From the Department of Immunology, Jerome Holland Labs, American Red Cross, Rockville, Maryland 20855
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ABSTRACT |
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The interleukin (IL)-4 receptor -chain
(IL-4R
) contains a sequence motif
(488PLVIAGNPAYRSFSD) termed the insulin IL-4 receptor
motif (I4R motif). Mutation of the central Tyr497 to Phe
blocks the tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) and diminishes proliferation in response to IL-4. Recent data
suggest that the I4R motif encodes binding sites for several protein
tyrosine binding (PTB) domain-containing proteins such as IRS1 and Shc
and potentially for the Src homology 2 domain of signal transducer and
activator of transcription 6 (STAT6). To analyze the function of the
I4R motif in regulating IL-4 signaling, we changed conserved residues
upstream and downstream of the central Tyr to Ala in the human
IL-4R
. We analyzed the ability of these constructs to signal the
tyrosine phosphorylation of IRS2 and STAT6, the induction of DNA
binding activity, and CD23 induction in response to human IL-4 (huIL-4)
in transfected M12.4.1 cells. Mutagenesis of residues downstream of
Tyr497, such as Arg498 or Phe500,
to Ala had no effect on any of these responses, suggesting that the I4R
motif may not be important for functional Src homology 2 domain
interactions. However, mutagenesis of Pro488 to Ala (P488A)
greatly diminished the tyrosine phosphorylation of IRS2 and abolished
tyrosine phosphorylation of STAT6, induction of DNA binding activity,
and CD23 induction in response to huIL-4. By contrast, a P488G mutant
signaled these responses to huIL-4. Mutagenesis of hydrophobic amino
acids previously shown to contact the PTB domain of IRS1,
Leu489 or Ile491, to Ala had only minimal
effects on responses to huIL-4. However, changing both
Leu498 and Ile491 to Ala greatly diminished the
tyrosine phosphorylation of IRS2 and abolished STAT6 activation. Taken
together, these results indicate the important role of the I4R motif in
regulating IRS docking and suggest that I4R docking to a PTB
domain-containing protein regulates activation of the STAT6
pathway.
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INTRODUCTION |
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Interleukin (IL)-41
evokes a wide variety of biological responses by binding to a high
affinity receptor complex (1). In murine lymphoid cells, the receptor
complex predominantly consists of a 140-kDa, high affinity binding
chain (IL-4R) and the common
-chain (
c) (2) that is also a
component of the receptors for IL-2, IL-7, IL-9, and IL-15 (3). Both
chains of the IL-4 receptor complex are members of the hematopoietin
receptor superfamily (4). These receptor subunits do not contain any
consensus sequences encoding tyrosine or serine/threonine kinases.
However, it has been shown that the IL-4R
associates with the Janus
family kinase JAK-1 (5) and the
c associates with JAK-3 (6, 7).
Binding of IL-4 to its receptor results in the tyrosine phosphorylation of several molecules, including JAK1, JAK3, and the IL-4R
(2).
IL-4 treatment also results in the tyrosine phosphorylation of the insulin receptor substrate-1 (IRS1) and IRS2 (8-10). IRS1 and IRS2 are large cytoplasmic proteins (170-180 kDa) that contain numerous potential tyrosine and serine/threonine phosphorylation sites. Tyrosine-phosphorylated sites within IRS1 and IRS2 associate with SH2 domains found in cytoplasmic signaling molecules, including the p85 subunit of phosphatidylinositol 3-kinase and the growth factor receptor-bound protein 2. IRS1 and IRS2 have been shown to regulate both the proliferation and the protection from apoptosis of a factor-dependent myeloid cell line 32D in response to IL-4 (10-12).
Deletional studies of the IL-4R have led to the identification of a
sequence motif
(488PLX4NPXYRSFSD) termed
the insulin IL-4 receptor motif (I4R motif) (13). Mutation of the
central Tyr497 to Phe blocked the tyrosine phosphorylation
of IRS1 and diminished proliferation in response to IL-4 in 32D cells.
It has been shown that IRS1 contains a protein tyrosine binding (PTB)
domain, also called a phosphotyrosine interaction domain (14-16),
which is important for the interaction of the I4R motif with IRS1.
