(Received for publication, September 24, 1996, and in revised form, December 9, 1996)
From the Department of Molecular Genetics and Microbiology,
University of Medicine and Dentistry of New Jersey, Robert Wood
Johnson Medical School, Piscataway, New Jersey 08854-5635 and the
Department of Biochemistry, St. Jude Children's
Hospital, Memphis, Tennessee 38105
Binding of interferon gamma (IFN-) causes
oligomerization of the two interferon
receptor (IFN-
R) subunits,
receptor chain 1 (IFN-
R1, the ligand-binding chain) and the second
chain of the receptor (IFN-
R2), and causes activation of two Jak
kinases (Jak1 and Jak2). In contrast, the erythropoietin receptor
(EpoR) requires only one receptor chain and one Jak kinase (Jak2).
Chimeras between the EpoR and the IFN-
R1 and IFN-
R2 chains
demonstrate that the architecture of the EpoR and the IFN-
R
complexes differ significantly. Although IFN-
R1 alone cannot
initiate signal transduction, the chimera EpoR/
R1
(extracellular/intracellular) generates slight responses characteristic
of IFN-
in response to Epo and the EpoR/
R1·EpoR/
R2 heterodimer is a fully functional receptor complex. The results demonstrate that the configuration of the extracellular domains influences the architecture of the intracellular domains.
The interferon (IFN-
)1 receptor
complex consists of at least two receptor components, a ligand binding
chain and a signal transducing chain, each of which is a member of the
class II cytokine receptor family (1, 2). Isolation of the two chains
of the interferon
receptor (IFN-
R) has permitted an analysis of
the contributions of each to the signal transduction mechanism. The first chain of the receptor (IFN-
R1) binds ligand (3-9). The second
chain of the receptor (IFN-
R2) does not bind ligand by itself but is
required for signal transduction (3, 10-16). A large body of
experiments has elucidated the involvement of the Jak-Stat pathway in
signaling by various cytokines (for reviews, see Refs. 17-22). The
Janus kinases (or Jaks) are a family of receptor-associated soluble
tyrosine kinases with four known members, Tyk2, Jak1, Jak2 and Jak3.
Two of the kinases, Jak1 and Jak2, are required for signal transduction
by IFN-
. Further analyses of the interactions have shown that the
IFN-
R1 chain binds Jak1 (16, 23, 24) and the intracellular domain of
the IFN-
R2 chain brings Jak2 into the signal transduction complex
(16). Upon binding of the ligand, IFN-
, to the IFN-
R1 chain,
activation of Jak1 and/or Jak2 by reciprocal transphosphorylation
causes the phosphorylation of IFN-
R1 (16, 25). Stat1
, a latent
cytoplasmic transcription factor (26), binds to the phosphorylated
IFN-
R1, undergoes tyrosine phosphorylation (27), and forms
homodimers that translocate to the nucleus and initiate transcription
of IFN-
inducible genes (for reviews see Refs. 17 and 21).
As with other cytokine receptors, oligomerization upon ligand binding
is the first step in the signaling cascade of IFN-. IFN-
is a
non-covalent symmetrical homodimer (28) that binds to IFN-
R1 with a
stoichiometry of 1:2 (29, 30). It is known that a species-specific
interaction between the extracellular domains of the IFN-
R1 and
IFN-
R2 subunits is essential for signaling (10-12, 31-33). The
IFN-
R2 subunit does not by itself bind the ligand, but can be
cross-linked to IFN-
when both IFN-
R1 and IFN-
R2 chains are
present (16). Several lines of evidence (16, 34) suggest that the
IFN-
signaling complex contains two IFN-
R1 chains, two IFN-
R2
chains and one IFN-
homodimer.
The erythropoietin (Epo) receptor, EpoR, is a member of the class I
cytokine receptor subfamily. A single chain encodes both ligand-binding
and signal-transducing functions. Epo induces homodimerization of the
receptor to initiate signal transduction (for reviews, see Refs. 18,
19, and 35). Jak2 is associated with the cytoplasmic domain of the EpoR
and is activated upon ligand-induced dimerization of the receptor (36).
