(Received for publication, March 26, 1995; and in revised form, May 22, 1995)
From the
Constituents of the Type 1 interferon (IFN) receptor (IFNABR)
identified to date include the and
transmembrane subunits
and the associated intracellular kinases, Jak 1 and Tyk 2. In this
report, we demonstrate that a human cell type that expresses both
subunits of IFNABR, together with Jak 1 and Tyk 2, exhibits a limited
binding capacity for and is only partially sensitive to the effects of
IFN-
/
, despite adequate levels of the cytoplasmic
transcription factors Stat1, Stat2, and Stat3. Specifically, a low
affinity interaction between IFN-
/
and cell surface receptors
results in ISGF3 (Stat1:2) activation and an antiviral response, yet no
IFN-inducible growth inhibition. Using a panel of murine cells that are
variably configured with respect to the human IFNABR-
/
subunits, we provide evidence that an additional component(s) encoded
on human chromosome 21 is required to confer high affinity binding and
IFN-inducible growth inhibition to cells that express the
and
subunits of the IFNABR. The data indicate that transcriptional
activation that leads to an antiviral response is mediated by
IFN-
/
activation of IFNABR-
and IFNABR-
in the
context of a low affinity interaction, yet a high affinity interaction
is necessary for signal transducing events that mediate growth
inhibition. We provide evidence that the extent of ISGF3 activation
correlates directly with the magnitude of an antiviral but not a growth
inhibitory response.
Type 1 interferons (IFN), ()comprised of
and
subtypes, are a family of biologically active related proteins
that exhibit target cell specificity(1, 2) . This
specificity is mediated by a high affinity interaction between the IFN
and its cell surface receptor(3, 4, 5) .
Accumulating evidence indicates that the Type 1 IFN receptor is
multicomponent. A cDNA coding for a human IFN-
receptor peptide
(IFNABR-
) was reported to code for a functional human IFN-
receptor when transfected into murine cells(6) . However,
sensitivity to the biological effects of human IFN was restricted to a
specific IFN-
species, IFN-
8. More recently, a distinct cell
surface receptor protein has been identified(7) . This
receptor, IFNABR-
, may represent the primary ligand recognition
chain of the IFN receptor complex. Studies from our laboratory
demonstrate that specific species of membrane glycosphingolipids
containing a terminal Gal
1-4Gal associate with IFNABR-
to create a functional receptor(8) .
The initial interaction
between a Type 1 IFN and its specific cell surface receptor apparently
leads to ligand-induced tyrosine phosphorylation of IFNABR- (9) and a rapid phosphorylation activation, in the absence of
protein synthesis, of a latent cytosolic transcription factor, ISGF3
(reviewed in (10) ). Complementation of IFN-resistant mutant
cell lines with two members of the Janus family of nonreceptor protein
tyrosine kinases (Jak), Tyk 2 (11, 12) and Jak 1 (13) , that are required for IFN-induced signal transduction
further defines a potential role for receptor-associated
phosphorylation events in the signaling cascade initiated by ligand
binding. Indeed, the data imply that Jak 1 associates with IFNABR-
(7) and Tyk2 with
IFNABR-
(9, 11, 12, 14) .
Current models for IFN-induced signaling invoke kinase-mediated
phosphorylation of the STAT proteins Stat1, Stat2, and
Stat3(15, 16, 17, 18, 19) .
These phosphorylated STATs dimerize via SH2-phosphotyrosyl interactions (20, 21) and translocate to the nucleus where they
bind to specific promoter sequences, thereby regulating gene
expression. Homo- and heterodimers of Stat1 and Stat3 bind the
palindromic IFN response element, pIRE,(22, 23) . A
heterodimer of Stat1 and Stat2 associates with a DNA-binding adapter
protein, p48, to form the Stat complex designated
ISGF3(24, 25) . ISGF3 transcriptionally activates a
subset of genes that contain an IFN-stimulated response element
(ISRE)(26) .
The existence of multiple, distinct signaling
pathways that effect different biological outcomes in response to a
single IFN suggests that component configuration is critical within a
multimeric transmembrane receptor complex. Members of the hematopoietic
growth factor family of cytokines, which include interferon-,
interleukins, erythropoietin, growth hormone, granulocyte
colony-stimulating factor, and granulocyte-macrophage colony
stimulating factor, mediate their pleiotrophic effects through
interactions with multicomponent receptors (27, 28, 29, 30, 31, 32, 33, 34, 35) .
