(Received for publication, December 1, 1994)
From the
The Type I interferon (IFN) receptor has a multisubunit
structure. The component of the receptor that has been most thoroughly
studied is the subunit. Expression of the
subunit in mouse
L-929 cells confers antiviral response to human IFN
8, but not to
human IFN
2 or IFN
. This antiviral effect is observed without
a significant increase in IFN binding. It has not been determined why
mouse cells expressing the human
subunit show different response
to the antiviral activity of distinct human Type I IFNs. In this
report, we demonstrate that the response to human Type I IFNs in mouse
cells expressing the
subunit is dependent on cross-binding to the
mouse receptor. This is supported by the finding that human IFN
8,
but not human IFN
2, cross-binds to the mouse receptor even in the
absence of expression of the human
subunit. We also demonstrate
that only mouse cells expressing the human
subunit are able to
tyrosine-phosphorylate p135
and JAK-1 and to
form the ISGF3 complex in response to human IFN
8. These results
demonstrate that the
subunit is essential for IFN
signaling
through the JAK kinases and ISGF3.
Type I Interferons (IFNs) ()exert a wide variety of
effects that include antiviral and antiproliferative effects,
histocompatibility leukocyte antigen induction, boosting of natural
killer activity, etc.(1, 2) . Type I IFNs specifically
bind to a multisubunit cell surface
receptor(3, 4, 5, 6) . To follow the
designation of other receptor subunits of the cytokine receptor
superfamily, the different subunits of the Type I IFN-R are designated
with Greek letters. Experiments performed with specific anti-Type I IFN
receptor (IFN-R) monoclonal antibodies (mAb) have demonstrated that
there are at least two receptor subunits designated as the
(110
kDa) and
(100 kDa) subunits(5, 6) . Two subunits
of the Type I IFN-R have also been cloned(7, 8) . The
subunit cloned by Uzéet al.(7) corresponds to the
subunit and is recognized by
the IFNaR3 mAb (9) . Thus, we will refer to the cDNA cloned by
Uzéet al.(7) as the
subunit of the Type I IFN-R(9) . The receptor subunit cloned by
Novick et al.(8) corresponds to the
subunit. (
)
The subunit was cloned by its ability to confer
antiviral response to human IFN
8 in mouse cells(7) .
Interestingly, expression of this subunit in mouse L-929 cells makes
them responsive to human IFN
8 without a significant increase in
human IFN binding(10, 11, 12) . More
importantly, knockout mice for the
subunit fail to respond to
different viral agents underscoring the role of this subunit in the
antiviral response(13) . However, the
subunit should also
play a role in Type I IFN binding as indicated by cross-linking of this
subunit to radioiodinated human IFN
2 (6) and a slight
increase in binding observed in Xenopus laevis oocytes
transfected with the cDNA encoding this subunit(14) .
We
have demonstrated that the subunit of the Type I IFN-R is rapidly
phosphorylated on tyrosine after IFN binding to the receptor (15, 16) . Furthermore, one of the Janus tyrosine
kinases involved in IFN
signaling, the p135
kinase, associates with and tyrosine phosphorylates the
subunit in vitro(9, 17) . We have
mapped the p135
binding domain to the 47
juxtamembrane amino acids of the
subunit. This region contains
two tyrosine residues (tyrosines 466 and 481) that are the putative
targets for p135
phosphorylation(9) .
In this report, we show that the subunit binds human
IFN
8, as well as IFN
2, IFN
7, IFN
, and IFN
,
when is part of the Type I IFN-R complex expressed in human cells.
However, binding of human IFN
8, but not human IFN
2, can be
detected in mouse cells even in the absence of the human
subunit.
In this context, the response to human IFN
8 in mouse L-929 cells
transfected with the
subunit is dependent on cross-binding of
human IFN
8 to the mouse receptor. Since the
subunit is
associated with and tyrosine phosphorylated by
p135
, we tested if this receptor subunit was
able to activate this kinase in mouse cells. Our data indicate that
activation of the p135
and JAK-1 tyrosine
kinases(20, 21, 22, 23) , and the
transcriptional factor
ISGF3
(21, 24, 25, 26, 27) ,
occurs in mouse cells transfected with the
subunit in response to
IFN
8. Since the JAK-1 tyrosine kinase does not associate with the
subunit, and it is not activated in cells transfected with vector
alone, these data raise the possibility that the JAK-1 tyrosine kinase
may be downstream of p135
.
