(Received for publication, June 21, 1995; and in revised form, July 14, 1995)
From the
The interferon receptor (IFN
R) or type I IFN-R
is formed by a 110-kDa
subunit or IFNAR and by a
subunit,
which has short and long forms (molecular masses of 55 and 95-100
kDa, respectively). In this report, we demonstrate that the
IFN
/
R cDNA recently cloned corresponds to the 55-kDa or short
form of the
subunit, while the 95-100-kDa species reported
here corresponds to a longer form of the IFN
/
R cDNA that is
probably produced by alternative splicing of the same gene. Stable
transfection of the
subunit with either form of the
subunit
results in the expression of low and high affinity receptors, while
expression of either form of the
subunit alone only produces low
affinity receptors. More important, only expression of the
and
long form of the human
subunits in mouse L-929 cells
reconstitutes the activation of the Jak kinases and the Stat factors,
as well as the antiviral response to human type I IFNs.
Characterization of the interferon receptor or type I
interferon receptor (IFN
R or IFN-R) (
)with monoclonal
antibodies (mAb) has revealed that this receptor is composed of at
least two chains: the
and
subunits (recognized by the
IFNaR3 and IFNaR
1 monoclonal antibodies,
respectively)(1, 2) . Using mAbs and affinity
cross-linking methods, we described two forms of the type I IFN-R:
normal and variant(2, 3) . The ``normal''
receptor is expressed in most cells and is composed of
and
subunits with molecular masses of 110 and 100 kDa, respectively. The
``variant'' receptor is expressed in monocytic cell lines and
normal bone marrow cells, and in contrast to the ``normal''
receptor, its
subunit has a molecular mass of 55
kDa(2, 3) . Two cDNAs encoding IFNAR (4) and
IFN
R (5) subunits of the type I IFN-R have been
cloned. We have recently demonstrated that the
subunit is encoded
by the IFNAR cDNA(6) , and its cytoplasmic domain directly
interacts with Tyk-2 tyrosine kinase.
The IFNR cDNA
product (5) has a similar M
as the variant
form of the
subunit (2) and is proposed to form a
disulfide-bonded dimer that it is only cleaved by high concentrations
of reducing agents(5) . The finding that the IFN
/
R
cDNA was cloned from a monocytic cDNA library led us to postulate that
a longer form of this protein may be expressed in other cell types. In
this report, we demonstrate that the IFN
/
R cDNA encodes what
was previously designated as the variant or short form of the
subunit (
)(2) . A long form of the
subunit (
) was cloned from a U-266 cDNA library. The
subunit and the IFN
/
R protein
(
) have identical extracellular and transmembrane
domains but only share identitity in the first 15 amino acids of the
cytoplasmic domain. Expression of the
subunit in
mouse L-929 cells resulted in a protein with a molecular mass of 100
kDa, while L-929 cells transfected with the
subunit
expressed a protein with a molecular mass of 55 kDa. L-929 cells
coexpressing the
and
subunits displayed the
affinity cross-linking pattern described for normal receptors, while
coexpression of the
and
subunits resulted in
the expression of variant receptors. Interestingly, only cells
transfected with the long form of the
subunit were able to
tyrosine phosphorylate the Tyk-2 and Jak-1 kinases, activate the ISGF3
and FcR
F1/2 transcription factors, and induce an antiviral state
in response to human type I IFNs.
