The signaling specificity for cytokines that have
common receptor subunits is achieved by the presence of additional
cytokine-specific receptor components. In the type I interferon (IFN)
family, all 14 subtypes of IFN
, IFN
, and IFN
bind to the same
and
L subunits of the type I IFN-R, yet
differences in signaling and biological effects exist among them. Our
data demonstrate that IFN
2 and IFN
utilize different regions of
the
L subunit for signaling. Thus, in contrast to other
cytokine systems, signal diversity in the type I IFN system can be
accomplished within the same receptor complex by utilizing different
regions of the same receptor subunits.
 |
INTRODUCTION |
Ligand binding to a receptor induces oligomerization of receptor
subunits, which results in activation of various signaling pathways.
Specificity in ligand receptor systems is achieved at the extracellular
level by the specific interaction of a ligand with its distinct
receptor components and at the intracellular level by the interaction
of the cytoplasmic domain of the receptor subunits with a distinct set
of signal transducing proteins. Many cytokine systems share receptor
subunits (1-3), and in these situations it is commonly accepted that
specificity is determined by the existence of additional
ligand-specific receptor chains. For instance, the
IL2R1 has a binding subunit
(
chain) and two signaling chains designated as
and
c. The
IL2R
c chain is also common to the IL4, IL7, and IL9 receptors and
functions in association with specific receptor subunits for each of
these cytokines (i.e. IL4R
, IL7R
, and IL9R
chains).
Therefore, these cytokines have the ability to produce both redundant
and distinct biological effects (1-3).
The type I interferon (IFN) family includes 14 subtypes of IFN
, as
well as one IFN
and IFN
, all of which bind to the same cell
surface receptor designated as IFN
R, IFN
R, or type I IFN-R (4). The type I IFN-R is composed of at least two subunits: the
chain or IFNAR1 (5-9) and the
subunit or IFNAR2, which has short
(
S) and long (
L) forms (10-14). Although
expression of the
chain with either
L or
S produces high affinity receptors in murine L-929
cells, only coexpression of
and
L allows activation of the Jak-Stat pathway and induction of an antiviral state in response
to stimulation by both huIFN
2 and huIFN
(13, 14). Interestingly,
although both of these human IFNs bind to the same receptor and
activate the same components of the Jak-Stat pathway, there are some
signaling and biological differences. IFN
signaling has two
distinctive features: (i) induction of a very strong association of the
and
L subunits of the type I IFN-R (15) and (ii)
transcriptional activation of the
-R1 gene (16). These signaling
differences could be responsible for the disparity in biological
effects among the members of the IFN
family. For example, IFN
is
more effective than other type I IFNs in the treatment of multiple
sclerosis (17, 18). However, unlike other cytokines, the differences in
signaling and biological activities between IFN
and IFN
do not
appear to result from the utilization of different receptor subunits.
This has been demonstrated by the finding that mouse L-929 cells stably
expressing the human
and
L subunits respond equally
well to the induction of an antiviral state by huIFN
and huIFN
(13), and yet only IFN
triggers the aforementioned association of
the
and
L chains (15).
In this report we show that IFN
2 and IFN
require distinct
intracytoplasmic regions of the
L chain to elicit an
antiviral response. L-929 cells expressing
L truncated
at amino acid 417 show a marked decrease in the antiviral response to
IFN
but not to IFN
2. However, no differences in the activation of
ISGF3 or SIF factors by IFN
2 or IFN
were detected. These data
suggest that other signaling components, in addition to the Stat
pathway, should be activated to obtain an antiviral response. Moreover, IFN
seems to activate this unknown pathway through a distinct mechanism that requires the 417-462 region of the
L
subunit.
 |
MATERIALS AND METHODS |
IFNs, Antibodies, and Antiviral Assays--
Human recombinant
IFN
2 and IFN
were kindly provided by Drs. Paul Trotta
(Schering-Plow) and S. Goelz (Biogen). The anti-phosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal antibodies against Jak1 and Stat1 were purchased from Transduction Labs. (Lexington, KY). The anti-Stat1 and
anti-Stat2 and anti-Jak1 sera were kindly provided by Drs. A. Larner
(Food and Drug Administration, Bethesda, MD) and J. N. Ihle (St.
Jude's Children's Hospital, Memphis, TN), respectively. Antiviral
assays were performed as described previously (19).
Expression of Different Deletions of the
L Subunit
of Type I IFN-R in Mouse L-929 Cells--
These constructs were made
by polymerase chain reaction using proofreading Vent polymerase and
primers with an early termination codon at positions 346, 417, and 462 (see Fig. 1), respectively (20). Transfectants were grown in medium
containing G-418 (500 µg/ml) and hygromycin B (500 µg/ml).
Immunoblotting--
Cells were treated with different
concentrations of the indicated IFNs for 15 min, rapidly centrifuged at
2000 × g for 30 s in an Eppendorf microfuge, and
subsequently solubilized in lysis buffer (20 mM Tris, pH
7.5, 50 mM NaCl, 10 mM sodium pyrophosphate, 20 mM NaF, 1 mM EDTA, 1 mM
MgCl2, 1 mM dithiothreitol, 0.5% Triton X-100,
10 µg/ml leupeptin, 10 µg/ml aprotinin, 100 mM
phenylmethylsulfonyl fluoride, 200 µM sodium
orthovanadate). Immunoprecipitation and immunoblotting were performed
as described previously (7).
Radioiodination of Type I IFNs, Competitive Displacements, and
Affinity Cross-linking--
Radioiodination of IFN
2 and competitive
displacement assays were performed as described previously (6).
EMSA--
Whole cell extracts were prepared as described by
Ghislain and Fish (21) and analyzed by EMSA using end labeled ISRE
oligonucleotides to detect ISGF3.
 |
RESULTS |
We hypothesized that the differences observed between IFN
and
IFN
signaling occur within the same receptor complex and not by an
additional ligand-specific subunit as in other cytokine systems (1-3).
Such a model assumes that a particular subtype of the IFN family will
use regions of the receptor complex that are not used by another
subtype. Thus, we studied the ability of huIFN
2 and huIFN
to
induce an antiviral state in mouse L-929 cells stably coexpressing the
wild type
subunit with a
L chain truncated at amino
acid 462, 417, or 346, respectively (Fig.
1). All cell lines established were able
to respond to huIFN
2 as well as to muIFN
(positive control),
indicating that the transfected receptor and the IFN
signaling
pathway are functional. However, cells expressing the
L
chain truncated proximal to amino acid 462 (Table
I, 
L417.7,

