(Received for publication, June 13, 1995; and in revised form, July 18, 1995)
From the
Interleukin-2 (IL-2) regulates numerous biological events,
including T lymphocyte proliferation. Interleukin-2 receptor
(IL-2R)-mediated signaling is triggered by ligand-induced
heterodimerization of the IL-2R and
subunits,
which results in the activation of signaling intermediates that are
associated with either IL-2R
or
. Previous
mutagenesis studies of the IL-2R
cytoplasmic tail demonstrated
that the partially conserved box 1 and box 2 motifs and specific
tyrosine residues are critical for growth signaling. By deletion and
alanine scanning mutagenesis, another set of residues that are critical
for IL-2R-mediated signaling has now been identified. These residues
lie within the divergent 35-amino acid ``spacer'' region
separating box 1 and box 2. The role of this receptor subregion in
early phases of IL-2R signaling was evaluated using BA/F3 stable cell
lines expressing three functionally impaired mutants from this region.
All three cell lines displayed substantially diminished growth
responsiveness to IL-2. Receptor-mediated STAT factor activation,
IL-2R
phosphorylation, and Janus kinase activation were also
markedly impaired. These findings indicate that this variable spacer
region, which we have termed the V-box, is essential for the initiation
of IL-2R-mediated signal transduction.
The interleukin-2 receptor (IL-2R) ()complex exists
in two functional forms: a high affinity heterotrimer comprising the
IL-2R
, IL-2R
, and
subunits, as well as an
intermediate affinity heterodimer containing the IL-2R
and
chains. In T lymphocytes, binding of IL-2 to either
form of the receptor activates an intracellular signaling cascade that
culminates in cellular proliferation(1, 2) . The
primary role of the IL-2R
subunit is to confer high affinity
binding of the ligand to the receptor complex(3, 4) .
Consistent with this concept, the cytoplasmic tail of IL-2R
is
only 13 amino acids long (5, 6, 7) and no
known signaling intermediates associate with this region. Instead,
intracellular signal transduction results from ligand-induced
heterodimerization of the IL-2R
and
subunits,
which have cytoplasmic tails of 385 and 86 amino acids, respectively.
Heterodimerization of these receptor chains results in the
concurrent activation of signaling intermediates (8, 9, 10) , ()some of which are
preassociated with either IL-2R
or
.
Specifically, two members of the Janus family of tyrosine kinases, JAK1
and JAK3, are bound to the cytoplasmic regions of the IL-2R
and
chains, respectively, in the absence of
IL-2(11, 12, 13) . These tyrosine kinases
become enzymatically activated following ligand binding, perhaps by
cross-phosphorylation. However, the precise molecular details of this
inductive event remain unclear. The JAK kinases in turn are thought to
activate members of the STAT family of transcription factors
(``signal transducers and activators of transcription'').
Upon phosphorylation, the STAT factors dimerize and translocate to the
nucleus, where they activate the transcription of specific target genes
(reviewed in (14) ). In the IL-2R system, the activation of
STAT5 has recently been demonstrated by a number of
laboratories(15, 16, 17) , and in some cases,
the activation of STAT1 and STAT3 has also been
observed(16, 18) .
The IL-2R and
chains are both members of the cytokine receptor
superfamily(19) . Receptors in this family share two
extracellular motifs, a set of four cysteines with canonical spacing
and a WSXWS sequence. Moreover, these proteins are also
characterized by two small, partially conserved, intracellular regions
called the box 1 and 2 motifs(20, 21) . These motifs
are separated by a region often called the ``spacer,'' which
varies in length from 13 to 49 amino acids in the various cytokine
superfamily members.
Early mutagenesis studies of IL-2R focused
on large deletions within the cytoplasmic tail. Analysis of the
IL-2R
SD mutant, which lacks residues 266-323, revealed that
amino acids located within and surrounding the box 2 motif of
IL-2R
are critical for growth signaling(21) . More refined
structure-function studies of the cytoplasmic tail of IL-2R
have
demonstrated that both the proximal box 1 and box 2 motifs, as well as
specific tyrosine residues in the distal receptor tail, play a critical
role in IL-2R-mediated growth signaling(1, 22) .
Deletion of either box 1 or box 2 of IL-2R
completely abrogated
IL-2-induced proliferation. However, alanine scanning mutagenesis
unexpectedly revealed that only two residues within boxes 1 and 2
contributed side chains critical for such signaling. These amino acids
are Asp-258 in the box 1 motif (1) and Leu-299 in the box 2
motif(1, 23) . Interestingly, Asp-258 is poorly
conserved in the box 1 elements of other cytokine superfamily members.
This finding raised the possibility that variable rather than conserved
residues might play a central role in growth signaling through the
IL-2R complex. This hypothesis was attractive because it could help
explain the specificity of signaling exhibited by various cytokine
superfamily members despite the presence of partially conserved motifs.
