The development and function of different cell lineages in
multicellular organisms is controlled in large part by response to a
number of secreted proteins known as cytokines. A subset of cytokines
with related three-dimensional structures, the four helix bundle
cytokines, mediate their biological activities through a group of
structurally related receptors, the hematopoietin receptor family (1, 2, 3) . This cytokine family is
characterized by pleiotropic and overlapping biological functions, the
latter explained in part by the sharing of common receptor subunits and
downstream signal transduction components. Interleukin-2 (IL-2) (
)and interleukin-15 (IL-15) are two helical cytokines with
an especially close functional relationship. IL-2, the first identified
T cell growth factor, is a product of activated T cells. The IL-2
receptor (IL-2R) is a heterotrimeric complex consisting of three chains
(for reviews, see (4) and (5) ). The
subunit
(IL-2R
) (6, 7, 8) binds IL-2 with low
affinity (K
= 10
M
) but does not contribute directly to
signal transduction. The IL-2R
is not a member of the
hematopoietin receptor family but instead contains two
``sushi'' domains related to structures found in a number of
proteins involved in the complement and coagulation
cascades(9) . The IL-2R
(10) and IL-2R
(11) chains are both members of the hematopoietin receptor
family and are required for signaling. IL-2 cannot bind to the
or
chains alone but can bind to a complex of
and
with
intermediate affinity (K
=
10
M
). Full high affinity
binding (K
= 10
M
) requires the additional presence of the
chain. The
chain is noteworthy as a component of the
receptors for IL-4, IL-7, and IL-9 and has been referred to as

, for common
chain (12, 13, 14, 15, 16) . It
also has been shown to be defective in X-linked severe
combined immunodeficiency(17) .
IL-15 is a recently
discovered cytokine that shares biological activities with IL-2, such
as the activation and proliferation of T cells and the costimulation of
B cells with CD40 ligand(18, 19) . Our previous work
demonstrated that the IL-2R
and -
chains are both essential
for IL-15 signaling but that IL-2R
does not interact with
IL-15(18, 20, 21, 22) . Although the
human 
heterodimer of the IL-2R can bind IL-15(20) ,
it is still unclear whether the 
heterodimer per se can transduce IL-15 signals in the absence of IL-15R
. In
contrast, we found that murine IL-2R
and -
cannot bind simian
IL-15, although murine T-cell lines can proliferate in response to this
cytokine, an observation which led us to the identification and
molecular cloning of a murine IL-15R
chain(21) . The
murine IL-15R
chain is related to the IL-2R
chain and can
reconstitute responsiveness of murine cells to simian IL-15 when
transfected into cells that express murine IL-2R
and -
,
demonstrating that it is indeed the
chain of the IL-15R. Despite
the overall similarities of the structures of the receptors for IL-2
and IL-15, there remain two important differences: the IL-15R
,
unlike the IL-2R
, has a broad distribution of expression in
different cell types, and the IL-15R
shows a very high affinity of
binding to IL-15, more than a 1000-fold higher than the affinity of
IL-2R
binding to IL-2.
In this paper, we continue the
comparison of the IL-2 and IL-15 receptors. We have identified three
alternately spliced forms of human IL-15R
, all of which bind IL-15
with high affinity. Analysis of genomic clones shows that the
intron-exon structures of the IL15RA and IL2RA genes
are similar and that these loci are closely linked in both human and
murine genomes. In addition, as seen in the IL-2/IL-2R system, the
cytoplasmic domain of the IL-15R
is dispensable for growth
signaling, and the human IL-2R 
heterodimer can transduce
IL-15 signals in the absence of IL-15R
.
EXPERIMENTAL PROCEDURES
Cell Lines, Growth Factors, and Culture
Conditions
Cell line 32D-01, an IL-2 non-responsive
derivative of the murine myeloid line 32D, and subline 32Dm
-5,
expressing the transfected murine IL-2R
chain, have been described (21) . Cell line JP111-6, a subline of human acute
lymphocytic leukemia T-cell line Jurkat, and JP
3, a derivative of
JP111-6 stably transfected with a plasmid expressing the human
IL-2R
cDNA (pSRB5; (23) ), were generous gifts from Dr.
Kazuo Sugamura (Tohoku University, Sendai, Japan). Cell lines were
maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 1%
penicillin, streptomycin, and glutamine under 5% CO
. Murine
myeloid 32D-01 cell cultures were also supplemented with 50 µM 2-mercaptoethanol and 30 ng/ml murine IL-3. Proliferation assays
were performed as described(21) . Human IL-2 was purchased from
Chiron. Murine IL-3 was purchased from Genzyme. Simian IL-15 (18) was expressed in yeast and purified at Immunex.