Several independent studies have demonstrated that the I4R motif
encodes a binding site for the PTB domain of IRS1/2 and Shc (17, 18).
More recently, solution structure analyses of the binding of a
phosphopeptide derived from the I4R motif of the huIL-4R
with the
PTB domain derived from IRS1 (19) have indicated that amino acid
residues at the
8 and
6-positions relative to Tyr497 in
the I4R motif (Leu489 and Ile491) make contacts
with residues in the PTB domain of IRS1.
In addition to IRS family members, IL-4 also induces the tyrosine
phosphorylation of a member of the signal transducer and activator of
transcription (STAT) family, STAT6 (20-23). The general model is that
after tyrosine phosphorylation, STAT6 dimerizes in the cytoplasm,
translocates to the nucleus, and subsequently binds to consensus
sequences (termed -activated sequences) found within the promoter
regions of IL-4-inducible genes. Recent studies of mice with a targeted
disruption of the STAT6 gene clearly demonstrate that STAT6 is
necessary for the induction of genes (CD23, major histocompatibility
complexes II and I
, and IL-4R
genes) in response to IL-4
(24-26). Several lines of evidence suggest that the IRS and STAT6
pathways are separate at the initiation phase of the signal. In 32D
cells lacking IRS expression, IL-4 treatment was able to activate the
DNA binding activity of STAT6 as well as in cells expressing IRS1 (27).
On the other hand, IL-4 was able to stimulate the tyrosine
phosphorylation of IRS2 in lymphocytes derived from mice deficient in
STAT6 expression as well as in lymphocytes derived from normal mice
(28).
A series of deletion, mutagenesis, and chimeric receptor studies of the
huIL-4R (13, 29, 30) demonstrated that a region containing three
tyrosine residues with a consensus sequence of GY(K/Q)XF 90 amino acids downstream of the I4R motif was necessary for maximal
IL-4-induced activation of STAT6 DNA binding activity and CD23
induction in M12.4.1. They also indicated that STAT6 activation, in the
absence of IRS activation, was sufficient to signal maximal CD23
induction in these cells. Furthermore, these same studies also
suggested that Tyr497 in the I4R motif could signal partial
STAT6 activation, implying that any one of the first four cytoplasmic
tyrosine residues could potentially act as a docking site for the SH2
domain of STAT6. Interestingly, mutation of Tyr497 to Phe,
known to consistently affect activation of the IRS pathway in response
to IL-4 (13, 29), also affected activation of the STAT6 DNA binding
activity in approximately half of expressing clones, while the others
responded normally (27, 31).
To analyze the function of the I4R motif in regulating IL-4 signaling in detail, we changed conserved residues upstream and downstream of the central Tyr to Ala. We analyzed the ability of these constructs to signal the tyrosine phosphorylation of IRS2 and STAT6, the induction of DNA binding activity, and CD23 induction in response to huIL-4 in transfected M12.4.1 cells. The results presented herein indicate the important role of the I4R motif in regulating IRS recruitment and suggest that I4R docking to a PTB domain-containing protein(s) regulates activation of the STAT6 pathway.
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EXPERIMENTAL PROCEDURES |
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Cells and Reagents--
The murine B cell lymphoma M12.4.1
(obtained from Dr. Richard Asofsky, NIAID, National Institutes of
Health) was maintained in RPMI (BioWhittaker, Inc., Walkersville, MD)
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, and 1 × 107 M 2-mercaptoethanol. Recombinant muIL-4
and recombinant huIL-4 were obtained from R & D Systems (Minneapolis,
MN).
Mutagenesis and Transfection--
For the mutagenesis, we cloned
the huIL-4R cDNA (provided by Dr. Melanie Spriggs, Immunex,
Seattle, WA) into the pAlter-1 vector obtained from Promega (Madison,
WI). Amino acid numbering begins with +1 as the initiator methionine of
the signal peptide of the huIL-4R cDNA. There are 25 amino acids
in the signal peptide. Oligonucleotide-directed mutagenesis was
performed according to the manufacturer's protocol using mutant
oligonucleotides that would convert the wild type codons to an A codon
(or as indicated a G). For example, the oligonucleotide used for the
L489A mutation was designed as follows: 5'-ACA GAG ACG CCC
GCC GTC ATC GCA GGC-3' (L489A). We prepared single-stranded
DNA containing the huIL-4R
using helper phage R408. The mutant
oligonucleotides were annealed with aliquots of the single-stranded DNA
along with an oligonucleotide that repairs the mutation in the
ampicillin resistance gene. Subsequently, synthesis and ligation were
performed to link them. The DNA was transformed into a repair minus
strain of Escherichia coli (BMH 71-18 mutS), and the cells
were grown in the presence of ampicillin. A second round of
transformation in JM109 and selection for ampicillin resistance was
performed. Bacterial colonies containing the desired mutation were
identified by sequence analysis of plasmid DNA. Mutant huIL-4R
was
then cloned into the EcoRI site of pME18s.