Generally, specific ligands exhibit low affinity binding to individual
receptor components, yet high affinity binding occurs when the intact
receptor complex is assembled. For certain cytokine receptors, such as
the interleukin-2 receptor complex, an intermediate affinity
interaction between the ligand and a single receptor component, the
interleukin-2
chain, may lead to a limited signal
transduction(36) . Thus, there may be some redundancy
associated with multimeric receptor complexes that allows for partial
responses when specific receptor components are not expressed at the
cell surface.
In agreement with affinity studies with I-IFN-
(37, 38, 39, 40, 41, 42, 43, 44) ,
here we report that IFNABR-
by itself is not sufficient for the
appropriate high affinity binding and complete biological responses
effected by IFN-
and -
. We provide evidence that a low
affinity interaction between IFN-
/
and the two receptor
chains IFNABR-
and IFNABR-
is associated with ISGF3
activation and antiviral responses. Cell surface expression of human
chromosome 21-encoded factors, which include IFNABR-
and
IFNABR-
forms, permits a high affinity interaction between the IFN
and the receptor complex, such that target cells exhibit both antiviral
and growth inhibitory activities.
MRC-5 DNA from the second PCR reaction was size-fractionated on a low melting point agarose gel, and the 992-bp band was extracted and ligated into the vector PCR II. The entire reverse transcription PCR from beginning to end was performed three individual times. Single strand sequencing was performed according to the Sequenase Version 2.0 DNA Sequencing kit. Sequences were checked against results obtained from automated DNA sequencing (Biotechnology Service Centre, University of Toronto).
Figure 1:
Antiviral and growth inhibitory
activities of IFN-Con and IFN-
in T98G and MRC-5
cells. A and B, antiviral activities. Cells in
microtiter wells were treated with the appropriate IFN dose for 24 h
and then challenged with 5
10
plaque-forming units
of encephalomyocarditis virus. Viral cytopathic effect was quantitated
directly by spectrophotometric determination. A, T98G; B, MRC-5. C and D, growth inhibitory
activities. Cells in microtiter wells were treated with the appropriate
IFN dose and then growth assessed after 96 h by spectrophotometric
determination. C, T98G; D, MRC-5. Values are the
means of triplicate determinations, and errorbars denote the S.D. from the means.
, IFN-Con
;
, IFN-
.
Figure 2:
Analysis of IFNABR- and IFNABR-
expression on MRC-5 cells. A-C,
I-IFN-Con
binding to receptors on MRC-5 and
T98G cells. IFN-Con
, as a consensus IFN-
, with a
specific activity of 3
10
units/mg protein, was
used. The steady-state receptor binding characteristics were
determined. 2.4
10
cells/ml were incubated with the
indicated concentrations of
I-IFN-Con
for 2 h
at 4 °C. The values shown were obtained by subtracting nonspecific
cpm bound from total cpm bound. Nonspecific binding was determined in
the presence of a 100-fold excess of unlabeled IFN. The points
represent the mean values of triplicate cultures and exhibit a S.D. of
±3% for the MRC-5 (A) data and ±6% for the T98G (B) data. Scatchard analyses of the binding data are indicated (C).
, MRC-5;
, T98G. D and E,
IFNABR-
expression in MRC-5 cells. Northern analysis of 10 µg
of RNA with a 992-bp IFNABR-
cDNA probe revealed two transcripts
of 1.55 and 4.5 kilobase pairs (D). 4 µg of whole cell
extracts from untreated MRC-5 and T98G cells were separated by gel
electrophoresis, transferred to filters, and then immunoblotted with a
mAb to IFNABR-
, and immunoreactive bands were visualized by the
ECL Western blotting system (E). F, flow cytometric
analysis of IFNABR-
mAb binding to native IFNABR-
on MRC-5
and T98G cells. 0.5
10
cells were incubated with
fluorescence-activated cell sorter buffer (negative control) or
IFNABR-
mAb for 45 min on ice, washed, and incubated with
biotin-SP-conjugated F(ab`)
rat anti-mouse IgG for an
additional 30-45 min. The cells were washed and then incubated
with R-phycoerythrin-conjugated streptavidin for an
additional 30 min. Immunofluorescence was analyzed with a Becton
Dickinson FACScan. Incubation with either medium alone or secondary and
tertiary reagents alone resulted in superimposable negative cytograms,
represented as open profiles; positive cytograms
(+IFNABR-
mAb) are represented as filled cytofluorograph
profiles. Mean fluorescent intensities: MRC-5,
47; T98G,
21.