Molecular Cloning
The 1.65-kb murine
IL-15R
cDNA (21) was labeled with
P and used
to probe a WI-26 VA4 cDNA library prepared in
gt10 (24) using standard techniques(25) . Briefly, plaque
lifts were hybridized with a random prime-labeled full-length murine
IL-15R
cDNA probe at 42 °C in a buffer containing 50%
formamide and 5
SSC and washed at 50 °C in 2
SSC,
0.1% SDS. Additional human IL-15R
cDNA clones were subsequently
isolated from an A-172 cDNA library by hybridization with pooled
plasmid inserts on Southern blots, essentially as described
previously(26) . The A-172 library was prepared in a plasmid
vector from double-stranded cDNA synthesized using random hexamer
primers and a commercial cDNA synthesis kit (Pharmacia Biotech Inc.).
DNA Sequencing
DNA sequencing was
performed using the Taq DyeDeoxy Terminator Cycle sequencing
kit on an automated ABI DNA sequencer (model 373A, Applied Biosystems).
In all cases, sequence was obtained from both strands of sample DNAs.
Generation of a Murine IL-15R
Cytoplasmic Domain
Deletion Mutant
A murine IL-15R
cytoplasmic domain
deletion mutant was constructed by PCR using a full-length murine
IL-15R
cDNA template and primers 5`-AATAGTCGACATGGCCTCGCCGCAGCTCCG
and 5`-ACAGCGGCCGCTTACCTTGATTTGATGTACCAGGC, resulting in an amplified
fragment encoding a membrane-bound form of the murine IL-15R
chain
lacking the last 32 amino acids of the predicted cytoplasmic domain.
This fragment was subcloned into a mammalian expression vector, its
sequence verified by DNA sequencing, and transfected into 32D-01 cells
as described below.
Stable Transfection of 32D-01 Cells with Full-length
and Truncated IL-15R
cDNA Clones
Transfection of
32D-01 cells with expression vectors encoding human IL-15R
,
truncated murine IL-15R
, and murine IL-2R
was performed by
co-electroporation of linearized plasmids and pSV2neo, followed by
selection in medium containing G418 (Life Technologies, Inc.), using
the method previously described(21) . Plasmids containing
murine and human IL-2R
cDNAs have been described
previously(20, 21) .
Receptor Binding
Receptor binding
analysis using
I-simian IL-15 was performed as described
previously(21) . Briefly, cells were incubated at 4 °C for
4-5 h with radiolabeled ligand over a concentration range
extending from 0.3 to 400 pM and separated by phthalate oil.
To maintain proper conditions of ligand excess, cells were diluted in
the incubation mixture with a carrier cell line (B lymphoblast line
Daudi (ATCC CCL 213)) that did not bind IL-15.
Tyrosine Phosphorylation of Jak1
For the
analysis of Jak1 activation, cells were starved for 1 h in media
containing 0.5% fetal bovine serum, followed by stimulation with or
without 450 ng/ml cytokines for 15 min. Cells were then solubilized
with lysis buffer (1% Nonidet P-40, 10 mM Tris-HCl (pH 7.5),
150 mM NaCl, 1 mM EDTA, 1% aprotinin, 2 mM
Na
VO
, 2 mM phenylmethylsulfonyl
fluoride, 0.1 mM Na
MoO
, 10 mM NaF, and 30 mMp-nitrophenyl phosphate) on ice
for 30 min. After centrifugation, the supernatants were precleared with
protein A-Sepharose CL-4B (Pharmacia), followed by immunoprecipitation
with anti-Jak1 polyclonal antibodies (Upstate Biotechnology Inc.). The
immunoprecipitates were separated by SDS-polyacrylamide gel
electrophoresis and transferred to Hybond-ECL membrane (Amersham). The
filter was processed according to the manufacturer's
instructions, using antiphosphotyrosine monoclonal antibody 4G10
(Upstate Biotechnology) at 1:1000, and peroxidase-conjugated sheep
anti-mouse Ig (Amersham) at 1:3000. The antibody bound to the membrane
was detected by enhanced chemiluminescence (ECL, Amersham).
RNA Analysis
Polyadenylated RNAs were
prepared, electrophoresed, and blotted to nylon membranes using
standard methods(25) . Blots containing mRNAs from various cell
lines and tissues were hybridized with radiolabeled RNA probes in a
buffer containing 50% formamide at 63 °C, washed in 0.1% SSC at 68
°C, and autoradiographed using standard methods, as described
previously (27) .
IL-15R
Gene Structure
Characterization
A genomic library prepared from human
placenta DNA in the bacteriophage vector
FixII (Stratagene) was
screened by standard methods (25) with a random prime-labeled
human IL-15R
cDNA probe. Inserts from candidate IL15RA genomic clones were subcloned into pBluescript (Stratagene) and
analyzed by hybridization to radiolabeled oligonucleotides specific for
5`-non-coding, extracellular, transmembrane, cytoplasmic, and
3`-non-coding regions of the human IL-15R
cDNA to determine the
identity and degree of overlap of the clones isolated. Intron positions
in the IL15RA gene were determined by sequencing across
intron-exon junctions using human IL-15R
cDNA-specific sequencing
primers.