Immunoprecipitation and Immunoblotting-- Analysis of phosphotyrosine-containing proteins was performed as described previously (32). Briefly, cells were deprived of serum in RPMI for 2 h at 37 °C. After washing, 107 cells were resuspended in RPMI with 50 µM Na3VO4 and incubated in the presence or absence of murine or human IL-4 (10 ng/ml) for 10 min at room temperature. The reaction was terminated by 10-fold dilution in ice-cold phosphate-buffered saline containing 100 µM Na3VO4. Cell pellets were lysed in HEPES lysis buffer (50 mM HEPES, 50 mM NaCl, 0.5% Nonidet P-40, 1 mM Na3VO4, 50 mM NaF, 10 mM pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture) and clarified. The soluble fraction was immunoprecipitated with a polyclonal rabbit anti-IRS (a generous gift of Drs. Ling-Mei Wang and Jacalyn Pierce, NCI, National Institutes of Health), anti-STAT6 (Santa Cruz, CA), or anti-JAK3 (Upstate Biotechnology, Inc., Lake Placid, NY). The precipitates were washed in lysis buffer and solubilized in SDS sample buffer. The samples were separated on 7.5% SDS-polyacrylamide gels before transfer to a polyvinylidene difluoride membrane. The membranes were then probed with a monoclonal anti-phosphotyrosine antibody, RC20-H (Transduction Laboratories, Lexington, KY). The bound antibody was detected using enhanced chemiluminescence (Amersham Pharmacia Biotech). Where indicated, the blots were stripped and probed with control antibodies. Band intensities were analyzed using the public domain software NIH Image.
Electrophoretic Mobility Shift Assay--
M12.4.1 cells
expressing huIL-4R constructs were incubated with media, 10 ng/ml
muIL-4, or 10 ng/ml huIL-4 as indicated for 30 min and washed with
phosphate-buffered saline. Total cell extracts were prepared exactly as
described (29) and stored at
70 until use. Extracts (4 µg) were
incubated with 1 ng of labeled double-stranded oligonucleotide (5 × 105 cpm) corresponding to the N4
-activated sequence
element found in the promoter of the C
gene (5'-CAACTTCCCAAGAACAGA)
at room temperature for 20 min as described previously (29). Where
indicated, unlabeled C
probe, anti-STAT3, or anti-STAT6 (1 µg,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were included in the
binding reaction. Protein-DNA samples were analyzed by electrophoresis on 4.5% polyacrylamide gels in 0.22× TBE followed by
autoradiography.
CD23 Induction Assay--
M12.4.1 cells expressing the various
constructs of the huIL-4R were incubated at 1 × 105/ml in media in the presence or absence of murine or
human IL-4 (10 ng/ml) for 48 h at 37 °C. Expression of murine
CD23 was tested by FACS analysis using fluorescein isothiocyanate-B3B4
(anti-murine CD23), a generous gift of Dr. Daniel H. Conrad (MCV,
Richmond, VA), in the presence of the anti-Fc receptor antibody 2.4G2
to block Fc binding before analysis on a FACScan
(Becton-Dickinson).
Binding and Cross-linking of 125I-huIL-4-- For binding and cross-linking studies, 125I-huIL-4 was purchased from Amersham Pharmacia Biotech. Saturation binding analyses were done using 25 ng/ml 125I-huIL-4 essentially as described previously (13). 125I-huIL-4 was cross-linked to the IL-4 receptor complex using 3 mM bis(sulfosuccidinimidyl)suberate (Pierce) as described previously (32).