Total RNA from MRC-5
cells, when reverse transcribed, contained cDNA that could be amplified
by PCR using IFNABR--specific primers(7) . Analysis of the
PCR products revealed an expected 397-bp band (data not shown). To
further examine IFNABR-
expressed in MRC-5 cells, a 992-bp
fragment was subcloned into the vector PCR II and sequenced. Our
results indicate that an IFNABR-
form expressed in MRC-5 cells is
encoded by a gene whose sequence is essentially identical to the
previously reported sequence, with one codon difference that would
result in a conservative change to the amino acid residue at position
216: Asp to Asn. In Northern blots, however, two transcripts of 1.55
and 4.5 kilobase pairs were revealed that may represent differentially
spliced products of the same gene (Fig. 2D). Whole cell
extracts from MRC-5 and T98G cells exhibit comparable levels of
IFNABR-
when Western blots are probed with a polyclonal Ab to
IFNABR-
(Fig. 2E). Flow cytometric analysis of
IFNABR-
mAb (Fig. 2F) binding to native
IFNABR-
on MRC-5 and T98G cells identifies similar levels of
IFNABR-
cell surface expression on these cell types. When viewed
together, the data suggest that IFNABR-
and IFNABR-
expression are not limiting factors that affect the binding capacity of
MRC-5 cells.
There is emerging evidence to suggest that the Jaks,
Tyk 2 and Jak 1, constitutively associate with the intracellular
regions of IFNABR- and IFNABR-
, respectively, and that this
association creates a productive receptor, both in terms of ligand
recognition and signal transduction. IFN-
/
-induced
receptor-mediated ISGF3 activation requires both Tyk 2 and Jak 1.
Because there is good evidence to suggest that transcriptional
activation that precedes IFN-induced responses is mediated by ISGF3
activation, we examined the extent of IFN-induced ISGF3 activation in
both T98G and MRC-5 cells. The results in Fig. 3A demonstrate that IFN-Con
/IFN-
treatment of both
T98G and MRC-5 cells resulted in specific activation of ISGF3. Antisera
to Jak 1 and Tyk 2, as well as monoclonal antibodies raised against
Stat1, Stat2 and Stat3, detected these factors in extracts from
unstimulated T98G and MRC-5 cells (Fig. 3B), indicating
that these are not limiting in the MRC-5 cells.
Figure 3:
STAT and Jak levels in MRC-5 cells. MRC-5
cells were either left untreated or treated with 10 ng/ml of
IFN-Con (con
) or IFN-
(
). T98G cells were similarly either left untreated or
treated with 1 ng/ml of IFN-Con
or IFN-
. A, 5
µg of whole cell extracts prepared as described under
``Experimental Procedures'' were reacted with 30,000 cpm of a
P-end-labeled ISRE, representing nucleotides -107 to
-87 of the human 2-5A synthetase gene, which contains a
functional ISRE. Complexes were resolved by using native gel
electrophoresis and visualized by autoradiography. Mobility of ISGF3 is
indicated. Specific complexes were identified by the addition of
100-fold excess of unlabeled ISRE (ISRE) or mutant ISRE (mut ISRE) to the reaction. FP, free probe. B, 4 µg of whole cell extracts from untreated MRC-5 and
T98G cells were separated by gel electrophoresis, transferred to
filters, and immunoblotted with monoclonal Abs to Stat1, Stat2, Stat3,
Jak 1, and Tyk 2. Immunoreactive bands were visualized by the ECL
Western blotting system. Stat1 exists as two isoforms, Stat2 exists as
a single isoform, and Stat3 exists as three isoforms. MRC-5 cells
constitutively express one isoform of Stat3, whereas T98G cells express
two isoforms of Stat3.