Human Chromosomal Localization of IL15RA by Metaphase
Fluorescence in Situ Hybridization (FISH)
Two IL15RA genomic clones, each approximately 15 kb in size, with an overlap
of approximately 2 kb and encompassing all but the first exon of IL15RA
(see below), were chosen for FISH analysis. FISH was performed as
described previously(28) . Briefly, plasmids containing
subcloned IL15RA genomic fragments were nick-translated
together using digoxigenin-11-UTP and hybridized overnight at 37 °C
to fixed metaphase chromosomes prepared from
bromodeoxyuridine-synchronized, phytohemagglutin-stimulated peripheral
blood lymphocytes obtained from a normal donor. Simultaneous
hybridization with a biotin-16-dUTP-labeled chromosome 10-specific
satellite DNA probe (Oncor) was performed to confirm chromosome
identity. Specific hybridization signals were detected by applying
fluorescein-conjugated sheep antibodies to digoxigenin (Boehringer
Mannheim) and avidin Texas Red (Vector Laboratories), followed by
counterstaining in 4,6-diamidino-2-phenylindole (Sigma). Fluorescence
microscopy was performed with a Nikon microscope equipped with a
CytoVision Probe image analysis system and a fluorescence filter wheel,
which sequentially captures the fluorescein, Texas Red, and
4,6-diamidino-2-phenylindole images and electronically superimposes
them (Applied Imaging, Santa Clara, CA).
Interspecific Mouse Backcross
Mapping
Interspecific backcross progeny was generated by
mating (C57BL/6J
Mus spretus)F
females and
C57BL/6J males as described(29) . A total of 205 N
mice was used to map the Il15ra locus. DNA isolation,
restriction enzyme digestion, agarose gel electrophoresis, Southern
blot transfer, and hybridization were performed essentially as
described(30) . All blots were prepared with
Hybond-N
nylon membrane (Amersham). The probe, a
1.65-kb BglII fragment containing the full-length mouse
IL-15R
cDNA, was labeled by nick translation with
[
P]dCTP; washing was done to a final
stringency of 1.0
SSCP, 0.1% SDS, 65 °C. Fragments of 11.5,
6.4, 3.4, 2.5, 1.8, 1.1, and 0.7 kb were detected in SphI-digested C57BL/6J DNA, and fragments of 20.0, 8.0, 3.4,
1.1, and 0.7 kb were detected in SphI-digested M. spretus DNA. The presence or absence of the 20.0 and 8.0 SphI M. spretus-specific fragments, which cosegregated, was
followed in backcross mice.A description of two of the probes and
restriction fragment length polymorphisms (RFLPs) for the loci linked
to Il15ra, including vimentin (Vim) and
spectrin 2, brain (Spna2), has been reported
previously(31) . Two probes have not been reported previously
for our interspecific backcross. The interleukin-2 receptor
(Il2ra) probe, a 1.55-kb PstI fragment of mouse cDNA,
detected major TaqI fragments of 6.0 (C57BL/6J) and 4.2 (M. Spretus) kb. The B lymphoma murine Moloney leukemia virus
insertion region 1 (Bmi1) probe, a 1-kb BamHI-EcoRI fragment of mouse genomic DNA, detected EcoRI fragments of 6.2 (C57BL/6J) and 12.0 (M.
Spretus) kb and MspI fragments of 3.8 (C57BL/6J) and 3.1 (M. Spretus) kb. Again, the presence or absence of M.
spretus-specific fragments was followed. The EcoRI and MspI data for Bmi1 were combined. Recombination
distances were calculated as described (32) using the computer
program Spretus Madness. Gene order was determined by minimizing the
number of recombination events required to explain the allele
distribution patterns.
RESULTS
Cloning of Alternate Forms of Human
IL-15R
Binding of
I-simian IL-15 was
used to identify candidate cell lines for the cloning of a human
IL-15R
cDNA. Simian IL-15 shares 97 and 73% amino acid identity
with human and murine IL-15, respectively, and binds to receptors on
both human and murine cells with equivalent
affinities(18, 20, 21, 27) .
Relatively large numbers of high affinity binding sites were detected
on the SV40-transformed lung epithelial line WI-26 VA4 (33) (1425 ± 742 sites, K
=
9.8 ± 4.2
10
M
;
see Fig. 2A) and the glioblastoma line A-172 (ATCC CRL
1620) (2053 ± 916 sites, K
= 5.3
± 2.6
10
M
;
data not shown). A WI-26 VA4 cDNA library prepared in the bacteriophage
gt10 vector was screened by cross-hybridization with a
P-labeled full-length murine IL-15R
probe. The insert
of a single hybridizing clone (W5) was subcloned, sequenced, and found
to share about 65% nucleic acid sequence identity with the murine
IL-15R
cDNA (Fig. 1A). Clone W5 lacked some of the
expected 5` sequences (about 125 bp compared to the murine cDNA),
indicating that it did not contain the initiator methionine and first
portion of the putative IL-15R
signal peptide coding region. The
only other positive clone isolated from the WI-26 library was identical
to clone W5. Since both clones lacked a complete 5`-end, a second cDNA
library prepared from A-172 cells was screened with the W5 insert to
isolate additional human IL-15R
cDNA clones. The insert from one
clone (A212) contained 129 bp of additional 5` sequence compared to
clone W5, including a putative initiator Met and full-length signal
sequence. The sequence shown in Fig. 1A is a composite
of clones A212 and W5 and includes the positions of introns determined
from the analysis of genomic IL15RA clones described below.