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RESULTS |
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Previous studies have shown that the central tyrosine in the I4R
motif (Tyr497) of the huIL-4R is critical for the
recruitment of IRS1 to the IL-4 receptor complex and the activation of
the IRS pathway. These studies also suggested that Tyr497
has some capacity to recruit STAT6 and to influence the activation of
the STAT6 pathway. Residues downstream of phosphotyrosines participate
in the binding to SH2 domains, especially the +1 and +3 residues (33).
It has recently become clear that residues upstream of phosphotyrosines
participate in the binding to PTB domains (19, 33). Therefore, we
changed conserved residues upstream and downstream of the central
tyrosine, those likely to participate in PTB or SH2 domain binding,
respectively, to alanine (Fig. 1) to
further understand the role of the I4R motif of the huIL-4R
. We
transfected these huIL-4R
constructs into the murine B lymphoma
M12.4.1 and screened for stable expression by FACS using monoclonal
anti-huIL-4R
(data not shown). Receptor expression was verified
using 125I-huIL-4 binding assays. All clones demonstrated
similar levels of expression of the transfected huIL-4R
ranging from
1000 to 4000 molecules/cell.
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We analyzed the ability of these mutant receptors to transmit signals
in response to huIL-4. Since M12.4.1 cells express endogenous muIL-4
receptors and human and murine IL-4 do not cross-bind, we used muIL-4
as a positive control for each transfectant. For each of the responses
assayed, we show representative results for each construct; at least
three independent clones expressing the indicated constructs gave
comparable results. We first analyzed the ability of these mutant
receptors to signal the activation of the IRS pathway in response to
huIL-4 by analyzing the induction of tyrosine phosphorylation of IRS2
(Fig. 2). All M12.4.1 cells demonstrated
the tyrosine phsophorylation of IRS2 in response to muIL-4. M12.4.1
cells expressing the wild type (WT) huIL-4R demonstrated potent
tyrosine phosphorylation of IRS2 in response to human IL-4, as expected
(29). Cells expressing receptors with mutations downstream of
Tyr497, such as R498A and F500A, also responded to huIL-4
treatment with potent tyrosine phosphorylation of IRS2 comparable with
the levels seen in muIL-4-treated cells. However, cells expressing the
P488A mutant showed greatly diminished tyrosine phosphorylation of IRS2
to levels that were <10% of the signal seen in WT-expressing cells in
response to huIL-4 or of the signal elicited by the muIL-4 control.
Proline residues can induce bends or kinks in protein structure, and
Pro488 is just upstream to the hydrophobic residues shown
to interact with residues found in a hydrophobic pocket of the PTB
domain of IRS1 (19) (Fig. 1). To determine whether Pro488
might allow the downstream residues of the I4R motif to recruit and
activate IRS2 in cells, we changed this residue to a glycine, an amino
acid without a side chain, thus allowing protein flexibility. Cells
expressing the P488G mutant demonstrated significant tyrosine phosphorylation of IRS2 in response to huIL-4, although slightly reduced compared with the response elicited by muIL-4, suggesting that
Pro488 participates in IRS recruitment by regulating the
availability of the downstream I4R motif residues to interact with the
PTB domain. Interestingly, cells expressing huIL-4R
with mutations in either of the hydrophobic residues shown to contact the PTB domain
of IRS1, Leu498 or Ile491, demonstrated
significant tyrosine phosphorylation of IRS2 in response to huIL-4,
although the L498A-expressing cells showed a slightly diminished level
(~60% of signal elicited of endogenous muIL-4). However, cells
expressing a construct with both Leu489 and
Ile491 changed to Ala (L,I-A) showed greatly diminished
tyrosine phosphorylation of IRS2 (<10% of WT) in response to huIL-4.
These results suggest that these hydrophobic residues participate in
the recruitment of IRS2, as shown in the solution structure analysis
(19), and that expression of either Leu489 or
Ile491 alone is sufficient to activate the IRS2
pathway.
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We next examined the ability of huIL-4 to activate the STAT6 pathway
and CD23 induction in M12.4.1 cells expressing the huIL-4R mutants
(Figs. 3 and
4). Treatment of cells expressing WT
huIL-4R
with human or mouse IL-4 induced a DNA binding activity
specific for the C
probe, indicating the activation of STAT6 (Fig.
