(Received for publication, March 30, 1995; and in revised form, June 20, 1995)
From the
Interferon (IFN-
) signals through a multimeric
receptor complex consisting of two different chains: the IFN-
receptor binding subunit (IFN-
R, IFN-
R1), and a transmembrane
accessory factor (AF-1, IFN-
R2) necessary for signal transduction.
Using cell lines expressing different cloned components of the
IFN-
receptor complex, we examined the function of the receptor
components in signal transduction upon IFN-
treatment. A specific
IFN-
R2:IFN-
cross-linked complex was observed in cells
expressing both IFN-
R1 and IFN-
R2 indicating that IFN-
R2
(AF-1) interacts with IFN-
and is closely associated with
IFN-
R1. We show that the intracellular domain of IFN-
R2 is
necessary for signaling. Cells coexpressing IFN-
R1 and truncated
IFN-
R2, lacking the COOH-terminal 51 amino acids (residues
286-337), or cells expressing IFN-
R1 alone were unresponsive
to IFN-
treatment as measured by MHC class I antigen induction.
Jak1, Jak2, and Stat1
were activated, and IFN-
R1 was
phosphorylated only in cells expressing both IFN-
R1 and
IFN-
R2. Jak2 kinase was shown to associate with the intracellular
domain of the IFN-
R2.
It has been shown that the active receptors for several
cytokines consist of at least two subunits (Jung et al., 1987;
Kishimoto et al., 1992; Miyajima et al., 1992; Akira et al., 1993; Taniguchi and Minami, 1993). The multicomponent
structure of the interferon receptor complex was proposed based
upon studies with human-rodent and mouse-hamster somatic cell hybrids.
Interferon
(IFN-
) (
)binds to the IFN-
receptor binding subunit (IFN-
R1), a species-specific cell-surface
receptor encoded on human chromosome 6 (Rashidbaigi et al., 1986) and mouse chromosome 10 (Mariano et al., 1987;
Kozak et al., 1990). However, although IFN-
R1 is the
binding subunit of the IFN-
receptor complex, it alone is not
sufficient for signal transduction (Jung et al., 1987, 1988;
Hibino et al., 1991). In addition to the binding subunit, an
additional factor encoded on human chromosome 21 (Jung et al.,
1987, 1988) and mouse chromosome 16 (Hibino et al., 1991) was
found to be required for responsiveness of the cells to IFN-
as
measured by MHC class I antigen induction. This additional factor was
recently cloned from human and mouse cells and has been designated as
the accessory factor (AF-1) or second chain of the IFN-
receptor
complex, IFN-
R2 (Soh et al., 1994; Hemmi et al.,
1994).
There is no intrinsic kinase motif within the intracellular
domains of either IFN-R1 or IFN-
R2. Two distinct regions of
the intracellular domain of IFN-
R1 have been found to be important
for signal transduction (Cook et al., 1992; Farrar et
al., 1991, 1992). The first, proximal to the transmembrane region,
is necessary for both receptor-ligand internalization and biological
responses (Farrar et al., 1991). The second region, near the
carboxyl terminus, includes Tyr-457 (Tyr-440, starting at the putative
first amino acid of the mature chain), Asp-458, and His-461, which are
required for biological responsiveness (Cook et al., 1992;
Farrar et al., 1992). After phosphorylation of Tyr-457,
Stat1
(Schindler et al., 1992) binds to this region
(Stat1
recruitment site) due to specific interaction between the
SH2 domain of Stat1
and phosphorylated Tyr-457 of IFN-
R1
(Greenlund et al., 1994), with resultant phosphorylation of
Tyr-701 of Stat1
(Shuai et al., 1993a). This
phosphorylation is probably caused by the IFN-
-activated tyrosine
kinases Jak1 and/or Jak2, members of the Janus kinase (Jak) family of
cytoplasmic protein tyrosine kinases (for review, see Ziemiecki et
al., 1994; Ihle et al., 1994, 1995; Ihle and Kerr, 1995).
Jak1 and Jak2 tyrosine kinases participate in signal transduction
initiated by IFN-
as shown in mutant cell lines defective in the
IFN-
signal transduction pathway suggesting that the IFN-
receptor components and the Jak kinases interact
(Müller et al., 1993; Silvennoinen et
al., 1993; Watling et al., 1993). In this report, we
elucidate the structure of the IFN-
receptor complex and the role
of the receptor components in the activation of the IFN-
signal
transduction pathway.