The A212 cDNA was missing exon 3 (99 bp between introns 2 and 3, Fig. 1, A and B). The human IL-15R
shares
54% amino acid identity with the murine IL-15R
, with 82% identity
in the putative sushi domain (exon 2, Fig. 1A). A
second isolate from the A-172 library, clone A133, contained an
incomplete cDNA insert that was missing most of the extracellular
coding domain but contained a 120-bp insertion in the cytoplasmic
domain predicted to encode an alternate cytoplasmic tail (at the
position of intron 6, Fig. 1A). In addition, an allelic
difference at nucleotide 627 resulting in an amino acid change was seen
between WI-26 clone W5 (ACC/Thr) and A-172 clones A212 and A133
(AAC/Asn). An independently isolated IL-15R
cDNA obtained from a
peripheral blood T cell cDNA library was sequenced and found to encode
ACC/Thr at this position. The peripheral blood T cell isolate also
contained the signal peptide coding sequence of clone A212 (data not
shown). Reverse transcriptase-PCR analysis was used to determine the
representation of the W5 (full-length), A212 (missing exon 3), and A133
(insertion at intron 6) forms of IL-15R
in other human mRNA
populations, using oligonucleotide primers designed to amplify across
the positions of either exon 3 or intron 6. Two amplified bands of the
expected sizes (±99 bp at exon 3; ±120 bp at intron 6)
were detected in first strand cDNAs generated from peripheral blood T
cells, the NK-like YT cell line (34) and the myelogenous
leukemia line K-562 (ATCC CCL 243). The same relative intensities of
the different forms (A133
W5 > A212) was seen in all three
samples (data not shown). These results suggest that all three
alternatively spliced forms of IL-15R
mRNA are present in these
cell populations.
Figure 2:
Equilibrium
I-IL-15 binding
to native and recombinant human IL-15R
. WI-26 cells (A)
and murine 32D-01 cells transfected with human IL-15R
cDNA clone
h15R (B) were assayed for binding as described under
``Experimental Procedures'' and data plotted in the Scatchard
coordinate system(48) . Conc., molar concentration of
radiolabeled IL-15.
Figure 1:
A,
nucleotide and predicted amino acid sequence of human IL-15R
clones A212, W5, and A133. The 5`-most 129 bp are from clone A212. The
5`-ends of clones W5 and A133 are nucleotides 130 and 485,
respectively. The 120-bp insertion of clone A133 is shown at the bottom of the figure. Indicated are the predicted signal
peptide (single underline), putative N-linked
glycosylation site (box), and transmembrane domain (double
underline). Also indicated are the intron locations (arrows) mapped on genomic IL15RA clones and
positions of primers used to confirm the lack of additional introns
upstream of intron 1 (dashed arrows). Exon 3 is missing in
clone A212. Nucleotide 627 is an Ala in W5, a Cys in A212 and A133,
encoding Thr in W5 and Asn in A212 and A133. B and S, BstXI andSphI sites used in generating full-length
expression constructs. B, schematic comparison of coding
regions of full-length forms of the three alternately spliced variants
isolated from WI-26 and A-172 cDNA libraries. The BstXI and SphI sites were used to construct full-length versions of the
three alternate forms as described in the text. Shown is the
relationship of full-length clone W5(h15R), the form most closely
homologous to the murine IL-15R
, to clone A212 (h15R
E3),
which lacks exon 3, and clone A133(h15RaltC), which contains a 120-bp
insertion at the position of intron 6 (panel A). The signal
and transmembrane coding domains are cross-hatched. Closed
triangles indicate the positions of the original 5`-ends of cDNA
clones A133 and W5.
Recombinant Human IL-15R
Expression and
Analysis
The function of the three alternate human
IL-15R
cDNAs was examined following stable transfection of
full-length expression constructs into a murine myeloid cell line. To
confirm that the 5`-ends of naturally occurring full-length forms of W5
and A133 were the same as that of the A212 isolate, PCR analysis of
WI-26 and A-172 first strand cDNAs was performed using primers designed
to amplify the entire coding region shown in Fig. 1A.