3A). The IL-4-induced complex can often be resolved into two
bands,2 where at other times
the two bands are not distinguished. This DNA-binding complex was
supershifted by anti-STAT6 antibody but not by anti-STAT3.
Interestingly, when we analyzed the cells expressing the mutant
constructs, we found that the pattern of responsiveness to huIL-4 was
similar to the pattern we observed for IRS2 tyrosine phosphorylation
(Fig. 3B); i.e. cells expressing the P488A mutant or the L,I-A mutant did not demonstrate STAT6 DNA binding activity in
response to huIL-4, while they clearly responded to muIL-4. Cells
expressing the P488G, L489A, I491A, and F500A mutants responded to
huIL-4 by inducing STAT6 DNA binding activity. In keeping with these
results, the pattern of CD23 induction in response to huIL-4 also
corresponded to the pattern we observed for STAT6 DNA binding activity
(Fig. 4). Treatment of cell lines expressing WT, P488G, L489A, I491A,
R498A, and F500A with huIL-4 resulted in CD23 induction to levels seen
in cells treated with the muIL-4 control. However, treatment of cells
expressing P488A or L,I-A with huIL-4 did not result in the induction
of CD23, while treatment with muIL-4 was able to induce its
expression.
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These results indicate that residues within the I4R motif that are
likely to be important for the interaction with PTB domains also
participate in the regulation of the STAT6/gene induction pathway. It
is possible that the I4R motif serves to regulate the activation of an
additional signaling pathway, such as a serine kinase (34, 35), which
would further modulate STAT6 DNA binding activity. On the other hand,
the I4R motif could regulate the tyrosine phosphorylation of STAT6
itself. Therefore, we examined the ability of huIL-4 to induce the
tyrosine phosphorylation of STAT6 (Figs.
5 and 6).
Again, the pattern of responsiveness to huIL-4 corresponded to the
pattern we observed for STAT6 DNA binding activity. Treatment of cell
lines expressing WT, P488G, L489A, I491A, R498A, and F500A with huIL-4
resulted in the tyrosine phosphorylation of STAT6 to levels seen in
cells treated with the muIL-4 control. However, treatment of cells
expressing P488A or L,I-A with huIL-4 did not result in the induction
of STAT6 tyrosine phosphorylation, while treatment with muIL-4 was able
to do so. To determine whether this difference was due to a change in
the kinetics of STAT6 activation, we performed the analysis at various
time points ranging from 1 to 60 min (Fig. 6). We observed that the
induction of STAT6 tyrosine phosphorylation in response to huIL-4 was
quite rapid in cells expressing WT, P488G, L489A, or R498A.
Phosphorylation was easily detected within 1 min and reached plateau
levels within 5 min, staying elevated up to 60 min. Cells expressing
P488A or L,I-A mutant did not show induction of STAT6 tyrosine
phosphorylation in response to huIL-4 at any of the time points.
Consistent with previous studies with Y497F mutants (13, 27, 29, 31), these results demonstrate that mutations of the I4R motif of the huIL-4R that affect IRS2 phosphorylation also affect STAT6
phosphorylation, DNA binding activity, and gene induction and suggest
that PTB domain-containing proteins participate in the regulation of
the STAT6 pathway.
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To rule out the possibility that the P488A and L,I-A mutations somehow
disrupt dimerization with c or JAK activation and therefore preclude
any signaling whatsoever, we performed several control experiments
(Fig. 7). We tested the ability of
125I-huIL-4 to bind and become cross-linked to the
characteristic components of the IL-4 receptor complex on cells
expressing WT, P488A, and L,I-A constructs (Fig. 7A). All
three cell lines showed cross-linking of 125I-huIL-4 to
characteristic proteins of 140 and 70 kDa, representing the huIL-4R
and
c, respectively (36), suggesting that these mutants are able to
bind huIL-4 and induce dimerization of huIL-4R
with the endogenous
murine
c. In addition, cells expressing WT, P488A, and L,I-A
constructs responded to huIL-4 with the tyrosine phosphorylation of
JAK3. As has been observed in other murine B cell lines (37), we did
not detect JAK1 phosphorylation in response to either murine or human
IL-4 in these cells (data not shown). These results indicate that the
P488A and L,I-A mutants are able to bind IL-4, dimerize with the
c,
and signal the activation of the tyrosine kinase, JAK3.