To search for a putative long form of the
subunit, we screened a human myeloma U-266 cDNA library using the
IFN
R cDNA as the probe. We initially screened 200,000
colonies from which six positive clones were obtained. Restriction
endonuclease mapping revealed that some inserts contained a BamHI site not present in the IFN
R cDNA. Thus, a BamHI-XbaI fragment (the XbaI restriction
site is contained in the polylinker of the pcDNA II vector) was used
for a second round of screening where 13 additional clones were
obtained. Sequencing of the longest clone (4A1, 2636 bp) revealed an
open reading frame of 515 amino acids (nucleotides 67-1611), a
polyadenylation signal (nucleotides 2295-2299), and a poly(A)
tail (nucleotide 2628) (Fig. 1A). Alignment of the
predicted amino acid sequence from clone 4A1 with the IFN
R
protein revealed identical amino acid sequences from residues
1-280, resulting in identical extracellular and transmembrane
domains but sharing identity in only the first 15 amino acids of the
intracellular region (amino acids 265-280). Thus, the open
reading frame encoded by clone 4A1 predicts a cytoplasmic domain of 251
amino acids (residues 265-515), while the form of the
subunit encoded by the IFN
R cDNA has a cytoplasmic region of
67 amino acids (265-331). For simplicity and to follow the
designation with Greek letters used for other cytokine receptors, we
will refer to the IFN
R and 4A1 cDNAs as short
(
) and long (
) forms of the
subunit, respectively. Northern blot analysis (Fig. 1C)
with a probe corresponding to the cytoplasmic domain of
detected mRNAs of 1.85 and 4.4 kilobases similar to what it was
previously reported for
(Fig. 1C and (5) ). Partial sequencing of another clone (clone 1A1)
demonstrated that this cDNA had the same coding region as the
form previously reported(5) ; however, 1A1
was preceded by a slightly longer 5`-untranslated region. The majority
of the clones sequenced were similar to the 4A1 clone, suggesting that
-coding transcripts may be only a fraction of the
receptors transcribed in U-266 cells. The differences in the size
of the 4A1 clone (2636 base pairs) and the mRNA observed in Northern
blots (4.4 kilobases) suggest that the coding region may be preceded by
a longer 5`-untranslated region than that present in the 4A1 clone. Fig. 1C also shows that a probe encoding the
cytoplasmic region of clone 4A1 recognizes the mouse homolog (Fig. 1C, 3T3T cells). This is not surprising, since
the human
has the ability to interact with the mouse
signaling proteins (see below). It is likely that the different forms
of the
subunit correspond to alternatively spliced transcripts
from the same gene. Searching of the data bases using the BLAST
computer program did not show significant similarities between the
distinct region of the cytoplasmic domain of
and any
known proteins. However, multiple alignment with other members of the
cytokine receptor superfamily, using the computer program
MACAW(13) , revealed that
but not
contains a cytoplasmic sequence proximal to the transmembrane
domain that resembles the BOX 1 motif observed in most cytokine
receptors (Fig. 1A, boxedsequence).
We also found six regions with high content of acidic residues whose
significance is under investigation (Fig. 1A, shadedsequences).
Figure 1:
Sequence of the long form of the
subunit of the type I IFN-R. A, the nucleotide (top)
and amino acid (bottom, three-letter code) sequences for the
long form of the
subunit are shown. The polyadenylation signal (doubleunderlined), the point where the
and
sequences are similar (between arrows), the transmembrane region (thickunderline), the box 1 motif (boxed), and the
acidic domains (shadedareas) are indicated. B, schematic representation of the IFN
R (homologous
to clone 1A1) and 4A1 clones. The identical (thicksolidline) and different parts of the 5`- and 3`-untranslated
regions (UTR) of
(thinsolidline) and
(dashedline) as well as the differences in the cytoplasmic
domain are shown. C, Northern blot analysis using probes
encoding the specific cytoplasmic regions of clone 4A1 and
IFN
R cDNA. Total RNA (20 µg) was used for Northern blot
analysis. kb,
kilobases.
Figure 2:
Expression of the different forms of the
subunit of the type I IFN-R. A, mouse L-929
transfectants were produced as described under ``Experimental
Procedures.'' LpZ
cells correspond to L-929 cells
transfected with the
subunit.