Strikingly an Arg Cys mutation in the extracellular domain of EpoR
results in ligand independent dimerization/oligomerization and
constitutive, ligand-independent activation of Jak2 and mitogenesis (37, 38).
In this study we used chimeric EpoR, IFN-R1, and IFN-
R2
constructs to investigate the differences between the architecture of
Epo and IFN-
receptor complexes and shed light on the requirement for one or two receptor-associated tyrosine kinases and the necessity for one or two distinct transmembrane chains for effective signal transduction.
Recombinant human erythropoietin was a gift from Dr.
Lawrence Blatt of Amgen. Restriction endonucleases were from Boehringer Mannheim and New England Biolabs; T4 DNA ligase was from U. S. Biochemical Corp.; [-32P]dCTP was from DuPont NEN. All
other reagents were of analytical grade and were purchased from
Sigma.
CHO-B7 cells represent the Chinese hamster ovary cell line (CHO-K1) containing a transfected human HLA-B7 gene (12). The 16-9 hamster × human somatic hybrid cell line is a CHO-K1 derivative containing a translocation of the long arm of human chromosome 6 and the human HLA-B7 gene (13). These cells were maintained in Ham's F-12 medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal bovine serum (Sigma). Transfections were carried out with the DOTAP transfection reagent (Boehringer Mannheim) according to the manufacturer's protocol and the transfected cells were maintained in F-12 medium containing 450 µg/ml Geneticin (antibiotic G418). Unless otherwise noted, experiments were performed with cloned cells expressing the various receptor subunits.
Construction of Chimeric ReceptorsThe EpoR expression
plasmid was made by cloning the EcoRI-AflIII
fragment of the human EpoR cDNA p18R (39) into the EcoRI and EcoRV sites of the eukaryotic expression vector
pcDNA3 (Invitrogen). The construction of plasmids expressing
Hu-IFN-R1 and Hu-IFN-
R2 chains from cDNA under the control of
cytomegalovirus promoter has been previously described (5, 12, 16, 40).
For ease of construction of the various chimeric receptors, the
polymerase chain reaction (PCR) was employed to incorporate a unique
NheI site at the 3
end of the extracellular domain (EC) and
at the 5
end of the transmembrane-intracellular domains (IC) of the receptors. The primers were designed to code for the three amino acids
Trp, Leu, and Ala, which are commonly found in the transmembrane domain
of several proteins, encompassing the NheI site. The
extracellular portions of EpoR, Hu-IFN-
R1, and Hu-IFN-
R2,
containing an NheI site (designated
EpoREC/NheI,
R1EC/NheI, and
R2EC/NheI) were generated by PCR from the
respective cDNAs as templates with the use of the T7 primer
(5
-TAATACGACTCACTATA-3
) and the internal primers
5
-GCC
CAGGGGTCCAGGTCGCTAGGCG-3
(corresponding to nucleotides 1874-1893 of p18R EpoR cDNA; Ref. 39),
5
-GTG
CAAGAACCTTTTATACTGCT-3
(corresponding to
nucleotides 779-785 of the Hu-IFN-
R1 cDNA; Ref. 4), and
5
-ATC
CATTGCTGAAGCTCAGTGGAGG-3
(corresponding to
nucleotides 1370-1390 of the Hu-IFN-
R2 cDNA; Ref. 14). The intracellular portions of the various receptors with the unique NheI site at the 5
end of the transmembrane domain
(designated EpoRIC/NheI,
R1IC/NheI, and
R2IC/NheI were generated by PCR on
corresponding cDNA templates with the use of the SP6 primer (5
-ATTTAGGTGACACTATA-3
) and the internal primers
5
-GTG
GACGCTCTCCCTCATCCTCG-3
(corresponding to nucleotides 1902-1921 of plasmid p18R),
5
-GTG
GATTCCAGTTGTTGCTGCTTTAC-3
(corresponding
to nucleotides 792-814 of the Hu-IFN-
R1 cDNA), and
5
-GTG
GATCTCCGTGGGAACATTT-3
(corresponding to
nucleotides 1398-1416 of the Hu-IFN-
R2 cDNA). The
NheI site in each primer is underlined. The PCR products
encoding the extracellular domains were incubated with T4 DNA
polymerase and dNTPs to generate blunt ends; then the PCR fragments,
which contained the vector multiple cloning sites, were subsequently
digested with the restriction endonucleases EcoRI
(EpoREC/NheI and
R2EC/NheI) or BamHI
(
R1EC/NheI), and cloned into the
EcoRV and EcoRI/BamHI sites of the
expression vector pcDNA3 (Invitrogen) to yield the plasmids
pEpoREC, p
R1EC and p
R2EC.