To further examine
the specific interaction between Type 1 IFN and IFNABR-, we
investigated the biologic effects of a number of different human Type 1
IFN in murine A9 cells that were stably transfected with the human
IFNABR-
. Northern blots confirmed that human IFNABR-
mRNA is
expressed in the transfectants (data not shown), and flow cytometric
analysis of IFNABR-
mAb binding to human IFNABR-
on A9-
transfectants containing plasmid only and the A9+ transfectants
containing IFNABR-
identified IFNABR-
cell surface expression
on the A9+ transfectants alone (Fig. 4). The results in Fig. 5demonstrate that murine A9+ cells exhibiting cell
surface expression of the human IFNABR-
are restricted in their
antiviral responsiveness to selected human Type 1 IFN. Whereas the
parental A9 cells are relatively insensitive to the antiviral effects
of human IFN-
2b (ID
=
2.5 µg/ml/5
10
units/ml), IFN-Con
(ID
=
50 ng/ml/1.5
10
units/ml), and
IFN-
(ID
=
100 ng/ml/2
10
units/ml) compared with murine IFN-
(60 ng/ml/90U/ml), when
challenged with the same infecting dose of encephalomyocarditis virus (Fig. 5A), the A9+ cells exhibit a 200-fold
increase in sensitivity to the antiviral effects of IFN-Con
(ID
=
0.5 ng/ml/1.5
10
units/ml) (Fig. 5B). The A9+ transfectants
remain relatively unresponsive to the antiviral effects of human
IFN-
2b and IFN-
, with dose-response curves similar to those
depicted in Fig. 5A. Examination of the sensitivity of
the transfectant A9+ cells to the growth inhibitory effects of
IFN-
2b, IFN-Con
, and IFN-
revealed that in
contrast to the partial antiviral responsiveness, the A9+ cells
remained refractory to the antiproliferative effects of each of these
IFN (Fig. 6). Gel retardation assays to determine IFN-induced
ISGF3 activation in the transfectant versus the A9-
cells showed that there was a direct correlation between IFN-induced
ISGF3 activation in the transfectants and IFN-induced antiviral
activity; whereas both IFN-
2b and IFN-
failed to induce ISGF3
activation in the A9+ cells and failed to elicit an antiviral
response in these cells, IFN-Con
induced ISGF3 activation
and an antiviral response in the A9+ transfectants (Fig. 7).
Figure 4:
IFNABR- cell surface expression on
A9-, A9+, and A9+21 cells. Flow cytometric analysis of
IFNABR-
mAb binding to native IFNABR-
on A9- transfectants
containing plasmid only, A9+ transfectants expressing IFNABR-
mRNA, and A9+21 cells that contain 1-5 copies of human
chromosome 21 on the murine A9 background. For details refer to Fig. 2. Incubation with either medium alone or secondary and
tertiary reagents alone resulted in superimposable, negative cytograms,
which are represented as open cytofluorograph profiles;
positive cytograms (+IFNABR-
mAb), represented as filled
cytofluorograph profiles, gave mean fluorescent intensities of 148
for A9+ and 139 for A9+21 cells.
Figure 5:
Antiviral activities of human IFN-s
and IFN-
in parental A9 cells, A9 cells transfected with human
IFNABR-
, and A9 cells containing 1-5 copies of human
chromosome 21. For experimental details refer to Fig. 1. Values
are the means of triplicate determinations, and error bars denote the S.D. from the means. A, approximate number of
parental A9/A9 cells transfected with plasmid alone; B,
approximate number of A9 cells stably transfected with IFNABR-
(A9+); C, approximate number of A9 cells containing
copies of human chromosome 21 (A9+21).
,
IFN-Con
;
, IFN-
;
, IFN-
2b;
,
murine IFN-
Figure 6:
Antiproliferative effects of IFN-s
and IFN-
in A9-, A9+ (A), and A9+21 (B) cells. 1.5
10
cells/ml were incubated
with the indicated doses of IFN for 96 h at 37 °C. IFN-induced
growth inhibition was recorded relative to the growth of untreated
cultures, as indicated. Values represent the means of triplicate
cultures and exhibited a S.E. of ± 4%.
