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(Q576R) on Human IL-4-Induced Signal Transduction1
*
Department of Immunology, Jerome Holland Laboratories, American Red Cross, Rockville, MD 20855; and
Department of Biology, Virginia Commonwealth University, Richmond, VA 23284
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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, Q576R, has been linked
with allergy in humans. Increased sensitivity of patients cells with
this mutation to IL-4 suggest that a Q576R change enhances IL-4
signaling. To directly test this hypothesis, we analyzed the ability of
huIL-4R cDNA bearing the Q576R and Y575F mutations to signal
tyrosine phosphorylation, DNA-binding activity,
proliferation, protection from apoptosis, and CD23 induction in
response to huIL-4 in murine cells. Responses generated by the Q576R
and Y575F mutants were similar to those of the wild-type receptor,
using various concentrations of huIL-4 and times of stimulation. These
results indicate that neither the Q576R nor the Y575F mutations have a
significant direct effect on IL-4 signal transduction, and that
hypersensitive induction of CD23 in cells derived from human allergy
patients may be due to different and/or additional alterations in the
IL-4 signaling pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RIß, IL-4, and, most
recently, the human (hu)3
IL-4R have been linked to atopy (5, 6, 7, 8, 9, 10).
IL-4 can participate in the allergic response at several levels. IL-4
regulates the differentiation of T cells to the Th2 type and directs
class switching to IgE (11, 12). In addition, IL-4 regulates the
adhesive characteristics of endothelial cells directing tissue
infiltration by allergic inflammatory cells, such as
eosinophils (13). IL-4 evokes these responses by binding to
a high affinity receptor complex (14). In lymphoid cells, the receptor
complex predominantly consists of a 140-kDa high affinity binding chain
(IL-4R) and the common 
-chain (c) (15). The
IL-4R associates with JAK-1, whereas the c associates
with JAK-3 (15). In cell types lacking c expression, the
IL-4 receptor complex consists of the IL-4R and the IL-13R1 chain
(16). Binding of IL-4 to either receptor complex results in the
tyrosine phosphorylation of several signal
transduction molecules including the insulin receptor substrates (IRS)
-1 and -2, and a member of the STAT family, STAT6 (14).
IRS1 and IRS2 are large cytoplasmic proteins (170–180 kDa) that
contain many tyrosine and serine/threonine
phosphorylation targets (17, 18). IRS1 and IRS2 have
been shown to regulate proliferation and protection from
apoptosis in the factor dependent myeloid cell line 32D in
response to IL-4. STAT6 becomes tyrosine phosphorylated,
followed by its dimerization and translocation to the nucleus where it
binds to consensus sequences (termed -activated sequences or GAS)
found within the promoter regions of IL-4-regulated genes (19). Studies
of mice with a targeted disruption of the STAT6 gene clearly
demonstrate that STAT6 is necessary for the induction of gene
expression (CD23, MHC II, I, and IL-4R) in response to IL-4
(reviewed in ref. 14).
A series of mutagenesis studies on the huIL-4R demonstrated that a
region containing three tyrosine residues with a consensus sequence of
GYK/QXF was necessary for maximal IL-4-induced activation of STAT6
DNA-binding activity and CD23 induction in M12.4.1 (20, 21, 22).
The second cytoplasmic tyrosine residue, Y575, was shown to be able to
act as a potent STAT6 recruiting site, even in the absence of the other
two (Y603, Y631) downstream STAT6 docking sites (21). Interestingly,
associations between incidence of atopy and mutations in the huIL-4R
have been found (9, 10). One mutation lies in the cytoplasmic region of
the huIL-4R at amino acid Q576 (9), the residue next to the STAT6
recruiting Y575 (21). This mutation changes the Src homology 2 (SH2)
domain binding site from GYQEF to GYREF (Q576R). Peripheral blood B
lymphocytes isolated from allergy patients bearing the Q576R mutation
demonstrated enhanced CD23 induction in response to huIL-4 as compared
with control B cells, suggesting the hypothesis that the Q576R mutant
receptor was hyperresponsive (9).
To directly analyze the effect of the Q576 to R substitution (Q576R) in
regulating IL-4 signaling, we transfected two different cell types with
the Q576R huIL-4R construct and analyzed the ability of this
construct, as well as others, to signal the tyrosine
phosphorylation of IRS and STAT6, the induction of
DNA-binding activity, proliferation, protection from apoptosis,
and CD23 induction in response to huIL-4. The results presented herein
indicate that neither the Q576R nor the Y575F mutations have a direct
effect on IL-4 signal transduction leading to proliferation or CD23
induction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The 32D cells expressing the huIL-4R wild type (WT), a
deletion at amino acid 657, Y497F, and Y713F mutants, have been
previously described (18, 23, 24). M12.4.1 cells expressing the
huIL-4R WT, d657, and Y575F have been previously described (21).
