstract] |
| 19. | Reinecker, H. C., MacDermott, R. P., Mirau, S., Dignass, A., and Podolsky, D. K. (1996) Gastroenterology 111, 1706-1713[CrossRef][Medline] [Order article via Infotrieve] |
| 20. | Tagaya, Y., Burton, J. D., Miyamoto, Y., and Waldmann, T. A. (1996) EMBO J. 15, 4928-4939[Medline] [Order article via Infotrieve] |
| 21. | Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[Medline] [Order article via Infotrieve] |
| 22. |
Magrangeas, F.,
Pitiot, G.,
Dubois, S.,
Bragado-Nilsson, E.,
Chérel, M.,
Jobert, S.,
Lebeau, B.,
Lethé, B.,
Mallet, J.,
Jacques, Y.,
and Minvielle, S.
(1998)
J. Biol. Chem.
273,
16005-16010 |
| 23. | Aruffo, A., and Seed, B. (1987) EMBO J. 6, 3313-3316[Medline] [Order article via Infotrieve] |
| 24. |
Schreiber, E.,
Matthias, P.,
Muller, M. M.,
and Schaffner, W.
(1989)
Nucleic Acids Res.
17,
6419 |
| 25. | Tejedor, F., and Ballesta, J. P. (1982) Anal. Biochem. 127, 143-149[CrossRef][Medline] [Order article via Infotrieve] |
| 26. |
Francois, C.,
Sorel, M.,
Cherel, M.,
Brailly, H.,
Minvielle, S.,
Blanchard, D.,
and Jacques, Y.
(1995)
Int. Immunol.
7,
1173-1181 |
| 27. |
Hoffmann, A.,
and Roeder, R. G.
(1991)
Nucleic Acids Res.
19,
6337-6338 |
| 28. |
Hori, T.,
Uchiyama, T.,
Tsudo, M.,
Umadome, H.,
Ohno, H.,
Fukuhara, S.,
Kita, K.,
and Uchino, H.
(1987)
Blood
70,
1069-1072 |
| 29. | Jans, D. A. (1994) FASEB J. 8, 841-847[Abstract] |
| 30. | Johnson, H. M., Torres, B. A., Green, M. M., Szente, B. E., Siler, K. I., Larkin, J. R., and Subramaniam, P. S. (1998) Biochem. Biophys. Res. Commun. 244, 607-614[CrossRef][Medline] [Order article via Infotrieve] |
| 31. |
Tagaya, Y.,
Kurys, G.,
Thies, T. A.,
Losi, J. M.,
Azimi, N.,
Hanover, J. A.,
Bamford, R. N.,
and Waldmann, T. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14444-14449 |
| 32. |
Cullen, B. R.,
Podlaski, F. J.,
Peffer, N. J.,
Hosking, J. B.,
and Greene, W. C.
(1988)
J. Biol. Chem.
263,
4900-4906 |
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