LpZC
.4 and LpZR
.11
correspond to mouse L cells transfected with the
and
subunits, while LpZR
.10 cells were
transfected with the
and
subunits. Affinity
cross-linking was performed as previously described(9) . The
high molecular weight complexes observed in U-266 cells and in L-929
transfected with the
and either
or
subunits correspond to associations of these subunits. The human
subunit is specifically detected by the IFNaR3 mAb in cells
cotransfected with the human
and either
or
subunits. Cross-linking of
I-IFN
2
to
in cells transfected with
alone
always resulted in faint bands even after long exposures of the
autoradiograms (data not shown).
We next studied the contribution of the different subunits to the
formation of the low and high affinity receptors. Table 1shows
that transfection of either or
results in the expression of only low affinity IFN
R (K
of approximately 450 pM).
Cotransfection of either form of the
subunit with the
subunit results in low and high affinity receptors (K
values of 1.4-6.5 nM and 26-114 pM for low and high affinity receptors, respectively) similar to
those observed in U-266 cells(14) . As previously reported, the
sole expression of the
subunit did not produce detectable
IFN
binding(7) . Thus, either form of the
subunit
determines the low affinity binding, while the
subunit, unable to
bind ligand independently, joins together with the
subunit in the
formation of high affinity receptor complexes.
Figure 3:
Contribution of the different subunits of
the type I IFN-R to signaling. A, anti-phosphotyrosine
immunoblotting. Mouse L-929 cells transfected with the different
subunits of the type I IFN-R were treated for 15 min with human
IFN2, murine IFN
, or left untreated. Immunoblotting was
performed with the antiphosphotyrosine mAb 4G10 (UBI). Low levels of
basal Jak-1 kinase activation are observed in most transfectants. EMSA
with GRR (B) and ISGF3 (C) probes is shown. Whole
cell extracts were obtained from mouse L-929 transfectants treated with
6,000 units/ml of IFN
2 or murine IFN
or left untreated. D, EMSA was performed using GRR and ISRE probes (12) in the presence of a 50-fold excess of unlabeled GRR or
ISRE oligonucleotides. A dilution of the indicated anti-Stat sera was
used for supershifts. The positions of FcRF
1, FcRF
2, ISGF3,
and free GRR and ISRE probes are indicated.
We have recently
reported that the cytoplasmic domain of but not
interacts with the Jak-1 tyrosine kinase, as well as
the Stat1 and Stat2 transcription factors. (
)To determine
whether activation of the Jak kinases was accompanied by formation of
the FcR
F1/2 and ISGF3 complexes, whole cellular extracts were
prepared from the various L-929 transfectants treated with human
IFN
2, murine IFN
, or left untreated. The
IFN
-dependent activation of the Stat1 and Stat2 transcriptional
regulators was assessed by EMSA with probes encoding the GRR present in
the Fc
1 receptor gene (Fc receptor for IgG) (12, 16) and the
ISRE(17, 18, 19) . Fig. 3B shows that high levels of FcRF
1/
2 and ISGF3 complexes
are observed in mouse L-929 cells transfected with
alone or cotransfected with the
and
subunits (Fig. 3, B and C, lanes6, 12, and 15). No retardation of the
GRR or ISRE probes in response to human IFN
2 treatment was
observed in cells transfected with the
or
subunits alone or in combination (Fig. 3, B and C, lanes3 and 9, and data not
shown). However, these cells did show retardation of the GRR and ISRE
probes after treatment with murine IFN
(Fig. 3, B and C,lanes2 and 8).
FcRF
1/
2 and ISGF3 DNA binding activity were blocked by an
excess of unlabeled GRR and ISRE oligonucleotides (Fig. 3D, lanes6 and 12),
respectively. The FcRF
1/
2 complexes were supershifted by
anti-Stat1 sera, indicating the presence of Stat1 in these complexes.
Similarly, the ISGF3 complex was supershifted by the anti-Stat1 sera
and to a lesser extent by the anti-Stat2 sera (Fig. 3D, lanes4, 10, and 11). These results
demonstrate that the presence of
is required for the
formation of the FcRF
1/
2 and ISGF3 complexes and confirms our
previous observation indicating that only
docks the
Stat1 and Stat2 transcription factors.