Analysis of the amplified products from these reactions confirmed that
the 5`-ends of all three forms are identical (data not shown). Since
only clone A212 was isolated as a full-length cDNA, full-length forms
of clones W5 and A133 were constructed by recombining the appropriate
cDNA fragments. To generate a full-length W5 construct (designated
h15R) most homologous to the murine IL-15R
cDNA, clone A212
sequence upstream of the BstXI site (Fig. 1A)
was fused with downstream W5 sequence. An expressible, full-length form
of the A133 cDNA (designated h15RaltC) was generated by substituting
the A133 sequence downstream of the SphI site (Fig. 1A) with the corresponding region in h15R. The
A212 cDNA (designated h15R
E3) could be expressed as is. These
three forms, shown schematically in Fig. 1B, were
subcloned into a mammalian expression vector and transfected into
murine myeloid 32D-01 cells alone and in combination with murine
IL-2R
. 32D-01 cells constitutively express murine IL-2R
and
-
, but not IL-2R
, and do not respond to either IL-2 or
IL-15(21) . The IL-2R
chain has been previously shown to
be required for IL-15
responsiveness(18, 20, 21) . We have
previously shown that simian IL-15 binds to murine IL-15R
with
high affinity (K
10
); however,
in the absence of IL-15R
, it is not capable of binding to murine
IL-2R 
complexes(21) . The murine IL-2R
chain
was therefore used for these transfection experiments because it
provides an ideal system to examine the effects of the human
IL-15R
chains directly. Analysis of IL-15 binding and
proliferation in these transfected clones is presented in Table 1. All three forms of human IL-15R
expressed in these
cells showed the same high affinity of IL-15 binding as native
IL-15R
expressed on WI-26 cells. Representative binding curves of
radiolabeled simian IL-15 on WI-26 cells (1475 ± 742 sites/cell, K
= 9.8 ± 4.2
10
M
) and 32D-h15R cells
expressing recombinant human IL-15R
(1.87 ± 1.1
10
sites/cell, K
= 1.2 ±
0.8
10
M
) are shown in Fig. 2. When transfected with murine IL-2R
, 32D-01 cells
respond to IL-2 but not to IL-15(21) . All three forms of human
IL-15R
conferred IL-15 responsiveness as measured by proliferative
ability when cotransfected with murine IL-2R
(Table 1).
These results show that the presence of the IL-15R
chain is
essential for IL-15 responsiveness in these cells. The results also
show that exon 3, which is absent in h15R
E3, is dispensable for
IL-15R
binding and function. This reinforces the concept that the
sushi structural domain (encoded by exon 2 in IL-15R
) is the
essential element for IL-15 binding. The allelic variation resulting in
the amino acid change Thr-152 (in clone h15R and h15RaltC) to Asn-152
(in clones h15R
E3) was neutral in regard to IL-15 binding and
proliferative responses in transfected 32D-01 cells.
The Cytoplasmic Domain of the IL-15R
Chain Is
Dispensable for IL-15 Signaling
Since we detected no
difference in the signaling ability in cells transfected with h15R or
h15RaltC, which encode alternate cytoplasmic domains of human
IL-15R
, we hypothesized that the IL-15R
cytoplasmic domain is
not essential for IL-15 signaling. To test this possibility, we
constructed a deletion mutant of murine IL-15R
(m15Rct
)
lacking the C-terminal 32 amino acids of the predicted cytoplasmic
domain and compared the proliferation of 32D-01 cells cotransfected
with murine IL-2R
and either full-length murine IL-15R
or
m15Rct
. As shown in Fig. 3, no difference in IL-15
responsiveness was detected between cells expressing full-length or
truncated murine IL-15R
, indicating that the murine IL-15R
cytoplasmic domain is dispensable for IL-15-mediated signaling, as
measured by mitogenesis.
Figure 3:
Deletion of the IL-15R
cytoplasmic
domain has no effect on IL-15 responsiveness. Proliferation of 32D-01
cells cotransfected with murine IL-2R
and either full-length
murine IL-15R
(panel A) or cytoplasmic truncation mutant
m15Rct
, lacking the 32 C-terminal amino acids (panel B).
Response was measured to murine IL-3 (
), human IL-2 (
), and
simian IL-15 (
). Results are the average of triplicate
wells.
The Human IL2R
and -
Chains Are Sufficient
for Simian IL-15 Binding and Signaling
We previously showed
that simian IL-15 could bind COS cells transfected with human
IL-2R
and -
chains(20) . To address the question of
whether the 
heterodimer can transduce IL-15-induced signals
in the absence of IL-15R
, we utilized the human T lymphocytic
leukemia line Jurkat, which is negative for
I-IL-15
binding (Table 2) and therefore does not express IL-15R
.