Recombinant murine (m) IL-4 and recombinant huIL-4 were obtained from
R&D Systems (Minneapolis, MN).
Mutagenesis and transfection
Mutagenesis was performed as described (25) using a mutant
oligonucleotide that would convert the WT codon for Q576 to an R codon
(5'-CCCACCAGTGGCTATCGAGAGTTTGTACATGCG-3'). We use
amino acid numbering beginning with +1 as the initiator methionine of
the signal peptide of the huIL-4R cDNA. There are 25 amino acids in
the signal peptide. This cDNA contains an I at position 75 (termed I50
under a different numbering system). 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.
Cells were transfected by electroporation and selected as described
(25). Neomycin-resistant cells were tested for the expression of
huIL-4R by FACS analysis using mAbs M8 and M10 (a generous gift from
Dr. Melanie Spriggs, Immunex, Seattle, WA), as previously described
(23). Receptor expression was confirmed and quantified by saturation
binding analysis. 125I-huIL-4 was purchased from Amersham
(Arlington Heights, IL). Saturation binding analyses were performed
using 25 ng/ml 125I-huIL-4 essentially as described (23).
Immunoprecipitation and immunoblotting
Analysis of phosphotyrosine-containing proteins was performed as previously described (23), except that all incubations before cell lysis were performed in the absence of any phosphatase inhibitors. Cell lysates were immunoprecipitated with a polyclonal rabbit anti-IRS (a generous gift of Drs. Ling-Mei Wang and Jacalyn Pierce, National Cancer Institute, National Institutes of Health) or anti-STAT6 (Santa Cruz Biotechnology, Santa Cruz, CA). The precipitates were washed in lysis buffer and solubilized in SDS sample buffer. The samples were subjected to Western blot analysis using anti-phosphotyrosine Ab, RC20-H (Transduction Laboratories, Lexington, KY). The bound Ab was detected using enhanced chemiluminescence (Amersham). Where indicated the blots were stripped and probed with control Abs. Band intensities were analyzed using the public domain NIH IMAGE software and expressed as integrated density.
Electrophoretic mobility shift assay
Cells expressing huIL-4R constructs were incubated with
medium or 1 ng/ml huIL-4 as indicated for 60 min and washed with PBS.
Total cell extracts were prepared exactly as described (25) and stored
at -70°C until use. Extracts (4 µg) were incubated with 1 ng of
labeled double-stranded oligonucleotide corresponding to the N4 GAS
element found in the promoter of the C gene (5'-CAACTTCCCAAGAACAGA)
as previously described (25).
Functional assays
Cellular proliferation studies were performed exactly as described (20). The percentage of apoptotic cells was determined by analyzing the nuclear DNA content with propidium iodide staining followed by flow cytometry (24). Expression of murine CD23 was tested by FACS analysis using FITC-B3B4 (anti-murine CD23), a generous gift of Dr. Daniel H. Conrad (Medical College of Virginia, Richmond, VA), as described (25).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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cytoplasmic region (Q576R) has been described that may cause enhanced
signaling to IL-4 (9). This mutation is adjacent to a STAT6-docking Y
residue (Y575) (21). It was proposed that one effect of the Q576R
mutation on IL-4-induced signaling might be the loss of recruitment of
a tyrosine phosphatase, such as SHP-1, that would act to down-regulate
IL-4-induced signal transduction (9). This proposal would predict that
either the Y575F or the Q576R mutants would signal enhanced STAT6
tyrosine phosphorylation in response to huIL-4 (9, 22).
To directly test the effect of the Q576R mutation on IL-4 signaling and
to test the role of this docking site in the regulation of IL-4
signaling, we prepared cDNA for the huIL-4R bearing this mutation.
This mutant receptor and the Y575F mutant were transfected into two
murine cell lines, the IL-3-dependent myeloid progentior cell 32D-IRS1
and the B lymphoma M12.4.1 line. Stable transfectants were screened for
receptor expression by FACS analysis and 125I-huIL-4
binding; all clones expressed between 1000 and 2000 receptors per cell
(data not shown). This level of receptor expression is consistent with
the levels expressed on normal lymphocyte populations (11).