Accordingly, we have now investigated the nonconserved 35-residue
spacer region separating Asp-258 in the distal portion of box 1 and
Leu-299 in the proximal part of box 2. Since this spacer region is
highly variable in amino acid composition in different cytokine
receptor members, the term variable (V) box will be used to refer to
it.
Figure 1:
Substitution and deletion analysis of
the IL-2R V-box. A, mutant IL-2R
chains were
screened for proliferation function in a transient transfection assay.
[
H]Thymidine incorporation (shown in counts/min)
was measured on days 7-14 post transfection.
WT,
wild-type IL-2R
;
V, IL-2R
, deleted of
residues 261-295 and replaced with a single methionine residue;
:
v, IL-2R
, deleted of residues 261-293
and replaced with residues 284-318 from the
receptor chain;
261-269, IL-2R
deleted of residues 261-269;
279-289,
IL-2R
deleted of residues 279-289;
290-294, IL-2R
deleted of residues
290-294. B, IL-2R
mutants were transfected into
COS-7 cells to demonstrate that IL-2R
chains of appropriate
lengths are synthesized. Cell lysates were subjected to SDS-PAGE,
followed by immunoblotting with an anti-IL-2R
antibody, 561. The
mutants analyzed are described in panelA.
To
further define V-box sequences needed for growth signaling, a series of
smaller deletion mutants was prepared and analyzed. Each of these
mutants encoded a IL-2R chain lacking 5-10 amino acids
within the V-box. Two of the mutants (IL-2R
261-269 and
IL-2R
279-289) failed to proliferate in the transient
proliferation assay (Fig. 1A), while a third mutant,
corresponding to the most C-terminal deletion,
IL-2R
290-294, proliferated in response to IL-2.
Expression studies in COS-7 cells confirmed that each of these various
V-box mutants was synthesized (Fig. 1B), indicating
that the observed lack of function observed was not due to a lack of
expression. Together, these results suggest that the IL-2R
V-box
plays an important role in growth signaling by the IL-2R complex and
that this function cannot be simply reconstituted by substitution of a
V-box from another cytokine superfamily receptor member.
Figure 2:
Alanine scanning mutagenesis of the
IL-2R V-box. A, schematic of IL-2R
V-box mutants
that were constructed. Mutants were assayed for function in the
transient proliferation assay as described in Fig. 1. Results
are summarized as the mean peak proliferative response obtained in a
representative assay and the standard error (S.E.) of triplicate
determinations. The peak incorporation was normalized by expressing the
peak incorporation as a percentage of the peak incorporation obtained
with a wild-type IL-2R
transfectant in each assay. B,
expression study in COS-7 cells. The experiment was performed as
described in Fig. 1, and the mutants are described
above.
Figure 3:
Surface expression of nonfunctional
IL-2R V-box mutants. BA/F3 stable cell lines encoding
nonfunctional IL-2R
V-box mutants were developed by transfection.
As a positive control, a cell line expressing a V-box mutation that had
no effect in the transient proliferation assay,
IL-2R
264-266A, was developed as well. Equilibrium
(Scatchard) binding studies were performed using
-I-IL-2
to determine expression and ligand binding characteristics of
IL-2R
/
receptor
complexes.
IL-2R-mediated growth signaling was first assessed in these
stable cell lines by measuring [H]thymidine
incorporation in response to varying doses of IL-2. The
IL-2R
261-263A, IL-2R
270A 272-2R, and
IL-2R
278-280A mutations all displayed markedly reduced
responsiveness to IL-2 in the assay. As expected, the
IL-2R
264-266A mutant proliferated normally (Fig. 4).
The IL-2R
270A 271-272R mutant conferred the smallest shift
in the dose-response curve, while the IL-2R
278-280A mutant
produced a more marked shift in response. The IL-2R
261-263A
mutant was essentially unresponsive to IL-2, even at very high doses.
Hence, these data confirmed the results obtained with the transient
proliferation assay and revealed more subtle differences between these
three mutants.
Figure 4:
Dose-response curves of nonfunctional
IL-2R V-box mutants. Proliferative responses to IL-2 were assessed
in these cell lines by measuring [
H]thymidine
incorporation 24 h after the addition of ligand.
, IL-2R
wild-type;
, IL-2R
261-263A;
,
IL-2R
264-266A;
, IL-2R
270A 271-2R;
,
IL-2R
278-280A.
Figure 5:
STAT5 induction in BA/F3 stable
transfectants expressing various IL-2R V-box mutations. Stable
cell lines were stimulated with IL-2, IL-3, or no cytokine for 10 min.