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DISCUSSION |
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Within the last few years, studies on the structure and signaling capacity of the IL-4 receptor have elucidated mechanisms by which IL-4 regulates its panoply of biological responses. However, a precise picture of how the IL-4 receptor recruits and activates its spectrum of signaling molecules that ultimately leads to a specific biological outcome has not been fully developed.
Previous studies defined distinct regions of the IL-4R that regulate
growth and gene expression by using a series of deletion and point
mutations of the huIL-4R
and a chimeric receptor approach (2, 13,
27-30). Transfection of truncation mutants of the human IL-4R
into
32D/IRS1 demonstrated that the region between amino acids 437 and 557 is important for human IL-4-induced IRS1 phosphorylation and growth in
these cells (13). This interval contains the I4R motif. Mutation of the
central tyrosine residue of the I4R motif to a phenylalanine in the
huIL-4R
impairs its ability to signal IRS1 phosphorylation and to
induce a proliferative response upon treatment with IL-4. In addition,
transfer of the region of the huIL-4R
containing the I4R motif to a
growth-impaired truncation mutant of IL-2R
conveyed both IRS1
phosphorylation and sustained proliferation in response to IL-2
(30).
In addition to growth, the ability of the huIL-4R constructs to
initiate gene expression and STAT-6 activation has been analyzed (27,
29). A construct terminating at amino acid 657 was fully capable of
stimulating gene expression and STAT-6 DNA binding activity. However, a
construct terminating at amino acid 557, one fully competent to signal
proliferation, was greatly impaired at stimulating gene induction and
STAT-6 DNA binding activity. These results indicate that the interval
between amino acids 557 and 657 is important for signaling the
induction of genes and full activation of STAT-6 in response to IL-4.
This region contains three tyrosine residues, falling in a general
consensus sequence of GY(K/Q)XF, any one of which is able to
activate the gene induction pathway maximally (29). This region of the
receptor can function independently of the I4R motif. Transfer of this
region of the huIL-4R
(containing amino acids 557-657) to a
truncated IL-2R
conveys both STAT-6 activation as detected by gel
shift assay and gene induction in response to IL-2 but no growth
promotion or IRS phosphorylation (30). These results suggested that
growth and gene induction in response to IL-4 are independently
controlled by distinct regions of IL-4R
.
However, several observations indicated that the I4R motif may
participate in the regulation of STAT6. The huIL-4R construct terminating at 557, although greatly impaired at inducing STAT6 DNA
binding activity, was still able to support modest STAT6 and CD23
induction, suggesting the hypothesis that Tyr497 could
recruit STAT6 (27, 29). In addition, several cell lines expressing the
Y497F mutant did not support the induction of IRS phosphorylation or
the induction of STAT6 DNA binding activity (27, 31). These results
lead us to analyze the contribution of the I4R motif to IL-4 signaling
by using a mutagenesis approach.
Mutagenesis of residues downstream of Tyr497, such as
Arg498 or Phe500 to Ala (in addition,
Ser499, Ser501, and Asn502; data
not shown) had no effect on any of the biochemical or biological responses analyzed. Residues at the +1 and +3-positions relative to the
phosphotyrosine have been shown to be important for SH2 domain
interactions in general (33), and residues in the +1 and +5-positions
of the STAT1 docking site of the IFN receptor are critical for binding
to the SH2 domain of STAT1 (38). The sequences surrounding the four
cytoplasmic tyrosines in the huIL-4R, which have been shown to
interact with (Ref. 22; GYKAFSS, and GYKPFQD)
or hypothesized to interact with STAT6 (Refs. 27 and 32;
AYRSFSN and GYQEFVH) suggest that the +1 and +3
residues would be important, since these are fairly well conserved
among these sites. These results presented herein suggest that the I4R motif may not play a critical role in activating important SH2 domain
interactions with STAT6 or some unknown protein(s).