Analogously, the PCR products encoding the intracellular domains of the
various receptors were treated with T4 DNA polymerase to generate blunt
ends, digested with XbaI restriction endonuclease, and
cloned into the EcoRV and XbaI sites of
pcDNA3 to yield the plasmids pEpoRIC,
p
R1IC, and p
R2IC. To introduce the
Stat1
binding site of Hu-IFN-
R1 into the cytoplasmic domain of
EpoR, two-step asymmetric PCR (detailed in Ref. 41) was carried out sequentially on Hu-IFN-
R1 cDNA and pEpoRIC cDNA
templates with vector primers and the internal primer
CTTGTCCTTCTGTTTTTATTTCagagcaagccacatagetggg. The uppercase letters
denote sequences corresponding to the Hu-IFN-
R1 cDNA, and the
lowercase letters represent sequences corresponding to the EpoR
cDNA. The Hu-IFN-
R2 chain with the Stat1
binding site of
Hu-IFN-
R1 was constructed by restriction enzyme digestion of
p
R2IC and IFN-
R1 cDNA with BspEI and
AvaI, respectively, followed by ligation. For construction
of the chimeric receptors, plasmids encoding the suitable extracellular
or intracellular domains were digested with NheI and
XbaI restriction endonucleases and ligated together. All
constructs were sequenced for verification of the entire nucleotide
sequence of the receptor. Sequencing was done in an Applied Biosystems
model 373 automated DNA sequencer with dideoxy dye-terminator
chemistry.
EMSAs were
performed with the 22-base pair sequence containing a Stat1 binding
site (5
-GATCGATTTCCCCGAAATCATG-3
) corresponding to the GAS element in
the promoter region of the human IRF-1 gene (42). Two oligonucleotides,
5
-GATCGATTTCCCCGAAAT-3
and 5
-CATGATTTCGGGGAAATC-3
, were annealed by
incubation for 10 min at 65 °C, 10 min at 37 °C, and 10 min at
22 °C, and labeled with [
-32P]dATP and the Klenow
fragment of DNA polymerase I by the filling-in reaction (43). Whole
cell extracts were prepared as follows (44). Cells were grown to
confluence in six-well plates, and harvested by scraping in ice-cold
phosphate-buffered saline. Cells from each well were washed with 1.0 ml
of cold phosphate-buffered saline, pelleted, and resuspended in 100 µl of lysis buffer (10% glycerol, 50 mM Tris·HCl, pH
8.0, 0.5% Nonidet P-40, 150 mM NaCl, 0.1 mM
EDTA, 1 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, 3 µg/ml aprotinin, 1 µg/ml
pepstatin, and 1 µg/ml leupeptin). After 30 min on ice, the extracts
were centrifuged for 5 min at full speed in a microcentrifuge and the
supernatant was recovered for use in the assay and stored at
80 °C.