These cells also express the IL-2R
chain but not the IL-2R
chain. Subline JP
3, derived from JP111-6 following
transfection with the human IL-2R
chain, showed several hundred
IL-15 binding sites with an intermediate binding affinity that was
approximately 50-fold lower than the observed affinity of IL-15 binding
to the IL-15R
chain (Table 2). To measure the transduction
of an IL-15-induced signal in these cells, we examined the
phosphorylation of Jak1 kinase in response to 450 ng/ml IL-2 or IL-15
stimulation (Fig. 4). Jak1 tyrosine phosphorylation, which has
been shown to be an early event in IL-2 signal
transduction(16, 35, 36, 37) , was
seen in JP
3 in response to IL-2 and IL-15 but not in the parental
line JP111-6. This demonstrates that a high concentration of
simian IL-15, like IL-2, is able to transduce a signal through a
complex of human IL-2R
and -
chains in the absence of its
specific receptor
chain.
Figure 4:
Simian IL-15 signals through human
IL-2R
and -
in Jurkat cells that lack IL-15R
. Tyrosine
phosphorylation of Jak1 (arrow) is seen in human
IL-2R
-transfected Jurkat subline JP
3 stimulated with 450
ng/ml (30 nM) IL-15 or IL-2 for 15 min but not in parental
line JP111-6. Precleared supernatants were immunoprecipitated
with anti-Jak1 polyclonal antibodies, with detection of phosphotyrosine
on Western blots as described under ``Experimental
Procedures.'' The migration of protein molecular weight standards
(in kilodaltons) is shown at the left of the
figure.
Differential Distribution of IL-15R
, IL-2R
,
-
, and -
RNA
A variety of cell lines and tissues
was examined for the expression of IL-15R
mRNA, as well as for
IL-2R
, -
and -
mRNA, to assess the relative potential of
each cell type to respond to IL-15 or IL-2. An IL-15R
mRNA species
of about 1.7-1.8 kb was detected in most of the cell lines and
all of the tissues examined by Northern blot analysis (Table 3).
IL-2R
expression was seen mainly in mRNA derived from T-cell lines
and tissues containing T cells, as well as peripheral blood monocytes
and the B lymphoblast line, Raji. IL-2R
mRNA was detected in most
of the tissue RNAs examined, but among the cell lines examined was
limited to T cell line HUT 102, bone marrow stromal line IMTLH,
myelogenous leukemia line K-562, and the SV40-transformed lung
epithelial cell line WI-26 (Table 3). In some of the cell lines
and tissues examined, a larger IL-2R
transcript was detected, the
structure of which is unknown. IL-2R
mRNA was abundant in lymphoid
and myeloid cell lines, and a low level was detected in prostate,
ovary, intestine, and colon. If the expression of the specific receptor
chain for IL-2 or IL-15 is a limiting factor for IL-2 or IL-15
responsiveness in cells that also express the IL-2R
and -
chains, then these results indicate that a greater variety of the cell
lines and tissues examined have the potential to respond to
physiological concentrations of IL-15 than to IL-2.
Analysis of IL15RA Genomic
Structure
Genomic IL15RA clones were isolated from
a human genomic library using a random prime-labeled human IL-15R
cDNA (W5) probe. Restriction mapping and probing with individual
radiolabeled oligonucleotides specific for 5`-non-coding, coding, and
3`-non-coding human IL-15R
cDNA sequences were used to determine
the overlap and extent of the genomic isolates. One isolate that
contained an approximately 15-kb insert and hybridized to all but the
5`-non-coding region oligonucleotide probe (
15Rgen19) was used to
map intron positions by sequencing across intron-exon boundaries using
IL-15R
cDNA-specific sequencing primers. Six introns in the IL15RA gene were identified (Fig. 1A and 5).
None of the isolated genomic IL15RA clones contained exon 1
sequences. We used exon 1-specific primers (dashed arrows, Fig. 1A) and PCR amplification with whole cell human
genomic DNA and full-length IL-15R
cDNA templates to detect any
additional introns upstream of intron 1. A single amplified fragment
was seen in all reactions (data not shown), indicating that no
additional IL15RA introns upstream of intron 1 were detected
in genomic DNA. Since the 5`-end of the cloned cDNA may not contain the
start of the IL-15R
mRNA, the possibility exists for additional
exons upstream of the sequence presented in Fig. 1A.
Analysis of the intron positions relative to the predicted amino acid
sequence shows that introns 1 and 2 delineate the boundaries of the
sushi coding domain of IL15RA, and introns 5 and 6 delineate
the transmembrane domain (Fig. 1A and Fig. 5).
The exon-intron boundaries relative to predicted structural domains of
IL-15R
and IL-2R
(shown schematically in Fig. 5) show
that the positions of introns 1, 2, 3, 5, and 6 of IL15RA are
in close agreement with the positions of introns 1, 2, 3, 6, and 7
previously determined for IL2RA(38, 39) .
Together with the similar roles of the IL-15R
and IL-2R
in
the IL-15 and IL-2 receptor complexes, the similarities in gene
organization of IL15RA and IL2RA also suggest close
evolutionary relatedness between these two genes.