To create an expression vector for the
glutathione S-transferase/Hu-IFN-R2 intracellular domain
fusion protein (GST/IFN-
R2
), the polymerase chain
reaction was performed with two primers
5`-TCCTGGATCCAAATATAGAGGCCTGATT-3` and 5`-GGGAATACTGGTCTCTGG-3` and
plasmid p
R2 as a template according to standard protocol (Sambrook et al., 1989). The polymerase chain reaction product was
digested with BamHI restriction endonuclease and ligated into BamHI and SmaI sites of the pGEX-2T vector
(Pharmacia).
The expression vectors
pR2, p
R2-C174S, p
R2t-285, and p
R2-Y294F were stably
transfected into CHO-B7 or 16-9 cells (1-3 µg of supercoiled
plasmid DNA per 10
-10
cells) with
LipofectAMINE
Reagent (Life Technologies, Inc.) according
to the manufacturer's instructions for stable transfection of
adherent cells. All cell lines transfected with plasmids carrying the neo
gene were selected and maintained in complete
F-12 medium containing 450 µg/ml antibiotic G418.
EMSA reactions contained 2.5 µl of nuclear
extract, 1 ng of P-labeled probe (specific activity
10
cpm/µg), bovine serum albumin (24 µg/ml),
poly(dI:dC) (160 µg/ml), 20 mM HEPES, pH 7.9, 1 mM MgCl
, 4.0% Ficoll (Pharmacia), 40 mM KCl, 0.1
mM EGTA, and 0.5 mM dithiothreitol in a total volume
of 12.5 µl. For the supershift assay, 1 µl of 1:10 dilution
(equivalent to 0.1 µl of the undiluted antibody) of anti-Stat1
antibodies was added to the EMSA reaction. Incubations were performed
at 22 °C for 20 min, then 4 µl of the reaction mixture were
electrophoresed at 400 V for 3-4 h at 4 °C on a 20
20
cm 5% polyacrylamide (19:1 acrylamide:bisacrylamide) gel. The dried gel
was exposed to Kodak XAR-5 film with an intensifying screen for 12 h at
-80 °C.
Figure 1:
Induction of HLA-B7 surface antigen.
Induction of HLA-B7 surface antigen by IFN- of the parental 16-9
cells, expressing only the IFN-
R1 chain (A and B); 16-9/IFN-
R2, 16-9 cells expressing both IFN-
R1
and IFN-
R2 chains (C and D); and
16-9/IFN-
R2t, 16-9 cells expressing the IFN-
R1 chain and the
truncated IFN-
R2t chain (E and F). HLA-B7
antigen was detected by treatment of cells with mouse anti-HLA
monoclonal antibodies (W6/32) followed by treatment with fluorescein
isothiocyanate-conjugated goat anti-mouse IgG. The cells were then
analyzed by cytofluorography. The dotted lines represent cells
not treated with IFN, and solid lines represent cells treated with 1000
units/ml of the indicated IFNs. A, C, and F show results after Hu-IFN-
treatment of cells, and B, D, and E, results after treatment with
Hu-IFN-
A/D. Relative fluorescence values are shown on a log scale
as described (Hibino et al.,
1992).
Figure 2:
Covalent cross-linking of
[P]IFN-
to the receptors. Cells were
harvested and incubated with [
P]IFN-
with
or without addition of a 200-fold excess of unlabeled IFN-
and
cross-linked as described under ``Experimental Procedures.''
The extracted ligand-receptor complexes were analyzed on a 7.5%
SDS-polyacrylamide gel. The cell lines were: CHO-B7/IFN-
R2, cells
expressing only the intact IFN-
R2 chain, lanes 1 and 2; 16-9 cells, lanes 3 and 4;
16-9/IFN-
R2 cells, lanes 5 and 6;
16-9/IFN-
R2t cells, lanes 7 and 8. The cell
lines are defined in the legend to Fig. 1. The arrows designate IFN-
IFN-
R1, IFN-
IFN-
R2,
and IFN-
IFN-
R2t complexes.