EMSA reactions contained 2.5 µl of the whole cell extracts, 1 ng
32P-labeled probe (specific activity approximately
109 cpm/µg), 24 µg/ml bovine serum albumin, 160 µg/ml
poly(dI·dC), 20 mM HEPES, pH 7.9, 1 mM
MgCl2, 4.0% Ficoll (Pharmacia Biotech Inc.), 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 a 1:10 dilution of anti-Stat1 antibody was included
in the reaction. Competition experiments contained a 100-fold excess of
the unlabeled oligonucleotide. Reactions were incubated at 24 °C for
20 min. Then 8 µl of the reaction mixture was electrophoresed at 400 V for 3-4 h at 4 °C on a 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.
Rabbit anti-Jak1 antibody was developed against
a synthetic peptide (KTLIEKERFYESRCRPVTPSC) corresponding to the end of
the second kinase-like domain of murine Jak1. Rabbit anti-Stat1
antibody, raised against the carboxyl-terminal region of Stat1
, was
a gift from James Darnell. Rabbit anti-Jak2 antibody (catalogue no.
SC-294) and rabbit anti-Stat5 antibody (catalogue no. SC-835) were from Santa Cruz Biotechnology. Monoclonal anti-phosphotyrosine antibody was
purchased from Sigma (catalogue no. P3300).
Cells were stimulated
with Hu-IFN- (1,000 units/ml) or Epo (100 units/ml) for 10 min at
37 °C. Immunoprecipitations and blottings were performed as
described (16, 40).
Cytofluorographic analysis of cells for surface expression of class I MHC antigens was performed as described previously (13, 41, 45) with mouse anti-human-HLA B-7 monoclonal antibody (W6/32) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG.
The schematic illustration
of the various chimeric receptor molecules that were produced is shown
in Fig. 1. In one set of chimeric constructs, the
extracellular domain of the EpoR was spliced to the transmembrane
domain and the cytoplasmic domain of each of the two IFN-R subunits.
In the other set of chimeras, the transmembrane and intracellular
domain of EpoR was fused to the extracellular domain of IFN-
R1 and
IFN-
R2.
Class I MHC Antigen Induction
To investigate the role of the
intracellular domain of IFN-R2 in the signal transduction complex of
IFN-
, we constructed a chimeric receptor chain consisting of the
extracellular domain of IFN-
R2 and the intracellular domain of EpoR.
This chimeric construct,
R2/EpoR, and the native IFN-
R2 subunit
were separately transfected into CHO-B7 as well as CHO-16-9 cells. The
ability of the transfected chimeric cDNA to transduce a signal upon
induction with Hu-IFN-
was assayed by measurement of enhanced MHC
class I antigen expression in the transfected cells and by activation of Stat1
. CHO-B7 cells transfected with IFN-
R2 or
R2/EpoR
cDNA showed no response to Hu-IFN-
as they lack the
ligand-binding receptor subunit, Hu-IFN-
R1 (data not shown).
Parental CHO-16-9 cells, which contain human chromosome 6q and express
the Hu-IFN-
R1 subunit, showed no induction of MHC class I antigens
in response to Hu-IFN-
(Fig. 2, panel A)
but when stably transfected with expression vectors encoding
Hu-IFN-
R2 cDNA or
R2/EpoR chimera, exhibited enhanced cell
surface expression of class I MHC antigens in response to Hu-IFN-
(Fig. 2, panels B and C). To assess how effectively the intracellular domain of EpoR could substitute for the
intracellular domain of the IFN-
R2 subunit, we measured the
induction of MHC class I antigens as a function of IFN-
concentration. As depicted in Fig. 3, there was a
slightly lower induction of MHC class I antigens in the cells
containing the chimeric
R2/EpoR than in the cells containing the
native Hu-IFN-
R2 chain at each concentration of Hu-IFN-
used.
Nevertheless, the fact that the EpoR intracellular domain can be
substituted for the Hu-IFN-
R2 intracellular domain shows that
another sequence that can recruit Jak2 into the signal transduction
complex can substitute for the intracellular domain of
Hu-IFN-
R2.