L417.9, 
L346.2, and

L346.4 cells) required significantly higher amounts
of huIFN
to obtain 50% protection against encephalomyocarditis
virus.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic representation of the human and
L chain constructs expressed in mouse L-929 cells.
The different interactions of the cytoplasmic domain of the
L subunit are indicated. Constructs were made by
polymerase chain reaction using primers with stop codons at the
indicated positions (20). The binding sites for the Tyk2 and Jak1
kinases are shown (7, 20, 33). Transfectants were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and selection drugs (500 µg/ml of hygromycin and 500 µg/ml of geneticin).
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Induction of an antiviral state by huIFN 2 and huIFN in L-929
cells expressing truncations of the L subunit and wild type
chain
Cytopathic effect assay was performed using a concentration of
encephalomyocarditis virus stock that produced 100% cytopathic effect.
The IFN concentrations (units/ml) shown represent the amount of IFN
required to inhibit cytopathic effect by 50%.
|
|
To demonstrate that the differences in the response to IFN
2 and
IFN
were not due to alterations in IFN
binding, we tested the
ability of unlabeled IFN
2 and IFN
to displace radioiodinated IFN
2 from the human receptor subunits in the 
L417
and 
L462 cell lines. If the defect in the induction
of an antiviral state by IFN
was a consequence of impaired binding
of this IFN to the receptor, then unlabeled huIFN
should be less
effective as a competitor for binding of 125I-IFN
2 to
the receptor than unlabeled IFN
2. Fig.
2 shows that in 
L417.7
and 
L462.2 cells, unlabeled IFN
was even more
effective in displacing 125I-IFN
2 from the receptor than
equivalent concentrations of unlabeled IFN
2. Thus, the lack of
induction of an antiviral state by IFN
in 
L417
cells is not due to impaired recognition of the receptor by this IFN
but rather by deletion of the 417-462 region of
L, indicating that this region may contain a specific IFN
response element.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Expression of L chain
truncated at amino acid 417 does not affect binding of huIFN to the
receptor. Increasing concentrations of unlabeled huIFN 2 or
huIFN (kindly provided by Drs. P. Trotta and S. Goelz, respectively)
were used to displace binding of radioiodinated IFN 2 to the receptor
expressed in  L417.7 and  L462.2
cells. The Kd values in  L417.7
cells were 33 and 5 pM for IFN 2 and IFN ,
respectively. In  L462.2 cells the
Kd values were 48 and 40 pM for IFN 2
and IFN , respectively. Affinities were calculated using the computer
program LIGAND (34). Radioiodinated huIFN 2 was selected for these
experiments, because it labels to a higher specific activity (96 µg/µCi) than huIFN and can induce an antiviral state in
 L417 and  L462 cells.
|
|
To further examine the differences observed in the antiviral response,
time course and dose response experiments were performed in