Since the
corollary of the IFN response is the induction of an antiviral
state, we tested the ability of human type I IFNs to induce the
antiviral response in different transfectants. Table 2shows that
only the mouse L-929 cells expressing human
subunit
(alone or in combinantion with the
subunit) respond to the
antiviral effect of human IFN
2 and human IFN
. The lack of
response to the antiviral effects of human IFN
2 or human IFN
in L-929 cells transfected with either
or
subunit alone or cotransfected with the
and
subunits is not due to defects in the initial steps of the
signaling pathway, since both the activation of the Jak kinases and DNA
binding activity were detected in cells treated with murine
IFN
.
Our results clearly demonstrate that expression of
the subunit is required for type I IFN-induced
antiviral activity. However, activation of the
subunit must be
required as indicated by the finding that knockout mice lacking the
subunit fail to elicit antiviral responses(20) . Thus, we
are cautious in the interpretation of the results obtained in mouse
cells expressing only the human
subunit, since we
cannot rule out that after binding of human IFN
2 and IFN
to
the
subunit these IFNs may also interact and activate
the mouse
subunit. This is supported by the finding that in cells
transfected with either
or
alone, a
weak band with electrophoretic mobility similar to the
subunit,
but not recognized by the IFNaR3 mAb (specific for the human
subunit), is cross-linked to human IFN
2 (data not shown).
The
results presented here provide novel information concerning the
structure and function of the type I IFN-R. First, we demonstrate that
the product of the IFNR cDNA corresponds to the previously
defined
subunit of the type I IFN-R. Second, there are two forms
of the
subunit probably derived from an alternative splicing of
the same gene. Thus, the 100-kDa form of the
subunit is encoded
by its own mRNA, and it is not a result of the dimerization of the
IFN
R (
) protein as suggested by Novick et al.(5) . Both forms of the
subunit have
identical extracellular and transmembrane domains but share only the
first 15 amino acids of the cytoplasmic domains. The long form of the
subunit also contains the box 1 motif present in other cytokine
receptors (Fig. 1A) and has the ability to interact
with Jak-1, Stat1, and Stat2.
We propose to designate the
different forms of the
subunit according to their size as
(515 amino acids) and
(331 amino
acids). Third, expression of the different forms of the
subunit
alone or in association with the
subunit demonstrated that the
subunit is the binding subunit, while the
subunit is
necessary to form high affinity receptors. Fourth,
is
absolutely required for the IFN
response. However, it should
be noted that despite the fact that
alone can
activate the signaling pathway and elicit an antiviral state in mouse
cells, both the
and
subunits are probably
required for IFN
and IFN
signaling as indicated by knockout
experiments(20) .
Although expression of alone or in association with the
subunit is not sufficient
to induce an antiviral state in response to type I IFNs, this does not
rule out the possibility that
plays a role in type I
IFN signaling by forming a complex with the
and
subunits. In this scenario, it is possible that L-929 cells
cotransfected with the human
and
subunits may
use the mouse
chain in the formation of the receptor
complex. Coexpression of the human
,
, and
subunits in mouse L-929 cells and reconstitution of
the human type I IFN-R in knockout mice for the different receptor
subunits will help to address these questions.
The formation of the
receptor complex is likely to involve not only the receptor subunits
but also the Jak kinases and Stat factors. Several lines of evidence
support this concept. First, mutant cell lines that lack the expression
of the Tyk-2 (21, 22) and Jak-1 (23) kinases
showed impaired binding for Type I IFNs. Second, it has been
demonstrated that there is a direct interaction between the Tyk-2 and
Jak-1 kinases with the and
subunits,
respectively ( (5) and (6) and data not shown).
Finally, the
subunit constitutively docks the Stat1
and Stat2 transcription factors.
Mutational analysis of the
intracellular domains of the different receptor subunits will define
the domains required for this interactions and type I IFN signaling.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29584[GenBank].