Various chimeric receptors between the EpoR and Hu-IFN-R1 and
Hu-IFN-
R2 subunits were constructed in order to gain an
understanding of the events leading to signal transduction. CHO-16-9
cells were stably transfected with expression vectors coding for EpoR,
EpoR/
R1, EpoR/
R2, the combination of EpoR/
R1 and EpoR/
R2,
and
R1/EpoR(p91). In response to Epo, the EpoR transfectants showed
no response (Fig. 2, panel D). The EpoR/
R1 transfectants
showed a slight enhancement of expression of MHC class I antigens (Fig.
2, panel E), which shows that the intracellular domain of
the Hu-IFN-
R1 chain, by itself, can recruit all the requisite
components for signal transduction. At lower concentrations of Epo
(less than 100 units/ml), there was little or no increased MHC class I
antigen expression in these cells (Fig. 4). The
transfectants containing both EpoR/
R1 and EpoR/
R2 chains
exhibited substantial expression of MHC class I antigens (Fig. 2,
panel F; Fig. 4). Cells transfected with the expression
vector coding for EpoR(p91) chimeric cDNA (EpoR with the p91
recruitment site from IFN-
R1) respond to Epo with enhanced
expression of class I MHC antigens, while the
R1/EpoR(p91) transfectants were unresponsive (Fig. 2, panels H and
G, respectively). Furthermore, the
R1/
R2(p91) receptor
chain is unable to transduce a signal upon binding
ligand,2 whereas the cells expressing the
EpoR/
R2(p91) chimeric receptor exhibited enhanced class I MHC
antigen expression in response to activation by Epo (Fig. 2,
panel I).
Activation of Stat Proteins
We analyzed Stat activation in
cells expressing wild-type and chimeric receptors in response to
IFN- and Epo. As shown in Fig. 5, IFN-
stimulation
resulted in Stat1
activation in transfected 16-9 cells expressing
native IFN-
R2 or
R2/EpoR chains. Similarly, Epo caused activation
of Stat1
in transfected cell lines expressing EpoR/
R1 and both
EpoR/
R1 and EpoR/
R2 receptor chains (Fig. 6).
Consistent with the small enhancement in surface expression of class I
MHC antigens in cells expressing EpoR/
R1 in response to Epo,
Stat1
activation was also lower in these cells compared to cells
expressing both EpoR/
R1 and EpoR/
R2 chains. Activation of p91 was
also observed in cells expressing the EpoR(p91) and EpoR/
R2(p91)
chains (Fig. 6). Furthermore, cells expressing those chimeric receptors
containing the EpoR intracellular domain, except
R1/EpoR(p91) and
R1/EpoR, exhibited activation of Stat5 in addition to Stat1
(Fig.
5; data with
R1/EpoR were negative similar to results with
R1/EpoR(p91)).3 Stat5 is phosphorylated
on tyrosine in response to Epo (19, 20). Both Stat1
and Stat5 are
supershifted by the addition of anti-Stat1
and anti-Stat5
antibodies, respectively. Addition of 100-fold molar excess of
unlabeled GAS oligonucleotide eliminates both Stat1
and Stat5
activated complexes.
Activation of Jak Kinases
IFN- activates Jak1 and Jak2
kinases (46), whereas Epo activates Jak2 (36) during signal
transduction. Thus, we tested the ability of the various chimeric
receptors to activate Jak1 and Jak2 kinases in response to binding of
ligand. Phosphorylation of Jak1 and Jak2 (Fig. 7) was
examined by immunoprecipitation of cellular lysates with
anti-phosphotyrosine antibodies, followed by a Western blot visualized
with specific anti-Jak1 and anti-Jak2 antibodies. Both Jak1 and Jak2
were phosphorylated in response to Hu-IFN-
treatment in 16-9 cells
expressing parental IFN-
R2 or chimeric
R2/EpoR receptors.
Induction with Epo phosphorylated both Jak1 and Jak2 kinase in the cell
line expressing both EpoR/
R1 and EpoR/
R2 chains. In the cell line
expressing only the chimeric EpoR/
R1 receptor, only Jak1 kinase was
phosphorylated in response to Epo. The cell line transfected with the
R1/EpoR chimeric receptor did not exhibit phosphorylation of either
Jak1 or Jak2 kinase upon IFN-
treatment.