L417.7 and 
L462.2 cells to
determine the length and intensity of tyrosine phosphorylation for
components of the Jak-Stat pathway. Fig.
3A shows that IFN
(lanes 3, 5, and 7) induced even
higher levels of Jak1 tyrosine phosphorylation than IFN
2
(lanes 1, 4, and 6) in

L417.7 cells at all time points studied. Intense
phosphorylation could be detected at 10 min and returned to base-line
levels by 90 min. Dose response experiments showed that activation of
Jak1 was achieved at doses as low as 300 units/ml of IFN
2 or IFN
(Fig. 3A, lower panel, lanes 2-5).
Similar results were obtained in time course and dose response
experiments performed with 
L462.2 (data not shown).
We also studied the activation of Tyk2 kinase in both cell lines. Fig.
3B shows that Tyk2 tyrosine phosphorylation was induced by
both IFN
2 and IFN
in 
L417.7 and

L462.2 cells at 10 min (lanes 2,
3, 9, and 10) and subsequently
decreased at 30 min (lanes 4, 5, 11,
and 12). The level of Tyk2 phosphorylation returned to
base-line levels by 90 min in 
L462.2 cells
(lanes 13 and 14) and significantly decreased in

L417.7 cells (lanes 6 and 7).
Tyk2 tyrosine phosphorylation was also detected with concentrations of
IFN
2 or IFN
as low as 300 units/ml (Fig. 3C, lanes 2, 3, 7, and 8). It
is worth mentioning that the level of tyrosine phosphorylation detected
in 
L462 cells were slightly higher than in

L417 cells (Fig. 3C, compare

L417 and 
L462 cells).

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 3.
Activation of the Jak-Stat pathway in
response to huIFN 2 and huIFN . A, cells were stimulated
with huIFN 2 or huIFN (20,000 units/ml) for the indicated periods
of time (top panel) or for 10 min with the indicated
concentrations of IFN (bottom panel). Cell lysates were
immunoprecipitated with an anti-Jak1 serum, resolved in a 8%
SDS-polyacrylamide gel electrophoresis, immunoblotted with the
anti-phosphotyrosine antibody 4G10, stripped, and reblotted with an
anti-Jak1 monoclonal antibody (Transduction Laboratories).
B, tyrosine phosphorylation of Tyk2 in response to IFN 2
or IFN . Cells were treated as described in A,
immunoprecipitated with an anti-Tyk2 antibody, and immunoblotted with
the anti-phosphotyrosine antibody 4G10. Stripping and immunoblotting of
the same filter with the anti-Tyk2 serum could not be performed due to
the high background produced by this antibody when used for
immunoblotting as described previously (13). C, experiment
similar to that in B in which lower doses of IFNs were used. Cellular
lysates were immunoprecipitated with an anti-Tyk2 antibody and
immunoblotted with the anti-phosphotyrosine antibody 4G10. The
electrophoretic mobility of Tyk2 and a background protein
(Bkgd) detected in some experiments are indicated.
IP, immunoprecipitation; WB, Western blot;
CT, control.
|
|
We next studied the activation of the Stat proteins. Fig.
4 shows that at all time points studied
there were no differences in the induction of tyrosine phosphorylation
of Stat1 by IFN
2 or IFN
in 
L417.7 (panel
A) or 
L462.2 (panel B) cells.
Tyrosine phosphorylation was maximal at 30 min and then progressively
declined. Fig. 4 (A and B) also shows that after
IFN
2 or IFN
treatment the anti-Stat1 serum coimmunoprecipitates
another tyrosine phosphorylated protein that has an electrophoretic
mobility similar to that of Stat2. The lower panels in
A and B of Fig. 4 show that equivalent amounts of
Stat1 were immunoprecipitated in all conditions. Similar results were
obtained when the activation of Stat1 and Stat2 (ISGF3) was studied by
EMSA with an ISRE probe. Fig. 4C shows that equivalent levels of the ISGF3 were induced by huIFN
2 and huIFN
in

L417.7 cells even though these cells are resistant to
the induction of an antiviral state by huIFN
but remain sensitive to
IFN
2. Similar results were observed with 
L462.2
cells (Fig. 4C, lower panel). Dose response
experiments were then performed to examine the ability of varying
concentrations of huIFN
2 or huIFN
to affect activation of Stat
proteins. Fig. 4D shows that Stat1 and Stat2 activation are
equivalent for human IFN
and IFN
at concentrations as low as 300 units/ml. The higher levels of tyrosine phosphorylation observed after
treatment with 300 units/ml of IFN
or IFN
in 
L462 cells are likely due to the higher amounts of
Stat1 protein precipitated in these lanes (Fig. 4D,
lower panel, anti-Stat1 immunoblot). The middle
panel in Fig. 4D shows an immunoblot with an anti-Stat2
antibody that identifies Stat2 as the protein coprecipitated by the
anti-Stat1 antibody. Coprecipitation of Stat2 is proportional to the
intensity of tyrosine phosphorylation as expected from a
SH2-phosphotyrosine interaction. In summary, the study of activation of
the Jak kinases and Stat factors in 
L417 and

L462 cells did not reveal significant differences
that would account for the impaired response to IFN
observed in

L417 cells.