The ability of huIL-4 to stimulate the tyrosine
phosphorylation of key substrates was analyzed in two
different cell types expressing WT, Y575F (Y2F), or the Q576R (QR)
forms of the huIL-4R (Fig. 1
). Since
mIL-4 and huIL-4 are species-specific, mIL-4 could be included as a
control for signaling through an endogenous WT mIL-4R. The levels of
IRS-1 (in 32D cells) or IRS-2 (in M12.4.1 cells) tyrosine
phosphorylation in transfected cells expressing WT,
Y2F, or QR mutants induced by huIL-4 were similar to the levels
observed after treatment with mIL-4 (Fig. 1A, values for
individual band densities are indicated in the figure legend). For
example, the integrated band intensities for IRS2 tyrosine
phosphorylation stimulated by mIL-4 and huIL-4,
respectively, as shown in Fig. 1A, center panel,
are 7.2 and 7.4 for WT, 9.4 and 9.9 for QR1, and 10.5 and 9.4 for QR2.
Furthermore, the levels of IRS tyrosine phosphorylation
induced in response to huIL-4 were comparable among cells expressing a
WT, QR, or Y2F huIL-4R.
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, B–D) using various treatment parameters. Similar
levels of STAT6 tyrosine phosphorylation were induced
in cells expressing WT, Y2F, and QR forms of the receptor after
treatment with huIL-4 at concentrations ranging from 0 to 10 ng/ml
(Fig. 1, B and C). For example, the integrated
band intensities for STAT6 tyrosine phosphorylation
stimulated by mIL-4 and huIL-4, respectively, as shown in Fig. 1C, are 2.9 and 4.9 for WT, 3.4 and 3.3 for QR1, 3.9 and 4.0
for QR2, 18 and 10 for the second WT, and 8.6 and 6.7 for Y2F. In
addition, similar levels of STAT6 tyrosine
phosphorylation were observed in cells expressing WT,
Y2F, and QR forms of the receptor after treatment with huIL-4 for
various times ranging from 15 min to 16 h (Fig. 1, B–D; and data not shown); similar kinetics of
STAT6 tyrosine phosphorylation were observed in human B
cells (data not shown). Thus, in these two IL-4-responsive cell types,
we found no evidence for an alteration in the sensitivity or in the
kinetics of responsiveness to huIL-4 by the Y2F or QR mutations.
Analysis of STAT6 activation was extended to the induction of
DNA-binding activity (Fig. 2). The
ability of huIL-4 to stimulate STAT6 binding to the GAS element found
in the promoter of the C gene, a GAS site specific for STAT6, was
evaluated (Fig. 2A) in cells expressing WT, Y2F, QR, and
other previously described huIL-4R mutants (Y1F, Y5F, deletion 657
(21, 23, 24), shown in Fig. 2B). The level
of huIL-4-induced DNA-binding activity was similar in cells expressing
WT, Y1F, Y2F, and QR forms of the receptor as determined by the band
intensity of the shifted complex. Also of note, cells expressing the
forms of the huIL-4R lacking the fifth cytoplasmic Y residue (Y5F
and a deletion at amino acid 657), a residue that in theory has the
potential to dock a phosphatase since it lies in a consensus ITIM motif
(26), showed levels of STAT6 DNA-binding activity similar to that
observed in cells expressing the WT receptor. It has previously been
shown that these receptor constructs are able to signal IRS1 and SHIP
(SH2-containing inositol phosphatase)
phosphorylation normally (24). These results indicate
that the Q576R mutation in and of itself does not alter the ability of
huIL-4 to stimulate tyrosine phophorylation events or induction of
STAT6 DNA-binding activity. In addition, a comparison of the
IL-4-induced STAT6 activation in cells expressing WT, Y2F, QR, and Y5F
suggest that neither Y2 nor Y5 act as the critical recruitment site for
a receptor-proximal protein tyrosine phosphatase that would act on
STAT6 in these cell types. However, these results do not preclude the
existence of such a phosphatase. Studies in other cell types using
pharmacologic inhibitors of tyrosine phosphatases suggest that an
IL-4R-proximal phosphatase can regulate STAT6 activation (27). It is
possible that multiple docking sites for such a phosphatase exist on
the huIL-4R so that altering a single site would not result in a
dramatic change in signaling or that such a phosphatase docks with JAK1
(26, 27).
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); in M12.4.1,
we analyzed CD23 induction (Fig. 4).
32D-IRS1 cells expressing the huIL-4R constructs WT, Y2F, QR, and
d657 showed proliferative responses peaking at 
1 ng/ml of huIL-4,
with plateau levels of [3H]thymidine incorporation
ranging from 20,000 to 38,000 cpm (Fig. 3A). Cells
expressing Y1F were greatly impaired in their proliferative response to
huIL-4 (23). Analysis of the ability of the huIL-4R constructs to
signal the protection of 32D-IRS1 cells from IL-3 withdrawal-induced
apoptosis demonstrated that cells expressing the WT, Y2F, and
QR constructs were protected from apoptosis by huIL-4 to the
same degree that they were protected by mIL-4. As expected, the cells
expressing the Y1F mutant were not protected from apoptosis by
huIL-4 (24).