The formation of an intermolecular disulfide
bridge between a Cys residue at position 174 of the human IFN-R2
and the corresponding Cys residue at position 167 of the human
IFN-
R1 (Soh et al., 1994) could account for the
interaction between these two subunits of the IFN-
receptor
complex. To test this hypothesis, we mutated IFN-
R2 at position
174 from Cys to Ser. However, the mutated IFN-
R2-C174S remained
fully active in class I MHC induction (data not shown), indicating that
Cys-174 is not essential for formation of an active IFN-
receptor
complex.
In cells expressing both chains
of the human IFN- receptor complex (16-9/IFN-
R2), IFN-
activated Jak1 and Jak2 as measured by the in vitro phosphorylation of Jak1 and Jak2 substrate peptide by
immunoprecipitated Jak1 and Jak2 kinases, respectively (Fig. 3).
However, we did not observe activation of Jak1 and Jak2 in the 16-9
cells expressing only one chain of the human IFN-
receptor complex (Fig. 3). The 16-9/IFN-
R2t cells, expressing the
IFN-
R1 and the truncated IFN-
R2t, failed to show activation
of the Jak kinases (Fig. 3). In addition, we observed
ligand-induced phosphorylation of Jak1 and Jak2 only in the cell line
16-9/IFN-
R2 expressing both chains of the human IFN-
receptor
complex (Fig. 4). Phosphorylation of Jak1 and Jak2 upon
IFN-
treatment was not detected in 16-9 and 16-9/IFN-
R2t
cells (Fig. 4).
Figure 3:
Activation of Jak1 and Jak2 tyrosine
kinases upon IFN- treatment. Top panel, the activation of
Jak1 and Jak2 was determined with an in vitro kinase assay as
described under ``Experimental Procedures'' with a peptide
substrate corresponding to the putative phosphorylation site of Jak2.
The cell lines were 16-9, 16-9/IFN-
R2,and 16-9/IFN-
R2t as
defined in the legend to Fig. 1. Bottom two panels,
equal loading of Jak proteins in the assay was demonstrated by using
one-third of the samples in Western blotting with Jak1 (middle
panel) and Jak2 (bottom panel)
antibodies.
Figure 4:
Tyrosine phosphorylation of Jak1 and Jak2
upon IFN- treatment. Untreated and IFN-
-treated cells were
lysed and immunoprecipitated with anti-Jak1 (first and second panels) or anti-Jak2 (third and fourth
panels) antibodies as described under ``Experimental
Procedures.'' The cell lines were: 16-9, lanes 1 and 2; 16-9/IFN-
R2, lanes 3 and 4;
16-9/IFN-
R2t, lanes 5 and 6. The cell lines are
defined in the legend to Fig. 1. Immunoprecipitates were
resolved on SDS-PAGE, transferred to PVDF membranes, and Western blots
were probed with anti-phosphotyrosine antibodies, first and third panels; with anti-Jak1 antibodies, second
panel; and with anti-Jak2 antibodies, fourth
panel.
Analogous results were obtained for
activation of Stat1. Only in 16-9/IFN-
R2 cells expressing
both receptor chains did IFN-
produce an active Stat1
as
measured by the electrophoretic mobility shift assay (Fig. 5A). The formation of the Stat1
DNA-binding
complex upon IFN-
treatment was suppressed after addition of an
excess of unlabeled oligonucleotides as a competitor (Fig. 5A). To show that this complex was formed by
Stat1
proteins, anti-Stat1
antibodies were added to the
nuclear extracts from IFN-
-treated 16-9/IFN-
R2 cells, and the
extracts were incubated with the same radiolabeled probe. The specific
DNA-binding complex was supershifted after addition of anti-Stat1
antibodies (Fig. 5B).
Figure 5:
Electrophoretic mobility shift assay
(EMSA). A, EMSAs were performed as described under
``Experimental Procedures'' with the 22-base-pair labeled
sequence containing the Stat1 binding site corresponding to the
GAS element in the promoter region of the human IRF-1 gene (Yuan et
al., 1994) with nuclear extracts from following cells: 16-9,
16-9/IFN-
R2, 16-9/IFN-
R2t, CHO-B7, and CHO-B7/IFN-
R2
cells as defined in the legends to Fig. 1and Fig. 2. In
addition, HEp-2 cells, a human epidermoid larynx carcinoma cell line,
were used as a positive control. B, the supershift assays were
performed as described under ``Experimental Procedures'' with
the 16-9/IFN-
R2 cell line. The position of the Stat1
DNA-binding complex and supershifted complex are indicated by the arrows. The same unlabeled oligonucleotides were used as a
competitor in 100-fold excess.