Following stimulation, nuclear extracts were prepared and subjected to
EMSA using a F
RI probe. Lanes 1-3,
IL-2R
261-263A; lanes4-6,
IL-2R
264-266A; lanes 7-9, IL-2R
270A
271-2R; lanes 10-12, IL-2R
278-280A.
Cells were stimulated with 10% WEHI 3B supernatant as a source of IL-3 (3), no cytokine(-), or 10 nM recombinant human
IL-2 (2).
Figure 6:
Tyrosine phosphorylation of IL-2R in
stable cell lines expressing IL-2R
V-box mutations. Following IL-2
stimulation of the BA/F3 stable cell lines, the IL-2R
chain was
immunoprecipitated with an anti-IL2R
monoclonal antibody,
subjected to SDS-PAGE, and immunoblotted with an antiphosphotyrosine
antibody. Lanes1 and 2, wild-type
IL-2R
; lanes3 and 4,
IL-2R
261-263A; lanes5 and 6,
IL-2R
264-266A; lanes7 and 8,
IL-2R
270A 271-2R; lanes9 and 10,
IL-2R
278-280A. Cells were stimulated with 10 nM recombinant human IL-2 (+) or incubated in the absence of
cytokine(-) for 10 min.
Figure 7:
JAK1 and JAK3 phosphorylation in stable
cell lines encoding nonfunctional IL-2R V-box mutants. Following
stimulation of the stable cell lines described in Fig. 3, JAK1
and JAK3 were serially immunoprecipitated and immunoblotted with an
antiphosphotyrosine antibody to assess kinase induction. A,
immunoprecipitations performed with an anti-JAK1 antibody. B,
an anti-JAK3 antibody was used. In both cases, lanes1 and 2, IL-2R
261-263A; lanes3 and 4, IL-2R
264-266A; lanes5 and 6, IL-2R
270A 271-2R; lanes7 and 8, IL-2R
278-280A. Cells were stimulated
with IL-2 (+) or incubated in the absence of cytokine(-) for
10 min.
Previous work from this and other laboratories has
demonstrated that membrane-proximal regions of the cytoplasmic tail of
IL-2R are critical for signaling by the IL-2R complex (for a
review, see (33) ). Surprisingly, only a single residue in each
of the box 1 and box 2 appears essential for growth signaling.
Furthermore, the critical residue within the box 1 motif (Asp-258) is
not well conserved among other cytokine receptor superfamily members.
This result led us to consider the hypothesis that variable amino
acids, rather than conserved residues, play an important role in
signaling by IL-2R
. Therefore, we focused on the spacer region
positioned between the box 1 and box 2 motifs that contains
predominantly nonconserved amino acids. Previous work had suggested
that this region, which we have termed the V-box, plays a role in
IL-2R-mediated growth signaling. The single critical residues
identified in the box 1 and box 2 motifs directly flank the intervening
V-box, i.e. Asp-258 is positioned at the C-terminal end of box
1, whereas Leu-299 is at the N-terminal end of box 2. Therefore, these
motifs might serve to expose the critical intervening variable region,
which could function as a binding platform for one or more signaling
intermediates.
The mutagenesis approaches presented in this paper
further support the hypothesis that the IL-2R V-box contains
elements critical for growth signaling. A gross deletion of this region
completely abrogated function in a transient assay of receptor
function. Furthermore, a mutant in which the IL-2R
V-box was
replaced with an analogous portion of the
receptor
also failed to support growth signaling. This result suggested that
V-box elements are not readily interchangeable, although additional
experimentation is required to prove the generality of this
observation. It should be noted that the analogous region for
substitution was defined on the basis of sequence homology, not
function, and these artificial sequence boundaries might in fact
interrupt functional domains. Thus, replacement of only the V-box
region of this domain could result in a hybrid domain that cannot
support growth signaling.
Finer mutagenesis studies, including
alanine scanning of the IL-2R V-box region, identified a number of
residues that are important for growth signaling in the transient
proliferation assay. Many of these critical amino acids do not have
side chains that can be phosphorylated or covalently modified in
another way following ligand binding. Consequently, it seems unlikely
that this region serves as an inducible binding site for a signaling
intermediate; rather, this V-box region might serve as a constitutive
docking site for one or more signaling molecules. Of note, replacement
of Trp-277 with alanine does not affect receptor function. Previous
mutagenesis studies had demonstrated that whereas a W277G mutation
results in impaired IL-2R
function(1) , a W277R mutation
does not affect signaling by the receptor complex(34) .
Consequently, Trp-277 may play an important role in the secondary or
tertiary structure of this region, rather than contributing a specific
side chain that participates directly in receptor-signaling
intermediate interactions.