,
IFN-Con
;
, IFN-
;
,
IFN-
2b.
Figure 7:
IFN-induced ISGF3 activation in A9-,
A9+, and A9+21 cells. Cells were either left untreated or
treated with 30 ng/ml (50 units/ml) of murine IFN-, 1 ng/ml (200
units/ml) of IFN-
2b, or 1 ng/ml (3000 units/ml) of
IFN-Con
. For details refer to Fig. 3and
``Experimental Procedures.'' Mobility of ISGF3 is indicated. FP, free probe.
In an attempt to restore complete sensitivity to
the full range of biologic responses inducible by Type 1 IFN, we
examined the influence of chromosome 21-encoded factors, in addition to
IFNABR- and IFNABR-
expression, on biological outcome. We
examined the biological effects of different human Type 1 IFN, both
IFN-
s and IFN-
, in murine A9 cells that contain 1-5
copies of intact human chromosome 21, designated A9+21. At the
outset we determined that the levels of cell surface expression for
IFNABR-
were comparable in A9+ and A9+21 cells (Fig. 4). These data confirmed that the extent of cell surface
expression for IFNABR-
was not a limiting factor in the A9+
transfectants. Next, we examined cells for IFNABR-
expression, and
our results demonstrate that in contrast to the
IFNABR-
-containing A9+ cells, A9+21 cells express
IFNABR-
RNA (Fig. 8).
Figure 8:
IFNABR- expression in A9+21
cells. A, using a PCR-based protocol, 2 ng of poly(A)+
RNA extracted from A9+ and A9+21 cells were reverse
transcribed, and the resultant cDNA probed for IFNABR-
using
specific primers(7) . Analysis of the PCR products revealed an
expected 397-bp band in the A9+21 and not the A9+ lane. B, Northern analysis of IFNABR expression in A9-,
A9+, and A9+21 cells. 10 µg of total RNA were separated
by gel electrophoresis and transferred to filters that were probed for
IFNABR-
by Northern hybridization. IFNABR-
cDNA was a 992-bp
insert in plasmid PCR II. The different lanes were probed for
-actin (2-kilobase pair insert in pUC18) to assess loading of
RNA.
In subsequent studies we
demonstrated that those additional factors encoded on chromosome 21
that are expressed in the A9+21 cells are sufficient to enhance
the antiviral effectiveness of IFN-Con by 150-fold over
that detected in the A9+ cells and to restore sensitivity to the
antiviral effects of IFN-
2b and IFN-
(Fig. 5).
Moreover, the A9+21 cells were fully responsive to the growth
inhibitory effects of the different human IFN-
s and IFN-
(Fig. 6). Whereas the growth inhibitory effects of murine
IFN-
remained unchanged in the parental A9 and A9+ cells
(ID
=
10
units/ml) due to the
rapid doubling time of the A9+21 cells (
12 h),
10
units/ml murine IFN-
are required to achieve an ID
in these cells (data not shown). Similarly, the effectiveness of
the human IFN in the A9+21 cells is apparently reduced by
approximately 1 order of magnitude compared with T98G cells. Of note,
our data indicate a correlation again between antiviral activity and
ISGF3 activation, because we were able to restore ISGF3 activation in
response to IFN-
2b and IFN-
in the A9+21 cells (Fig. 7).
Figure 9:
Competitive displacement of I-IFN-Con
from A9+21 cell surface
receptors. 1.2
10
cells were incubated at 4 °C
for 2 h with 10 ng/ml of
I-IFN-Con
containing
no unlabeled competitor (100% bound) or the indicated concentrations of
different IFN. The values shown were obtained by subtracting
nonspecific cpm from total cpm bound. Nonspecific binding was
determined in the presence of a 100-fold excess of unlabeled IFN. The
points represent the means of triplicate cultures, and error bars denote the S.D. from the means.
, IFN-Con
;
, IFN-
;
, IFN-
2b.
Our data
would suggest that different Type 1 IFN, both and
, exhibit
poor affinity for IFNABR-
that is not associated with an intact,
functionally competent receptor complex; A9+ murine cells that
lack human IFNABR-
do not bind these IFN with high affinity.