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A. A summary of results from several cell lines is
shown in Fig. 4B. Cells expressing the WT, Y2F, and QR forms
of the huIL-4R responded to various huIL-4 concentrations by
inducing equivalent levels of CD23 in response to huIL-4 (Fig. 4A). We also obtained such results with an incubation period
of 72 h (data not shown). Maximal responses to huIL-4 ranged from
85 to 104% of the maximal response to the mIL-4 control (Fig. 4B).
These results indicate that the QR mutation in the cytoplasmic domain
of the huIL-4R does not cause a significant change in the signal
transduction capacity of the receptor complex in the context of two
murine cell lines. The Y2F mutation also had no major effect on IL-4
signaling, suggesting that this site alone does not recruit a critical
negative regulator of IL-4 signal transduction. Nevertheless, B cells
from allergy patients bearing the QR mutation showed enhanced CD23
induction in response to huIL-4 as compared with B cells lacking the QR
change (9), and they demonstrated a 2- to 3-fold enhancement in the
tyrosine phosphorylation of STAT6 (Dr. G. Hershey,
personal communication). It is possible that for an effect of the QR
change to be observed, an additional mutation in the huIL-4R or a
mutation in, or amplification of, one of its signaling molecules (such
as JAK1, JAK3, STAT6, or a PTPase (protein tyrosine
phosphatase)) must also occur. The potential for a role of
altered IL-4 signaling in the predisposition to atopy is readily
apparent (7, 8, 9, 10); the determination of the mechanisms by which this may
occur will require further characterization of the IL-4R in allergic
patients and careful biochemical and functional analyses.
Note Added in Proof. During review of this manuscript, another study by Mitsuyasu et al. (28) also showed that the Q576R mutation had no effect on STAT6 DNA-binding activity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Achsah D. Keegan, Immunology Department, Jerome Holland Laboratories, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail address:
3 Abbreviations used in this paper: hu, human, IL-4R, IL-4R -chain; JAK, Janus family kinase; IRS, insulin receptor substrate; GAS, -activated sequence; m, murine.
Received for publication November 18, 1998. Accepted for publication January 29, 1999.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-subunit of the Interleukin-4 receptor. N. Engl. J. Med. 337:1720. upregulates IgE synthesis and associates with atopic asthma. Nat. Genet. 19:119.[Medline]
to selectively enhance endothelial cell adhesivenss for T cells: the contribution of VCAM-1-dependent and -independent binding mechanisms. J. Immunol. 146:592.[Abstract]
chain. Immunity. 4:113.[Medline]
: Y497 and Y713, but not the STAT6-docking tyrosines, signal protection from apoptosis. J. Immunol. 161:859. in regulating activation of the IRS2 and STAT6 pathways: analysis by mutagenesis. J. Biol. Chem. 273:9898.-chain in IgE synthesis. J. Immunol. 162:1227.This article has been cited by other articles:
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 A. Q. Ford, N. M. Heller, L. Stephenson, M. R. Boothby, and A. D. Keegan An Atopy-Associated Polymorphism in the Ectodomain of the IL-4R{alpha} Chain (V50) Regulates the Persistence of STAT6 Phosphorylation J. Immunol., August 1, 2009; 183(3): 1607 - 1616. [Abstract] [Full Text] [PDF]
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 D. S. Faffe, T. Whitehead, P. E. Moore, S. Baraldo, L. Flynt, K. Bourgeois, R. A. Panettieri, and S. A. Shore IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L907 - L914. [Abstract] [Full Text] [PDF]
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R. P. Andrews, M. B. Ericksen, C. M. Cunningham, M. O. Daines, and G. K. K. Hershey Analysis of the Life Cycle of Stat6. CONTINUOUS CYCLING OF STAT6 IS REQUIRED FOR IL-4 SIGNALING J. Biol. Chem., September 20, 2002; 277(39): 36563 - 36569. [Abstract] [Full Text] [PDF]
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K. A. Risma, N. Wang, R. P. Andrews, C. M. Cunningham, M. B. Ericksen, J. A. Bernstein, R. Chakraborty, and G. K. K. Hershey V75R576 IL-4 Receptor {alpha} Is Associated with Allergic Asthma and Enhanced IL-4 Receptor Function J. Immunol., August 1, 2002; 169(3): 1604 - 1610. [Abstract] [Full Text] [PDF]
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