In studies designed to dissect where the
V-box mutations exert their inhibitory effects within the signaling
cascade, we examined a relatively distal effect of ligand-induced
signaling, STAT activation. In the IL-2R system, mutants that display
impaired STAT activation also fail to proliferate normally in response
to IL-2, suggesting that STAT factors may play a role in growth
signaling. In the stable cell lines expressing various V-box mutants,
STAT activation is only barely detectable in the cell lines expressing
IL-2R270A 271-2R or IL-2R
278-280A and is
undetectable in the cell line expressing IL-2R
261-263A.
Consequently, the V-box mutations must affect a signaling step proximal
to STAT factor activation but distal to ligand binding.
Subsequent
studies focused on earlier steps in the signaling cascade, in order to
define more precisely the contribution of these critical V-box residues
to IL-2R-mediated signaling. Phosphorylation of IL-2R tyrosines is
thought to allow additional signaling intermediates to be recruited
into the activated receptor complex, probably through SH2-mediated
interactions. This phosphorylation is rapid and may contribute to
effective growth signaling(22) . Since STAT activation is
likewise thought to depend upon receptor-based phosphotyrosine
residues, the integrity of IL-2R
tyrosine phosphorylation was
examined in these mutant cell lines. Importantly, ligand-induced
tyrosine phosphorylation of IL-2R
was inhibited in cell lines
expressing each of the three nonfunctional V-box mutant receptors.
Notably, a minor degree of IL-2R
phosphorylation was detected in
the IL-2R
270A 271-2R line, but phosphorylation was
undetectable in the IL-2R
278-280A cell line. In contrast, as
discussed above, a small degree of STAT activation was detectable in
both the IL-2R
270A 271-2R and IL-2R
278-280A cell
lines. These results may simply reflect a small difference in
sensitivity of these two assays, with the gel shift analysis of STAT
activation being the more sensitive of the two. Nonetheless, in both
cases, IL-2R
phosphorylation and STAT activation are minimal, as
compared to the receptor-matched positive control cell line.
The
diminution of IL-2R phosphorylation was particularly intriguing
because it suggested that membrane-proximal mutations can affect
signaling events occurring at more distal sites within the IL-2R
chain. Moreover, this result suggests that these mutations affect an
early signaling event known to be important for a full mitogenic
response. The V-box therefore might be critical for the association of
the cytoplasmic receptor tail with either a kinase that phosphorylates
the IL-2R
tail directly or a protein that activates such a kinase.
In the erythropoietin receptor system, Aoki and co-workers (35) have recently shown that the association of JAK2 with the
erythropoietin receptor is abrogated when a tryptophan residue within
the V-box of this receptor is mutated to arginine. This suggests that
the V-box of the erythropoietin receptor is also critical for the
association of JAKs, although in the IL-2 system the association of
these kinases appears to be constitutive rather than induced by ligand
binding (11, 12, 13) .
In studies of JAK
activation, neither JAK1 nor JAK3 was induced in any of the three
nonfunctional V-box mutants studied, as measured by tyrosine
phosphorylation. Others have mapped the JAK1 binding site on IL-2R
to the SD region of the receptor, which is immediately downstream of
IL-2R
261-263A and overlaps with IL-2R
270A
271-272R and IL-2R
278-280A(12) . We have
co-expressed IL-2R
mutants with JAK1 in both COS-7 and CV-1 cells
and have been unable to map the JAK1 binding site to any one domain of
the receptor chain, including the SD region. (
)It is of
interest that JAK3, which is generally thought of as a
-associated molecule, fails to be activated in the
context of IL-2R
mutations. This finding strongly argues for
cross-talk between the IL-2R
and
through the two
associated JAK kinases. Alternatively, we and others have detected an
association between IL-2R
and JAK3 in overexpression systems (13) .
This association may simply be the result of
an intrinsic affinity of the JAK1 binding site for all kinases within
the JAK family, and may be enhanced by the concomitant overexpression
of both the receptor chain (containing the JAK1 binding site) and a JAK
kinase. This result may suggest a more complex kinase activation
mechanism in which
donates JAK3 to the IL-2R
chain following ligand-induced heterodimerization of IL-2R
and
. This event might in turn permit the simultaneous
activation of the JAK1 and JAK3 kinases.
In summary, these studies
suggest that the IL-2R V-box region contains determinants that are
critical for a very proximal step in the growth signaling cascade
initiated by the IL-2R complex. Our findings further support the
hypothesis that variable, rather than conserved amino acids, are
essential for this process. Although this result is at first
surprising, it provides an explanation for specificity of signaling by
various cytokine superfamily members. The conserved residues may
provide a secondary or tertiary structural motif that permits the
variable residues to be exposed; these variable residues may in turn
contact signaling intermediates in a very specific manner. In this way,
each cytokine receptor could contact specific signaling intermediates
despite common motifs. Further analyses of other cytokine receptor
superfamily members will be needed to test the generality of this
hypothesis.