Accordingly, we examined the ability of different IFN to bind to a
fusion protein that comprised the extracellular portion of IFNABR-
linked to the Fc region of IgG1. The conformational integrity of the
IFNABR-
/Fc fusion protein is suggested by the ability of 2
distinct mAbs, which recognize native cell surface IFNABR-
in flow
cytometry (51) , to immunoprecipitate the fusion protein (data
not shown). Differential recognition of the IFNABR-
fusion protein
by these antibodies, together with the ability of the fusion protein to
associate with a specific glycosphingolipid species normally associated
with cell surface IFNABR-
(8) , suggests that this fusion
protein is conformationally similar to native cell surface
IFNABR-
. Using
I-IFN-Con
we were unable
to identify specific, competable binding to the fusion protein to any
significant extent, nor were we able to demonstrate that IFN-
2b or
IFN-
exhibited any appreciable affinity for this fusion protein
(data not shown). Our approach was to incubate the fusion protein for 2
h with varying doses of
I-IFN-Con
at 4
°C, as per our standard binding reaction, in the presence or the
absence of a 100-fold excess of unlabeled IFN-Con
or
varying doses of competitor IFN, IFN-
2b, or IFN-
. The amount
of
I-IFN-Con
specifically bound to the fusion
protein was then determined by either (i) conjugating the fusion
protein to protein A-Sepharose and measuring bound cpm in the pellet
fraction separated by centrifugation or by (ii) covalently
cross-linking bound
I-IFN to the fusion protein with
disuccinimidyl suberimidate, affinity purifying the fusion protein on
protein A-Sepharose, and then visualizing the salt-eluted fraction by
SDS-polyacrylamide gel electrophoresis. Failure to detect
I-IFN-Con
bound to the fusion protein by
either method supports our findings that IFNABR-
alone does not constitute a binding receptor. Apparently contradictory
reports that suggest that IFNABR-
is a binding receptor (52, 53) did not evaluate the binding capacity of the
receptor chain alone.
Human IFNABR- stably expressed in murine cells
apparently confers sensitivity only to select human IFN-
s,
IFN-
8(6, 44, 54, 55) , and
IFN-Con
(our data, 9, 55). In contrast, a monoclonal
antibody to human IFNABR-
inhibits the biologic activity of
several species of human IFN-
, IFN-
, and IFN-
in human
cells(56) , suggesting a structural heterogeneity of the
IFNABR-
that would allow for differential affinities amongst the
different Type 1 IFN, dependent on the presentation/accessibility of
IFNABR-
on the cell type in question. The implications are that
additional components influence the interaction between different Type
1 IFN and IFNABR-
. Studies from this laboratory have identified a
requirement for glycosphingolipid modification of
IFNABR-
(8) . The relation of IFNABR-
to IFNABR-
is unclear, yet accumulating evidence indicates that co-expression is
essential for IFN-induced activation of Tyk 2 and Jak 1.
There is
considerable sequence identity between the murine and human
IFNABR- peptides (and bovine IFNABR-
) and between the murine
and human Type 1 IFN, yet species specificity is determined at the
level of receptor recognition. An earlier report from this laboratory
suggested that within the Type 1 IFN molecule are two epitopes
associated with receptor recognition: one that is conserved among the
different species of IFN-
s and IFN-
s and the other that may
be variable and associated with species specificity and separation of
IFN-
/
properties(57) . It is likely that the
different Type 1 IFN share a similar structural conformation and that
the IFNABR-
peptide, when optimally configured at the cell surface
in association with IFNABR-
, is able to accommodate for the
minimal structural variations. The preceding data would suggest that
the origin of the cell type dictates the accessibility of IFNABR-
to the different Type 1 IFN. Interestingly, the IFNABR-
mAb that
we employed for flow cytometry is able to distinguish human
IFNABR-
, regardless of the host cell, implying that the epitope(s)
to which it is directed on the extracellular portion of IFNABR-
is
unaffected by cell type yet is specific for human IFNABR-
alone.
We infer that the IFN recognition epitope(s) on IFNABR-
are
influenced by cell factors.