It was shown that
overexpression of Jak1 and Jak2 by transient transfection leads to
tyrosine autophosphorylation of Jak1 and Jak2 and activates Stat1,
as measured by DNA binding (Silvennoinen et al., 1993).
However, overexpression of Jak1 and/or Jak2 by stable transfection of
16-9 cells, expressing only Hu-IFN-
R1, did not permit IFN-
to
induce MHC class I antigens. (
)Furthermore, the activation
of Stat1
was not detected in these cells. Thus, both receptor
chains are necessary and sufficient for activation of Jak1, Jak2, and
Stat1
upon IFN-
induction.
Figure 6:
Tyrosine phosphorylation of IFN-R1
and coimmunoprecipitation of Jak2 kinase with antibody to IFN-
R1.
The immunocomplexes were precipitated with anti-IFN-
R1 antibodies
from lysates prepared from untreated and IFN-
-treated cells as
described under ``Experimental Procedures.'' The cell lines
were: 16-9, lanes 1 and 2; 16-9/IFN-
R2, lanes 3 and 4; 16-9/IFN-
R2t, lanes 5 and 6. The cell lines are defined in the legend to Fig. 1. Immunoprecipitates were resolved on SDS-PAGE,
transferred to PVDF membranes, and Western blots were probed with
anti-phosphotyrosine antibodies, first panel; with anti-Jak2
antibodies, second panel; and with anti-IFN-
R1
antibodies, third panel.
From this observation we hypothesized that the intracellular
domain of IFN-R2 associates with Jak2 and is responsible for
recruitment of Jak2 to the IFN-
receptor complex after IFN-
binding. To examine the interaction between Jak2 and the intracellular
domain of IFN-
R2, we prepared a glutathione S-transferase/IFN-
R2 intracellular domain fusion protein
(GST/IFN-
R2
). Lysates from Sf9 insect cells infected
with baculovirus producing Jak1 and Jak2 were incubated with the
GST/IFN-
R2
or GST proteins immobilized on
glutathione-Sepharose. The material bound to the glutathione-Sepharose
was eluted, analyzed by SDS-PAGE, blotted, and then probed with
anti-Jak1 and anti-Jak2 antibodies (Fig. 7). The Jak1 and Jak2
protein concentrations were equalized and monitored from total cellular
baculovirus lysates (Witthuhn et al., 1993). The
GST/IFN-
R2
fusion protein was found to bind Jak2
kinase (Fig. 7), demonstrating that the intracellular domain of
the IFN-
R2 chain of the IFN-
receptor complex associates
directly with Jak2.
Figure 7:
Association of Jak2 kinase with the
IFN-R2 intracellular domain. The Sf9 cell lysates with
baculovirus-produced Jak1 and Jak2 were incubated with the
GST/IFN-
R2 intracellular domain fusion protein or with GST alone
immobilized on glutathione-Sepharose. The proteins associated with GST
or GST/IFN-
R2
were resolved on the SDS-PAGE,
transferred to PVDF membranes, and Western blots were probed with
anti-Jak1 and anti-Jak2 antibodies. The Jak1 and Jak2 protein
concentrations were equalized with total cellular lysates (TCL).
It has been shown that binding of IFN- to the receptor
leads to formation of a receptor-ligand complex consisting of two
molecules of IFN-
(a dimer of IFN-
) and two molecules of
IFN-
R1 (Fountoulakis et al., 1992; Greenlund et
al., 1993). No other receptor components were found in
cross-linked complexes in previous experiments (Greenlund et
al., 1993). However, the interaction between the extracellular
domains of IFN-
R1 and IFN-
R2 was suggested in studies with
mouse-human and hamster-mouse IFN-
R1 chimeras (Hibino et
al., 1992; Hemmi et al., 1992; Gibbs et al.,
1991; Kalina et al., 1993). To investigate the interaction
between the chains of the IFN-
receptor complex and IFN-
, we
carried out cross-linking experiments with a number of cell lines
expressing different cloned components of the human IFN-
receptor
complex (Fig. 2). Our cross-linking experiments, showing that
IFN-
can be cross-linked to IFN-
R2, can be explained most
plausibly by postulating a close physical association between the
IFN-
R1 and IFN-
R2 chains of the IFN-
receptor complex.