Figure 5:
Schematic comparison of IL-15R
and
IL-2R
intron-exon positions relative to predicted structural
domains. Intron positions are indicated with arrows, and exons
are numbered. Also indicated are the locations of the signal
peptide (Sig.), sushi domains, and transmembrane regions (tm).
Human Chromosomal Localization of IL15RA by
FISH
To map the IL15RA locus, we performed FISH of
metaphase chromosomes prepared from peripheral blood lymphocytes of a
normal male donor using IL15RA genomic clones
15Rgen19
(described above) and
15Rgen11, which also contains a 15-kb insert
and overlaps about 2 kb of
15Rgen19 (5`-end of
15Rgen11,
3`-end of
15Rgen19). A representative hybridization of IL15RA clones
15Rgen11 and
15Rgen19 to a metaphase chromosome
preparation is illustrated in Fig. 6. Fluorescence signal
observed with these clones was specific to the distal tip of the short
arm of a group C chromosome (chromosomes 6-12 and X).
Simultaneous hybridizations of clones
15Rgen11 and
15Rgen19
with
satellite DNA probes specific to the various group C
chromosomes identified chromosome 10 as the chromosome containing the IL15RA locus. Specific labeling at the distal tip of the short
arm was observed with the IL15RA clones on 83 of 98 chromatids
(84.7%) of chromosomes hybridizing with the chromosome 10-specific
satellite DNA probe (25 metaphases analyzed). No other non-random
signals suggestive of alternative localizations or of
cross-hybridization with related sequences were seen in any of the
metaphase spreads hybridized. Based upon the presence of the IL15RA-specific hybridization in the extreme distal portion of
the short arm of chromosome 10, we have assigned IL15RA to
bands p14-p15, which is the same location determined for IL2RA(40) . This finding further reinforces the
relatedness of these two genes.
Figure 6:
The human IL15RA locus maps to
chromosome 10p14-p15. Fluorescence in situ hybridization to a
normal human metaphase chromosome spread with two overlapping IL15RA genomic clones is illustrated. The position of the IL15RA locus on the short arm of chromosome 10, in the region
p14-p15, is indicated by the arrows. The identity of the
chromosomes was verified by simultaneous hybridization with a
chromosome 10-specific
satellite DNA probe (arrowheads).
Mouse Chromosomal Localization of Il15ra by
Interspecific Backcross Mapping
The mouse chromosomal
location of Il15ra was determined by interspecific backcross
analysis using progeny derived from matings of [(C57BL/6J
M. spretus)F
C57BL/6J] mice. This
interspecific backcross mapping panel has been typed for over 1900 loci
that are well distributed among all the autosomes as well as the X
chromosome(29) . C57BL/6J and M. spretus DNAs were
digested with several enzymes and analyzed by Southern blot
hybridization for informative RFLPs using a mouse cDNA probe. The 20.0-
and 8.0-kb SphI M. spretus RFLPs (see
``Experimental Procedures'') were used to follow the
segregation of the Il15ra locus in backcross mice. The mapping
results indicated that Il15ra is located in the proximal
region of mouse chromosome 2 linked to Il2ra, Vim,
and Spna2. Although 93 mice were analyzed for every marker and
are shown in the segregation analysis (Fig. 7), up to 182 mice
were typed for some pairs of markers. Each locus was analyzed in
pairwise combinations for recombination frequencies using the
additional data. The ratios of the total number of mice exhibiting
recombinant chromosomes to the total number of mice analyzed for each
pair of loci and the most likely gene order are centromere Il2ra-0/147-Il15ra-1/182-Vim-1/132-Bmi1-5/142-Spna2.
The recombination frequencies (expressed as genetic distances in
centimorgans ± S.E.) are
[Il2ra,Il15ra]-0.6 ±
0.6-Vim-0.8 ±
0.8-Bmi1-3.5 ± 1.6-Spna2. No
recombinants were detected between Il2ra and Il15ra in 147 animals typed in common, suggesting that the two loci are
within 2.0 centimorgans of each other (upper 95% confidence limit).
Figure 7:
Il15ra maps in the proximal region of
mouse chromosome 2. Il15ra was placed on mouse chromosome 2 by
interspecific backcross analysis. The segregation patterns of Il15ra and flanking genes in 93 backcross animals that were
typed for all loci are shown at the top of the figure. For
individual pairs of loci, more than 93 animals were typed. Each column represents the chromosome identified in the backcross
progeny that was inherited from the (C57BL/6J
M.
spretus) F
parent. The shaded boxes represent
the presence of a C57BL/6J allele, and white boxes represent
the presence of M. spretus allele. The number of offspring
inheriting each type of chromosome is listed at the bottom of
each column. A partial chromosome 2 linkage map showing the
location of Il15ra in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in
centimorgans are shown to the left of the chromosome, and the
positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in
this study can be obtained from GDB (Genome Data Base), a computerized
database of human linkage information maintained by The William H.