IFNABR- expressed on human MRC-5
cells that also express IFNABR-
is able to interact with
IFN-
/
to invoke ISGF3 activation and an antiviral response,
yet the limited binding capacity of MRC-5 cells and the low affinity
ligand-receptor interaction apparently precludes their sensitivity to
the growth inhibitory effects of the same IFN. The implications are
that MRC-5 cells express functionally deficient Type 1 IFN receptors.
We observed that a 10-fold increase in IFN-
/
dose is required
in MRC-5 cells to achieve a similar level of ISGF3 activation to that
obtained in T98G cells and that there is a direct correlation between
the extent of IFN-induced ISGF3 activation and the magnitude of an
antiviral response. Using monoclonal antibodies directed against
individual Jaks and STATs, we have shown that cytoplasmic extracts from
MRC-5 cells express Jak 1, Tyk 2, Stat1, Stat2, and Stat3, thus we
infer that none of these factors are limiting in the MRC-5 cells.
Apparently, the receptor configuration in the MRC-5 cells precludes the
appropriate IFN-induced activation of at least Stat1
and Stat2.
Furthermore, our data indicate that ISGF3 activation mediated by
IFN-
/
-induced activation of IFNABR-
and IFNABR-
does not necessarily invoke an antiproliferative response. When viewed
solely in the context of the affinity characteristics of the
ligand-receptor interaction, the implications are that the threshold of
sensitivity to the antiviral effects of IFN is lower than that required
to induce a growth inhibitory response. We have demonstrated that MRC-5
cells express comparable levels of cell surface IFNABR-
and
IFNABR-
with Daudi (8) and T98G cells that are responsive
to the growth inhibitory effects of Type 1 IFN. These observations
suggest that accessory factors directly influence the binding capacity
of cells for IFN
and
, perhaps through an interaction with
IFNABR-
and/or IFNABR-
, thereby affecting signal transduction
events that mediate an antiproliferative response. In a similar manner
to the Jaks, the accessory component may contribute to the overall
affinity of the IFN receptor complex through an association with an
intracellular domain in either IFNABR-
and/or IFNABR-
. The
implications are that this accessory component mediates signal
transduction that leads to a growth inhibitory response.
Our results
with murine cells transfected with the human IFNABR- indicate that
the subtle structural variations among the different human IFN-
s
and IFN-
cannot be accommodated readily when human IFNABR-
is
expressed on murine cells and, indeed, receptor recognition is
dramatically restricted. Not sursprisingly, only the low affinity
signaling pathway associated with an antiviral response is effected in
the murine transfectants. Human IFNABR-
, perhaps associated with
murine specific components, will accommodate IFN-Con
. This
may be a consequence of the inherent higher specific affinity of
IFN-Con
for the Type 1 IFN receptor, per se, that
allows recognition of IFNABR-
on the murine background. By
contrast, IFN-
8, which has been shown to be active on murine cells
transfected with human
IFNABR-
(6, 52, 55) , is distinct from
the other human IFN-
species in that region associated with
species-specific receptor recognition (5, 57) and may
indeed resemble a murine IFN in this region of the molecule. Full
biological sensitivity was restored to IFNABR-
by complementation
with chromosome 21-encoded factors that include IFNABR-
. From our
experimental results we infer that distinct signaling pathways invoke
antiviral and growth inhibitory responses.
The challenge is to
elaborate those regions on IFNABR- and IFNABR-
that interact
with the ligand IFN, that are possibly involved in receptor-receptor
interactions, and that associate with accessory factors (e.g. Jaks) that are necessary for full ligand-receptor activation. Our
data indicate that co-expression of IFNABR-
and IFNABR-
is
not sufficient to invoke a complete biological response to
IFN-
/
. Apparently, ISGF3 activation that leads to an
antiviral response is mediated by IFN-
/
activation of
IFNABR-
and IFNABR-
in the context of a low affinity
interaction, yet a high affinity interaction is necessary for signal
transducing events that mediate a growth inhibitory response. Moreover,
we confirm that IFNABR-
alone is not a binding receptor chain. The
present study provides evidence that a fully productive Type 1 IFN
transmembrane receptor complex is comprised of at least one other
accessory factor encoded on chromosome 21 in addition to IFNABR-
,
IFNABR-
, Tyk 2, and Jak 1.