Several studies have not been able to demonstrate a direct interaction of STAT6 with the I4R motif. Hou et al. (22) showed that a phosphopeptide derived from the I4R motif could not block STAT6 dimer formation, while phosphopeptides derived from the downstream residues (Tyr603 and Tyr631) were able to do so (22). Interaction studies in the yeast two-hybrid system have also been unable to demonstrate a direct association between the I4R motif and STAT6.3 Furthermore, we were unable to precipitate STAT6 with a phosphorylated I4R motif peptide coupled to agarose beads.4 Together these results suggest that the I4R motif is not a direct docking site for STAT6 and that its influence on STAT6 activation may be via an indirect mechanism (Fig. 8).
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Interestingly, mutagenesis of Pro488 to Ala greatly diminished the tyrosine phosphorylation of IRS2 and STAT6 and abolished induction of CD23 and DNA binding activity in response to huIL-4. By contrast, a P488G mutant was competent to signal these responses to huIL-4. The observation that the Pro488 to Ala change was detrimental to function and that the Pro to Gly was not suggests that Pro488 regulates the availability of the I4R motif to PTB domain-containing proteins. Mutagenesis of both Leu489 and Ile491 to Ala also greatly diminished the tyrosine phosphorylation of IRS2 and STAT6 and abolished induction of CD23 and DNA binding activity in response to huIL-4. Mutation of one these residues to Ala was not enough to affect signaling function, indicating that potential interactions from either residue is sufficient to recruit and phosphorylate IRS2.
These findings are consistent with, but not identical to, studies on
the interaction of isolated phosphopeptides derived from the I4R motif
with the PTB domain of IRS1 (17). Wolf et al. (17) found
that a phosphopeptide derived from the I4R motif of the IL-4R
(LVIAGNPApYRS; where pY represents phosphotyrosine) would inhibit the
binding of an iodinated phosphopeptide derived from the I4R motif of
the insulin receptor (LYASSNPpYLSASDV) to the PTB domain of IRS1 with
an IC50 of 6.2 µM. This inhibition was
dependent on the phosphotyrosine. Changing Arg498 or
Ser499 to Ala had little effect on the IC50
(8.8 and 12 µM, respectively). However, they found that
changing the Leu489 or Ile491 to Ala
significantly altered the IC50, to 24 and 28 µM, respectively. A mutant phosphopeptide with both
Leu489 and Ile491 changed to Ala was not
evaluated. Direct interaction studies in the yeast two-hybrid system
have shown that mutations in Pro488 or Leu489
of an isolated I4R motif diminish IRS1 interaction.3 It is
difficult to predict how these in vitro measurements
translate to the interaction of a complete IRS2 molecule to a complete
huIL-4R
in intact cells. We have been unable to show coprecipitation
of IRS2 with either the endogenous murine or the WT human IL-4R
, likely due to the low levels of receptor and IRS2 in these cells. Our
Western blot assay for the tyrosine phosphorylation of IRS2 does not
distinguish between those mutants that no longer bind IRS2 and those
that may still recruit IRS2 to the receptor but are not competent to
allow the tyrosine phosphorylation of IRS2. However, taking into
consideration the published interaction studies (17, 18), our results
suggest that a 2-4-fold reduction in relative binding affinity does
not seriously affect the ability of the huIL-4R
to recruit and
phosphorylate IRS2 in cells.
Based on the reported structure of the interaction of the
phosphopeptide with the PTB domain of IRS1 (19), the role of
Pro488 is likely to regulate the availability of the I4R
motif to interact with the PTB domain of IRS or other PTB
domain-containing proteins. The PTB domain of IRS1 has been shown to
form a cleft between a -sandwich and an
-helix with the
phosphopeptide LVIAGNPApYR inserted in an extended conformation. The
NPApY sequence makes a Type I
-turn, and the amino-terminal
hydrophobic residues make up the extended portion. Although not
directly studied, Pro488 would be just outside of this
domain, potentially contributing to an important bend in the huIL-4R
protein. We found that a Gly residue is tolerated at this position
(position
9 relative to the Tyr) in the huIL-4R
. Interestingly, a
Pro at the
9 position is conserved among the I4R motifs derived from
the IL-4 and insulin receptors (13). In the insulin-like growth
factor-I receptors, it is replaced with Val or Thr; however, a Gly is
conserved at the
10 position. It will be of interest to determine
whether the Gly at
10 or Pro at
9 in the insulin and insulin-like
growth factor-I receptors is important for PTB domain docking.