Figure 1:
Expression of the
subunit in mouse L-929 cells. L-929 cells transfected with the
pZipNeo/IFNR construct (SVX.2 cells) or pZipNeo vector (SV.1 cells)
were surface-iodinated, and labeled proteins were immunoprecipitated
with the anti-
subunit mAb IFNaR3 and the rabbit serum
411-557 (against the cytoplasmic domain of the
subunit).
It has been reported
previously that mouse cells that express the subunit of the Type
I IFN-R show an exclusive or more significant antiviral response to
human IFN
8 than to other Type I IFN subtypes (7, 10, 18) . Table 1and Table 2show that SVX.1 and SVX.2 cells (transfected with the
subunit cDNA), but not SV.1 cells (transfected with vector
alone), display a more significant antiviral response with human
IFN
8 than with human IFN
2 for either encephalomyocarditis
virus or vesicular stomatitis virus.
In order to explain the
differences observed with these two human Type I IFNs in mouse cells
expressing the subunit, we considered the following
possibilities: 1) the
subunit is specific for human IFN
8; 2)
since expression of this subunit in mouse cells does not increase human
Type I IFN binding(10) , the ability of a given human Type I
IFN to trigger an antiviral response was conditioned by its ability to
cross-bind to the mouse receptor.
To test these possibilities, we
first studied the ability of the subunit to bind different Type I
IFNs in human cells. We affinity-cross-linked radioiodinated IFN
2,
IFN
7, IFN
8, IFN
, and IFN
to the Type I IFN-R
expressed in human cells. Fig. 2(A and B)
shows that the IFNaR3 mAb precipitates an IFN-receptor complex with an
molecular mass of 130-150 kDa (
subunit), as well as the
high molecular mass complex corresponding to the association of the
and
subunits previously reported(4, 6) .
The complexes were more prominent with radioiodinated IFN
2,
IFN
8, and IFN
. IFN
7 and IFN
were cross-linked to
the receptor with lower efficiency than other IFNs. However, a weak
band corresponding to the
subunit cross-linked to radioiodinated
IFN
7 and IFN
is detected by the IFNaR3 mAb. These results
suggest that the
subunit is not specific for human IFN
8 and
that it binds different Type I IFNs, in association with other receptor
components, when expressed in human cells. Although the
subunit-radioiodinated IFN complex (130-150 kDa) resolved in most
experiments as a wide band ( Fig. 2IFN
2, IFN
8, and
IFN
), in some experiments the
subunit also resolved as a
doublet (Fig. 3, U-266 cells, and data not shown).
Figure 2:
Different radioiodinated Type I IFNs are
cross-linked to the subunit. Type I IFNs were iodinated and
cross-linked to the Type I IFN-R expressed in the human myeloma cell
lines U-266 and H-929. In A two different concentrations of
IFN
2, IFN
7, and IFN
8 were used for affinity
cross-linking. Immunoprecipitation with an anti-IFN
serum was used
as a positive control. In B an aliquot of the cross-linked
lysate was directly analyzed as positive control for IFN
and
IFN
(left side). The same lysates were used for
immunoprecipitation with the anti-
subunit mAb IFNaR3 (center). Immunoprecipitations of IFN
and IFN
(control for that experiment) cross-linked to the
subunit are
also shown (right side).
Figure 3:
Human IFN8 binds to the mouse Type I
IFN-R. Radioiodinated human IFN
2 and human IFN
8 were
cross-linked to the receptor expressed in mouse SV.1 and SVX.2 cells.
Lysates were precipitated with an anti-IFN
serum or with the
anti-
subunit mAb IFNaR3. Cross-linking of radioiodinated
IFN
8 to U-266 cell was used as a positive control. The bands
corresponding to the
and
subunits are indicated. The
difference in electrophoretic mobility observed in SV.1 and SVX.2 for
the IFN
8-receptor complex are due to an artifact produced when the
gel was dried.