In response to treatment with IFN-, the IFN-
R1 chain can
dimerize and internalize in the absence of the IFN-
R2 chain
(Greenlund et al., 1993; Farrar et al, 1991), but
these events are insufficient for signal transduction. To investigate
the functional importance of the intracellular domain of IFN-
R2
for IFN-
signal transduction, we introduced a stop codon after
amino acid 285 of IFN-
R2. The IFN-
IFN-
R2t complex
was still observed in cross-linking experiments (Fig. 2).
Similar behavior of the intact and truncated IFN-
R2 in
cross-linking experiments demonstrated that the intracellular domain of
IFN-
R2 is not necessary for ligand binding or for association of
the extracellular domains of IFN-
R2 and IFN-
R1. However,
truncated IFN-
R2t lacking the COOH-terminal 51 amino acid residues
was unable to elicit signal transduction for class I MHC antigen
induction in respond to IFN-
treatment (Fig. 1), indicating
that the intracellular domain of IFN-
R2 is involved in signal
transduction. The fact that the expression of human IFN-
R2 and
IFN-
R1 in hamster cells, and mouse IFN-
R2 and IFN-
R1 in
human cells, is sufficient to reconstitute the signaling pathway upon
treatment with human and mouse IFN-
, respectively, for MHC class I
induction (Soh et al., 1994; Hemmi et al., 1994)
suggests that the IFN-
R2 intracellular domain interacts with the
hamster and mouse signal transduction components.
The participation
of the protein kinases Jak1 and Jak2 as well as Stat1 in the
IFN-
signal activation pathway has been described (Darnell et
al., 1994; Ihle et al., 1994; Ziemiecki et al.,
1994). It has been shown that, upon IFN-
treatment, four
participants of the IFN-
signaling pathway (the IFN-
R1, Jak1,
Jak2, and Stat1
) are phosphorylated on tyrosine and that the
tyrosine phosphorylation occurs rapidly (less then 1 min) after
IFN-
treatment (Igarashi et al., 1994; Greenlund et
al., 1994). However, the sequence of events leading to activation
of all components and the particular role of each participant in the
IFN-
signaling pathway is still unknown. Interaction between
intracellular domains of the subunits of the IFN-
receptor complex
and components of the signaling pathway was proposed
(Müller et al., 1993; Watling et
al., 1993). Indeed, association of Jak1 tyrosine kinase with
IFN-
R1 prior to IFN-
treatment and recruitment of Jak2 only
upon IFN-
binding has been shown by coimmunoprecipitation
experiments (Igarashi et al., 1994). We elucidated the role of
the second receptor chain IFN-
R2 in signal transduction with cell
lines expressing different components of the human IFN-
receptor
complex.
The IFN-R2 chain renders cells expressing the
IFN-
R1 chain responsive to IFN-
. None of the components of
the signal transduction machinery (Jak1, Jak2, Stat1
) were
activated upon IFN-
treatment in cells expressing only one chain
of IFN-
receptor complex or IFN-
R1 and the truncated
IFN-
R2t (Figs. 3-5). No phosphorylation of the IFN-
R1,
Jak1, or Jak2 was observed in these cell lines even after a longer film
exposure ( Fig. 4and Fig. 6). However, we observed
coimmunoprecipitation of Jak2 with antibodies to IFN-
R1 in cells
expressing both chains of the IFN-
receptor complex after
IFN-
treatment. To a lesser extent, we observed coprecipitation of
Jak2 with antibodies to the IFN-
R1 in cells expressing only
IFN-
R1 and only after IFN-
treatment (Fig. 6).