An intriguing observation is that changing amino acids in the I4R motif affects not only the IRS pathway but also the STAT6 pathway. Similar results were variably observed for cells expressing the Y497F mutant (27, 31). One possible explanation for this finding is that by changing residues in the I4R motif we have disrupted the overall receptor structure and function. Based on the 125I-huIL-4 cross-linking data and the induction of JAK3 tyrosine phosphorylation (Fig. 7) the mutant receptors P488A and L,I-A are at least partially functional. Although we and others have demonstrated the importance of JAK1 in IL-4 signaling in human fibroblasts (28, 39), activation of JAK1 by IL-4 in murine cells of B lineage is not readily detected and could not be used as a criterion for receptor activation in these cells (37).4
Another possibility is that the changes disrupt important protein
structure in the STAT6 docking site ~90 amino acids downstream of the
I4R motif (Fig. 8, model II). Although we cannot formally rule this out without structural data on the IL-4R cytoplasmic domain, we do not favor this explanation. Only those mutations that
affected IRS2 phosphorylation also affected STAT6 phosphorylation, DNA
binding activity, and gene induction. These changes include the Y497F,
P488A, and L,I-A mutations. No other changes significantly affected
either IRS or STAT6 (including P488G, L489A, L489R, I491A, I491R,
P495A, R498A, S499A, F500A, and S501A; this report and data not shown).
The greater number of mutations of the I4R motif that had no effect on
receptor signaling seems to make a structural effect on the downstream
STAT6 domain less likely. Rather, we favor a third possibility, that
the I4R motif recruits some PTB domain-containing protein(s) that
participates in the recruitment of STAT6 to the receptor complex and/or
tyrosine phosphorylation of STAT6 (Fig. 8, model I). In this
regard, we have previousy shown that the region of the receptor
containing the I4R motif precipitates tyrosine kinase activity (13).
If model I proposed in Fig. 8 is correct, we should be able to identify a PTB domain-containing signaling molecule whose expression is required for the IL-4-induced STAT6 tyrosine phosphorylation in murine B cells expressing physiologic levels of receptors. Previous studies in 32D cells demonstrated that IRS expression was not essential for STAT6 activation by IL-4, seemingly ruling out IRS1 and IRS2 as potential candidates (27). However, the specific role of IRS2 in regulating STAT6 in B cells has not been examined. There are a number of other proteins characterized to date with PTB domains including IRS3, Shc, FRIP-1, p62DOK, X11, and FE65 (15, 16, 40-42 )5; the latter two bind an NPXY sequence in amyloid precursor protein without a strict requirement for a phosphotyrosine residue in their docking site. It remains to be determined whether one of these proteins or a novel protein will be important for the IL-4-induced activation of STAT6.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Jacalyn H. Pierce for anti-IRS2 antisera and Drs. Jacek Hawiger and Dan Lawrence for suggestions on mutagenesis, Drs. Paul Rothman and John Ryan for electrophoretic mobility shift assay protocols, and Dr. Keats Nelms for sharing unpublished results and for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by U.S. Public Health Service Grant AI38985 and by the American Red Cross.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.
To whom correspondence should be addressed: Immunology Dept.,
Jerome Holland Laboratories, American Red Cross, 15601 Crabbs Branch
Way, Rockville, MD 20855. Tel.: 301-517-0326; Fax: 301-517-0344; E-mail: keegana{at}hlsun.redcross.org.
1
The abbreviations used are: IL, interleukin;
IL-4R, IL-4 receptor
-chain; JAK, Janus family kinase; STAT,
signal transducer and activator of transcription; IRS, insulin receptor
substrate; I4R motif, insulin IL-4 receptor motif; PTB, protein
tyrosine binding;
c,
-chain; huIL-4, human IL-4; huIL-4R
,
human IL-4R
; muIL-4, murine IL-4; SH2, Src homology 2; FACS,
fluorescence-activated cell sorting; WT, wild type.
2 H. Wang, J. Zamorano, and A. D. Keegan, unpublished observations.
3 K. Nelms, personal communication.
4 A. D. Keegan, unpublished observation.
5 K. Nelms, A. Snow, J. Hu-Li, and W. E. Paul, submitted for publication.
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REFERENCES |
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