We next
studied the ability of mouse SV.1 and SVX.2 cells to bind human
IFN2 and human IFN
8. Since it is not possible to measure
human IFN
2 or human IFN
8 binding in these mouse cell
lines(10) , we performed affinity cross-linking experiments. Fig. 3shows that radiolabeled human IFN
8 can be
cross-linked to the mouse receptor expressed in both SV.1 and SVX.2
cells even in the absence of the human
subunit. This is supported
by the finding that the IFN
8-receptor complexes are precipitated
by an antibody against the ligand (anti-IFN
serum) and not by the
anti-
subunit antibody IFNaR3. It should be noticed that very weak
precipitation of the human
subunit cross-linked to human
IFN
8 was detected by the IFNaR3 mAb in SVX.2 cells in long
exposures of the autoradiograms (data not shown). Interestingly, no
detectable amounts of IFN
2 cross-linked to the receptor were
detected using this method. The detection of the
subunit as a
doublet in human U-266 cells (positive control) could correspond to the
different electrophoretic mobility species previously reported for
IFN
8 (28) or to the presence of two binding sites in this
chain of the receptor as previously proposed by Bazan(29) . The
binding of IFN
8, but not IFN
2, to the mouse cells indicates
that the mouse receptor is involved in human IFN
8 binding. Thus,
the human IFN
-induced antiviral response observed in L-929 mouse
cells expressing the human
subunit is dependent on cross-binding
of a particular human Type I IFN to the mouse receptor. In this
context, the human
subunit provides for species specificity.
Figure 4:
Human IFN8 activates
p135
and JAK-1 in mouse cells expressing the
human
subunit. SV.1 and SVX.2 cells were stimulated (10 min. at
37 °C) with murine IFN
, human IFN
8, or left
untreated. Cells were lysed, and the lysates immunoprecipitated with
the indicated antibodies. Western blots were performed using
anti-phosphotyrosine antibodies (anti-pTyr). The position of
the p135
protein in the anti-phosphotyrosine
immunoblot is indicated by a dash. Blots were then stripped
and reprobed with anti-p135
or anti-JAK-1
antibodies (lower panel). Note that in the
anti-p135
immunoblot the heavily phosphorylated
bands that correspond to p135
in the upper
panel show as a ``negative'' image (arrow)
probably due to incomplete stripping of the
filter.
We also determined if
transmembrane signaling by the subunit was enough to activate the
transcriptional factor ISGF3. We obtained nuclear extracts from SV.1
and SVX.2 cells after treatment with murine IFN
, human
IFN
2, and human IFN
8 for 1 h. Human IFN
8 specifically
induced formation of the ISGF3 complex in SVX.2 cells, but not in SV.1
cells (Fig. 5A), whereas murine IFN
induced
ISGF3 formation in both SV.1 and SVX.2 cells. Fig. 5B shows that the formation of the ISGF3 complex is blocked by a
50-fold excess unlabeled oligonucleotide (Fig. 5B, lane
3), and by polyclonal serum against human Stat1
(38) (lane 6), but not by normal rabbit serum (lane
7), further indicating that the complex corresponds to ISGF3. A
weak band observed with human IFN
2 in SVX.2 cells is a background
band that comigrates with ISGF3, but it does not react with anti-ISGF3
antibodies (data not shown).
Figure 5:
Human IFN8 induces formation of the
ISGF3 complex in mouse cells that express the human
subunit of
the Type I IFN-R (A). SV.1 and SVX.2 cells were treated as
described in the legend to Fig. 4. Nuclear extracts were used
for electrophoretic mobility shift assay. The migration of the ISGF3
complex is indicated. B, the formation of the ISGF3 complex is
specifically blocked a 50-fold molar excess of unlabeled (Unl.) oligonucleotide, and by an anti-Stat1
antiserum (2
µl). Only partial blocking was achieved with the anti-Stat1 serum,
because it was raised against human Stat1, and it has a lower affinity
for the mouse counterpart. This is supported by the finding that this
serum completely inhibits ISGF3 formation in human
extracts(38) . Lanes 1 and 4 correspond to
nuclear extract from untreated SVX.2 cells; lanes 2, 3, 5, 6,
and 7 correspond to nuclear extracts from IFN
8-treated
SVX.2 cells in the presence of a 50-fold molar excess of unlabeled ISRE (lane 3), 2 µl of anti-Stat1 serum (lane 6), or 2
µl of normal rabbit serum (lane
7).
Mouse cells expressing the subunit of the Type I IFN-R cloned
by Uzéet al.(7) are protected
against vesicular stomatitis virus challenge by human IFN8. This
subunit of the receptor corresponds to a receptor component recognized
by the IFNaR1, IFNaR2, and IFNaR3 mAbs that we previously designated as
the
subunit(9) . Expression of this subunit in mouse
cells confers an antiviral response to human IFN
8, but not to
human IFN
2 or IFN
(7, 10, 18) .
However, the increase in antiviral activity is not accompanied by a
corresponding increase in human IFN binding(10) . Unlike rodent
cells transfected with the
subunit, rodent cells carrying human
chromosome 21 are able to bind and respond to different human Type I
IFNs(30, 31, 32, 33, 34, 35) .