For hormones, growth factors and cytokines, the conversion of the
extracellular ligand-binding event to the intracellular signal involves
a change in the oligomeric structure of the receptor. Depending on the
ligand, this can take the form of receptor homodimers (Epo, growth
hormone), heterodimers (ciliary neurotrophic factor, leukemia
inhibitory factor), homotrimers (tumor necrosis factor), and more
complex assemblies (reviewed in Ref. 47). In the case of IFN-, the
oligomerization involving IFN-
R1 and IFN-
R2 initiates the signal
transduction events: activation of Jak1 and Jak2, phosphorylation of
IFN-
R1 on Tyr-457 (16, 25), followed by phosphorylation and
activation of Stat1
(27). A major function of receptor dimerization
is to bring two receptor-associated kinases together for
transactivation and phosphorylation of the receptor chains. The
cytoplasmic domain of the IFN-
R2 subunit serves to bring Jak2 kinase
into the signal transduction complex (16). This is a crucial event
since deletion of the membrane-proximal region of the intracellular
domain of the IFN-
R2 chain, which encompasses the Jak2 association
site, completely abrogates its ability to transduce signals in response
to IFN-
(16), and cells lacking Jak2 do not respond to IFN-
(46).
This is further supported by the observation that the IFN-
R2/EpoR
chimeric receptor, which recruits Jak2, is almost as effective as the
native IFN-
R2 chain in supporting signal transduction in response to
IFN-
(Figs. 2, 5, and 7). The IFN-
R2 subunit is a helper receptor
subunit with a Jak2 association site, but no Stat recruitment site; its intracellular domain can be substituted with the cytoplasmic domain of
any receptor subunit that can bring a Jak kinase to the IFN-
receptor complex to support signal transduction (40).
The requirement for two distinct Jak kinases in the IFN- signaling
pathway was demonstrated with the use of kinase-deficient cell lines
(46, 48). Based on our results with the chimeric erythropoietin-interferon
receptors, we propose that this reflects two features characteristic of the IFN-
receptor complex: the unique
properties of the receptor relative to the positioning of the Jaks, and
the idea that Jak1 is relatively ineffective in one or more of the
following phosphorylation steps (trans-phosphorylation of itself,
phosphorylation of IFN-
R1, and activation of Stat1
). The presence
of Jak2 facilitates effective phosphorylation of the above steps. In
contrast to the growth hormone receptor (49) and the EpoR (37)
complexes, when one IFN-
homodimer binds two IFN-
R1 molecules,
the two receptor subunits do not interact with one another and are
separated by 27 Å (50) at their closest point. Therefore, although the
IFN-
R1 chain possesses both a Jak1 association site and a Stat1
recruitment site, alone it is unable to transduce a signal on
homodimerization as the two Jak1 kinases are not in physical proximity
to permit transphosphorylation (Fig. 8A).
Crystallographic analysis of the IFN-
·IFN-
R1 complex suggests
that each monomer of the IFN-
homodimer binds one IFN-
R1 and one
IFN-
R2 subunit (50). Thus the signal-transducing complex of IFN-
consists of the IFN-
homodimer bound to two IFN-
R1 and two
IFN-
R2 chains, which recruit Jak1 and Jak2, respectively (16, 40);
and Jak2 phosphorylates Jak1, following which either kinase
phosphorylates Tyr-457 of the IFN-
R1 chain (Fig. 8B; see also Refs. 25, 45, and 51). The phosphorylated segment of each
IFN-
R1 chain recruits Stat1
, which is then phosphorylated by Jak1
or Jak2, then released to dimerize and form the active Stat1
. In
contrast, with the EpoR/
R1 dimer, two Jak1 kinases are brought
sufficiently close together to activate one another (Fig.