View larger version (74K):
[in this window]
[in a new window]
|
Fig. 4.
Activation of Stat proteins by IFN and
IFN . A and B, time course experiments.
Antiphosphotyrosine immunoblotting (upper panels) were
performed as described in Fig. 3, but immunoprecipitations were
performed with an anti-Stat1 antibody instead. The same filters was
stripped and then probed with anti-Stat1 antibody (lower
panels) to determine the amounts of Stat1 protein loaded in each
lane. C, EMSA to detect ISGF3 induction. Whole cell extracts
were obtained from  L417.7 and
 L462.2 transfectants treated with huIFN 2 (20,000 units/ml), huIFN (20,000 units/ml), or left untreated for the
indicated times at 37 °C as described previously (21). EMSA was
performed using an ISRE probe (21) for the detection of the ISGF3
(arrows). D, dose response experiment. Tyrosine
phosphorylation of Stat1 and Stat2 was studied after stimulation of
 L417.7 and  L462.2 cells with
different concentrations of huIFN 2 and huIFN . Cell protein
homogenates were immunoprecipitated with anti-Stat1 serum and then
sequentially immunoblotted with anti-phosphotyrosine (upper
panel), anti-Stat2 (middle panel), and anti-Stat1
(lower panel) antibodies.
|
|
 |
DISCUSSION |
Cytokine systems that share subunits usually show both overlapping
and distinct biological effects that result from activation of common
and discrete receptor chains, respectively (3). In the type I IFN
system, however, several lines of evidence indicate that the different
subtypes of IFN
, IFN
, and IFN
utilize a common type I IFN-R.
For example, all type I IFNs compete for binding to the same receptor
(reviewed in Refs. 4 and 22) and specific monoclonal antibodies that
recognize the
and
chains, block binding, and inhibit biological
activity of several type I IFNs (11, 23). Other anti-
subunit
antibodies precipitate radiolabeled huIFN
2, huIFN
7,
huIFN
8, huIFN
, and huIFN
cross-linked to the
subunit
(24). Finally, expression of the human wild type
and
L subunits in mouse L-929 cells gives them the ability to mount an antiviral response after treatment by either huIFN
2 or
huIFN
(13). Our data for the type I IFN system indicate that binding
of different ligands to the same receptor chains produces changes that
go beyond simple dimerization of receptor subunits. IFN
2 and IFN
apparently utilize different regions of the intracellular domain of the
L subunit to generate an antiviral state. One possible
explanation is that each IFN promotes distinct conformational changes
in the receptor that affects signaling through the 417-462 region of
L, similar to the mechanism proposed for the Tar protein
(25, 26). This model theoretically increases the number of distinct
biological responses that can be promoted by different ligands
utilizing a common set of receptor subunits, which may explain how
different type I IFNs exert slightly different biological responses
(27). The presence of two binding sites on the
subunit of the type
I IFN-R receptor (28) may contribute to the diverse biological
responses induced by different subtypes of type I IFNs. This is
supported by the finding that the splice variant of the
subunit,
which lacks the N-terminal binding site, preferentially binds certain
type I IFNs (29).
Finally, the differences in induction of an antiviral state by IFN
and IFN
do not correlate with differences in activation of the
Jak-Stat pathway. This is not surprising because mutant U4 cells
complemented with kinase deficient Jak1 can induce IFN
-activating factor and the interferon-stimulated gene but not an antiviral state in
response to IFN
(30). Therefore, although activation of these DNA
binding complexes is required (31, 32), it is not sufficient to elicit
an antiviral effect. Similarly, the IFN
response is partially
conserved in mutant cells that lack Tyk2 (35, 36). These findings
suggest that additional signaling mechanisms should be triggered by
IFNs. It is tempting to speculate that the IFN
-induced activation of
the IFN
response element (IBR) region of
L is
responsible for the formation of other DNA binding complexes that
interact with an element different from the ISRE or IRE. The existence
of such elements has been also proposed by others to explain the
differences in induction of the IFN
-specific gene
R-1 (16).
We thank Dr. J. N. Ihle for providing us
with the anti-Jak1 serum and Dr. Andrew Larner for the anti-Stat1 and
anti-Stat2 sera.