Welch Medical Library of The Johns Hopkins University
(Baltimore).
DISCUSSION
IL-15 is a member of a family of molecules with similar
predicted three-dimensional structures, the helical cytokine family,
whose members interact in some fashion with cell surface molecules that
are members of the hematopoietin receptor
superfamily(3, 41) . A common feature of these
families is overlapping or redundant functions arising from the shared
usage of receptor components and thus, intracellular signaling
pathways. The IL-2 and IL-15 systems are examples of this type of
relationship: both IL-2 and IL-15 have similar activities and signal
through the same receptor complex (the IL-2R
and -
chains).
Perhaps the most striking difference between the IL-2 and IL-15
systems is the 1000-fold higher affinity of the IL-15R
for IL-15
than IL-2R
for IL-2. As was shown for murine
IL-15R
(21) , human IL-15R
binds IL-15 with the same
high affinity (K
10
M
), whether IL-2R
is co-expressed or
not ( Fig. 2and Table 1). By contrast, high affinity
binding is seen in the IL-2 system only when all three IL-2 receptor
chains are present(11) . In addition to this difference, the
widespread expression patterns of IL-15 (18) and IL-15R
( (21) and Table 3) contrast with the narrow expression
patterns of IL-2 and IL-2R
(reviewed in (5) ). The more
ubiquitous distribution of IL15RA mRNA may indicate that a
wider variety of cell types is responsive to IL-15 than to IL-2.
However, since IL-2R
and -
have been shown to be essential
for IL-15 signaling in cells of hematopoietic origin (18, 20, 21, 22) and their
expression is more limited than that of IL-15R
, IL-15R
expression alone may not be a sufficient indicator of IL-15
responsiveness. However, it remains a possibility that the IL-15R
subunit may associate with as yet unidentified receptor subunits to
signal in different cell types. In this regard, the Northern expression
data from Table 2may suggest that an extremely broad range of
cellular targets have the capability of responding to IL-15.
Alternatively, widely distributed IL-15R
expression in the absence
of IL-2R
and -
may provide a source of cell-associated or
soluble receptor to act as a decoy to remove circulating IL-15.
Despite these differences in expression pattern and binding
affinity, the IL-2 and IL-15 systems share many similarities. The
cytoplasmic domain of IL-15R
, like that of IL-2R
, appears to
be dispensable for signaling. Indeed, we found that a high
concentration of IL-15 can bind and transduce a signal in cells
expressing only the IL-2R
and -
chains, as can
IL-2(42) . As shown for the IL-2 system(43) , this
ability is species-specific in that simian IL-15 measurably binds human
but not murine (21) IL-2R 
complexes, whereas it can
clearly bind both human and murine IL-15R
chains. Whether
concentrations of the cytokine needed for signaling through 
alone are achieved in vivo remains unknown.
The human IL15RA locus maps to chromosome 10 in bands p14-p15, the same
region that contains the IL2RA locus(40) . There are
only a few human diseases mapped to the 10p14-p15 region that are
potential candidates for IL15RA mutation. Rare cases of
DiGeorge syndrome, a congenital anomaly characterized by the absence of
the thymus and parathyroid glands usually associated with partial
monosomy of chromosome 22, have been associated with the deletion of
the distal 10p region(44, 45) , which could encompass
the IL15RA locus. Rare but recurrent non-random chromosomal
translocations involving the distal 10p region occur in acute
lymphocytic and acute myeloid leukemias (t(10;11)(p14;q14-q21) and
t(10;17)(p13;q12-q21)) and may be candidates for IL15RA and/or IL2RA involvement(46) .
Murine Il15ra mapped to mouse chromosome 2. We have compared our interspecific
map of mouse chromosome 2 with a composite mouse linkage map that
reports the map location of many uncloned mouse mutations (compiled by
M. T. Davisson, T. H. Roderick, A. L. Hillyard, and D. P. Doolittle and
provided from GBASE, a computerized data base maintained at The Jackson
Laboratory, Bar Harbor, ME). The murine Il15ra gene mapped in
a region of the composite map that lacks mouse mutations with a
phenotype that might be expected for an alteration in this locus (data
not shown). The proximal region of mouse chromosome 2 shares regions of
homology with human chromosomes 10p and 9p (summarized in Fig. 7). In particular, IL2RA maps to human 10p15-p14
and Vim to 10p13. The assignment of IL15RA to human
10p15-p14 and Il15ra to mouse 2, tightly linked to Il2ra(47) , confirms and extends the region of
homology between human 10p and mouse chromosome 2.
The observation
that these genes are tightly linked in both mouse and humans suggests
that they arose through a random gene duplication event that predated
the separation of mouse and man. Consistent with this possibility is
the observation that IL2RA and IL15RA are similar
both with respect to gene size and organization. Additional studies to
determine their physical separation on murine chromosome 2 and human
chromosome 10 are warranted to further examine their relationship.