8C), albeit inefficiently. In the case of the
EpoR/
R1·EpoR/
R2 dimer, one Jak1 and one Jak2 are in close
apposition for Jak2 to phosphorylate Jak1 and initiate efficient
downstream signaling events (Fig. 8D). Cells expressing the
EpoR/
R2(p91) chimeric receptor (Fig. 2I) exhibit a
stronger biological response than cells expressing both EpoR/
R1 and
EpoR/
R2 (Fig. 2F) or even the native IFN-
receptor
(
R2, Fig. 2B), which supports a modulating role for Jak1
in the IFN-
R complex. In cells expressing both EpoR/
R1 and
EpoR/
R2 chains, binding of Epo can induce the formation of three
types of receptor dimers: EpoR/
R1 homodimers, EpoR/
R2 homodimers,
and EpoR/
R1·EpoR/
R2 heterodimers. The EpoR/
R1 homodimer is
barely active (Figs. 2E and 4), and the EpoR/
R2 homodimer is inactive. The major functional receptor complex therefore must be
the EpoR/
R1·EpoR/
R2 heterodimer (Fig. 8D).
That Jak1 is relatively ineffective in transphosphorylation is
supported by the observation that cells expressing the EpoR/R1 chimera show a smaller response than the cells expressing both EpoR/
R1 and EpoR/
R2 or the EpoR/
R2(p91) chimeric receptor
chains. Thus, even though homodimerization of the EpoR/
R1 receptor
by Epo brings the cytoplasmic domains of the two
R1 subunits into close proximity (Fig. 8C), the data of Figs. 2, 4, and 7
indicate that Jak2 is more effective at phosphorylating Jak1 than the
latter is at cross-phosphorylating itself. This is consistent with the results of Briscoe et al. (52), who reported that a Jak1
molecule with an inactive kinase domain can replace the normal Jak1 in signal transduction by IFN-
and suggested a structural role for Jak1
in the receptor complex.
As noted above, the Jak kinases do not mediate Stat selectivity and are
promiscuous in their activity; each of the Jak kinases can substitute
for Jak2 in signal transduction by IFN- (40). Selectivity is likely
maintained from the extracellular receptor-ligand interaction to the
final signal transduction mechanism by other regions of the
intracellular domains. For example, Heim et al. (53)
suggested that the SH2 recognition domain of Stat1
maintains some of
the specificity. It remains to be established, however, how Stat1
can be activated by many different cytokines and maintain specificity
through transcription. Other molecules that interact with Jaks and
Stats may contribute to the specificity of the interaction (54).4
We propose that the multichain cytokine class II receptors have two
major chains exemplified by the IFN- receptor complex (Fig.
8B). The ligand binding chain (IFN-
R1) and the accessory chain (IFN-
R2; helper receptor) serve as a foundation for the functional IFN-
R complex (16, 40). The geometry of the IFN-
R1 chain is such that its homodimerization yields a non-functional intracellular receptor complex. The accessory chain completes this
function (Fig. 8A). The question arises: why should two
separate chains have evolved when one in the correct configuration
would suffice? We postulate that the presence of two distinct chains provides for more effective control and fine tuning of responses to
ligand. For example, the differences in response of TH1 and TH2 cells to IFN-
result from the lack of expression of
the IFN-
R2 chain in the TH1 subset (55-57) and allows
exquisite fine tuning of sensitivity to IFN-
. It is also possible
that receptors with multiple chains could recruit additional factors
into the complex to generate a wider variety of intracellular signals.
This could explain how receptors with multiple subunits could activate
a greater number of specific pathways and signals than those with fewer
elements in the receptor complex. Our experiments begin to provide an
insight into these possibilities.
We thank Brian Pollack for providing the
pR2EC cDNA, Dr. Lawrence Blatt of Amgen for the gift
of recombinant human erythropoietin, and Dr. Simon Jones for the EpoR
cDNA. We are grateful to Dr. James Darnell, Jr. for the
anti-Stat1
antibodies. We thank Dr. Jerry Langer for critical review
of the manuscript and Eleanor Kells for its preparation.