As expected, the gene for the
subunit is localized on human
chromosome 21(36) . The finding that rodent cells transfected
with the
subunit do not show an increase in Type I IFN binding
and only respond to IFN
8 suggested that there should be a second
receptor component in this region of human chromosome 21. With the help
of a new specific monoclonal antibody against what it was designated as
the
subunit of the receptor, we demonstrated that two receptor
components are localized on the same area of human chromosome
21(5) . The presence of a second Type I IFN-R component in this
human chromosome has been recently confirmed using hamster cells
transfected with a yeast artificial chromosome that contains the q22.1
region of human chromosome 21(11, 12) . A cDNA
different than the
subunit has been recently cloned by Novick et al.(8) . It remains to be confirmed if this cDNA
maps to human chromosome 21.
It has not been elucidated why there
are differences in the antiviral response to human IFN2, IFN
,
and IFN
8 in cells transfected with the
subunit of the Type I
IFN-R. One possibility would be that the
subunit is specific for
IFN
8. This idea has been challenged by two findings: 1) a
monoclonal antibody produced against the recombinant protein produced
in COS7 cells is able to block the biological activity of different
Type I IFNs(19) , and 2) the
subunit can be cross-linked
to IFN
2 in human cells(4, 6) . Furthermore, when
we performed affinity cross-linking experiments with different
radioiodinated Type I IFNs followed by immunoprecipitation with the
specific anti-
subunit mAb IFNaR3, our results indicated that the
subunit can be cross-linked to different Type I IFNs when
expressed in human cells. However, similar experiments performed in
mouse L-929 cells demonstrated that human IFN
8, but not human
IFN
2, was able to bind to the mouse Type I IFN-R even in the
absence of the human
subunit. Mouse cells expressing the
subunit respond to human IFN
8, because this IFN
, unlike
IFN
2, binds to the mouse receptor. In this context the human
subunit is necessary for the species specificity of the antiviral
response. Our results also indicate that the
subunit expressed in
mouse L-929 cells has an molecular mass similar to that observed in
human cells. This result confirms a previous report (37) that
indicated that the
subunit is heavily glycosylated.
Since
expression of the human subunit in mouse L-929 cells suffices to
trigger an antiviral effect in response to IFN
8, we sought to
determine which components of the IFN
signaling pathway were
activated by transmembrane signaling through this subunit. We
previously reported that p135
associates and
tyrosine-phosphorylates the
subunit(9, 17) , but
we have not detected association of this subunit with JAK-1. Moreover,
it has been reported that the JAK-1 kinase associates with a different
receptor component recently cloned by Novick et
al.(8) . Our results indicate that IFN
8 specifically
triggers the transmembrane signaling by the
subunit with the
consequent activation of the p135
and JAK-1 tyrosine
kinases. The activation of these kinases was accompanied by formation
of ISGF3 as detected by electrophoretic mobility shift assay. Our data
further support a direct relationship between activation of the JAK
tyrosine kinases, induction of the ISGF3 complex, and antiviral
response in mouse cells transfected with the
subunit. The
presence of functional JAK-1 and p135
kinases appears to
be necessary for tyrosine phosphorylation of p135
and
JAK-1 in response to IFN
, suggesting that these tyrosine kinases
may cross-activate each other simultaneously or in a
cascade(20) . Since p135
associates with the
subunit, the activation by tyrosine phosphorylation of this JAK
tyrosine kinase is not surprising. However, JAK-1 does not seem to be
activated through the mouse
subunit as indicated by the lack of
tyrosine phosphorylation of JAK-1 in response to IFN
8 in cells
transfected with vector alone. Thus, tyrosine phosphorylation of the
JAK-1 tyrosine kinase should be explained by a different mechanism: 1)
JAK-1 is downstream of p135
in the IFN
pathway. In
this case the activation of p135
leads to the direct or
indirect activation of JAK-1. 2) JAK-1 is activated through the
subunit in an p135
-independent pathway. In this case,
the JAK-1 tyrosine kinase may associate to the
subunit through an
intermediary protein such as the adapters associated to other cytokine
receptors. Since binding of human IFN
8 to the mouse receptor is
involved in the antiviral effect of cells expressing the human
subunit, the contribution of each receptor to the IFN response cannot
be determined. To answer these questions, we are developing hybrid
cytokine receptors in which the binding domain of
subunit of the
IL2R is linked to the transmembrane and cytoplasmic domain of the
subunit of the IFN-R. In this system, IL2 should induce transmembrane
signaling exclusively through the cytoplasmic domain of the
subunit of the Type I IFN-R.