We
proposed that IFN-R2 may associate directly with Jak2 and that the
major IFN-
-induced recruitment of Jak2 to the IFN-
receptor
complex is the result of association of IFN-
R2 with IFN-
R1
after IFN-
binding. To investigate the possibility of association
of IFN-
R2 with Jak2, we used the GST/IFN-
R2 intracellular
domain fusion protein. The specific association of Jak2 with
GST/IFN-
R2
was observed (Fig. 7) indicating
that Jak2 associates with the intracellular domain of the IFN-
R2
directly in the absence of the IFN-
ligand. The coprecipitation of
Jak2 with antibodies to IFN-
R1 after IFN-
induction in cells
expressing only IFN-
R1 may be explained by the existence of a low
affinity Jak2 binding site on dimerized IFN-
R1 chains upon
IFN-
binding or by the ligand-induced interaction between Jak2 and
Jak1, which is bound to the IFN-
R1 chain. We propose that one
region of Jak2 interacts with IFN-
R2 and a second region of Jak2
with the IFN-
R1 dimer or with Jak1 attached to the IFN-
R1. An
analogous situation has been observed with the IL-2-induced association
of the IL-2R
chain with Jak3: it was shown that Jak3 primarily
associates with the
chain of the IL-2 receptor
complex, but after IL-2 stimulation is weakly coprecipitated with the
IL-2R
chain which primarily associates with Jak1 (Russell et
al., 1994). In the presence of the truncated IFN-
R2t chain,
IFN-
-dependent coprecipitation of Jak2 with IFN-
R1 chain was
not seen (Fig. 6). This may be due to different conformations of
the IFN-
receptor complex formed upon IFN-
binding with and
without the IFN-
R2 chain. Another explanation may be that the
endogenous hamster IFN-
R2 in the cell lines expressing the human
IFN-
R1 chain can still perform some function, such as bringing
Jak2 to the complex, but is not sufficient for signal transduction. To
test this possibility, cells with a deletion of the IFN-
R2 gene
will be required. It should be noted, however, that we have not been
able to detect binding of Jak2 in vitro to a
GST/Hu-IFN-
R1 cytoplasmic domain fusion protein (data not shown).
Based on the results, we propose a model for the cascade of events
in IFN- signaling (Fig. 8). There are at least two receptor
subunits: IFN-
R1, the primary ligand binding subunit, and
IFN-
R2, the second chain of the IFN-
receptor complex. Both
are required for signal transduction. Upon primary binding of the
IFN-
dimer to the IFN-
R1, all the components of the receptor
signaling complex are brought together due to association of IFN-
with the IFN-
R1 and the association of IFN-
R1 with
IFN-
R2 (Hibino et al., 1992). The most likely
stoichiometry of this complex is two molecules of IFN-
R1, two of
IFN-
R2, and one of the IFN-
dimer. Jak1 is associated with
IFN-
R1 before IFN-
treatment (Igarashi et al.,
1994). IFN-
R2 associates primarily with Jak2 and brings Jak2
kinase to the IFN-
receptor complex upon IFN-
treatment.
After oligomerization of the receptor chains, Jak1 and Jak2 tyrosine
kinases associated with the intracellular domains of IFN-
R1 and
IFN-
R2, respectively, are brought together and reciprocally
activated by phosphorylation. This interaction of Jak1 and Jak2 kinases
results in their activation, probably via heterodimerization of the
kinases, and consequently in phosphorylation of Tyr-457 of the
IFN-
R1 chain, which comprises the Stat1
recruitment site
(Greenlund et al., 1994). Recruitment of Stat1
to the
receptor complex due to the specific interaction between the Stat1
SH2 domain and the phosphorylated Tyr-457 of the IFN-
R1 (Greenlund et al., 1994) results in phosphorylation of Tyr-701 of
Stat1
(Shuai et al., 1993a) and in subsequent Stat1
homodimerization (Shuai et al., 1994). Most likely, the
proximity of two recruitment sites after IFN-
R1 dimerization
facilitates Stat1
dimerization and dissociation of the dimer from
the receptor complex. Existence of two recruitment sites for Stat IL-4
in close proximity on one receptor chain of the IL-4 receptor complex
substitutes for the necessity to bring two separate chains, such as
IFN-
R1, together to form a STAT dimer (Hou et al., 1994).
The Stat1
dimer formed in response to IFN-
then translocates
to the nucleus and interacts with the GAS element in the promoter
regions of IFN-
-inducible genes, the process that begins the
induction of this family of genes.
Figure 8:
Model of the IFN- receptor complex
and signal transduction. In the three-dimensional structure it is
likely that the extracellular domains of both IFN-
R1 and
IFN-
R2 chains contact the ligand,
IFN-
.