A functional role of the membrane-proximal extracellular domains
of the signal transducer gp130 in heterodimerization
with the leukemia inhibitory factor receptor
Andreas Timmermann, Andrea Ku¨ ster, Ingo Kurth, Peter C. Heinrich and Gerhard Mu¨ ller-Newen
Institut fu
¨
r Biochemie, Rheinisch-Westfa
¨
lische Technische Hochschule Aachen, Germany
gp130 is the common signal transducing receptor subunit of
interleukin (IL)-6-type cytokines. gp130 either homodimer-
izes in response to IL-6 and IL-11 or forms heterodimers
with the leukemia inhibitory factor (LIF) receptor (LIFR) in
response to LIF, oncostatin M (OSM), ciliary neurotrophic
factor (CNTF), cardiotrophin-1 (CT-1) or cardiotrophin-
like cytokine resulting in the onset of cytoplasmic tyrosine
phosphorylation cascades. The extracellular parts of both
gp130 and LIFR consist of several Ig-like and fibronectin
type III-like domains. The role of the membrane-distal
domains of gp130 (D1, D2, D3) and LIFR in ligand binding
is well established. In this study we investigated the func-
tional significance of the membrane-proximal domains of
gp130 (D4, D5, D6) in respect to heterodimerization with
LIFR. Deletion of each of the membrane-proximal domains
of gp130 (D4, D5andD6) leads to LIF unresponsiveness.
Replacement of the gp130 domains by the corresponding
domains of the related GCSF receptor either restores weak
LIF responsiveness (D4-GCSFR), leads to constitutive
activation of gp130 (D5-GCSFR) or results in an inactive
receptor (D6-GCSFR). Mutation of a specific cysteine in D5
of gp130 (C458A) leads to constitutive heterodimerization

sites for signalling molecules that, in most cases, also
become phosphorylated. Most importantly, STAT (signal
transducer and activator of transcription) factors are
recruited to the receptor, dimerize upon phosphorylation
and translocate into the nucleus to induce expression of
target genes [5].
Based on the architecture of the extracellular part,
hematopoietic cytokine receptors can be subdivided into
two groups. The extracellular parts of short cytokine
receptors like erythropoetin recepter (EpoR), growth
hormone receptor (GHR), prolactinR, IL-2Rb or IL-4R
consist of only a single cytokine binding module (CBM).
The CBM is made up of two fibronectin type III-like
(FNIII) domains containing some characteristic conserved
motifs in their primary structures. Several structures of
CBMs of short cytokine receptors bound to their ligands
have been solved showing that in the active receptor dimer
the membrane-proximal domains are juxtaposed in a well-
defined orientation [6,7].
The extracellular parts of complex cytokine receptors like
gp130, LIFR, leptinR or GCSFR contain at least one CBM
and additional FNIII- and Ig-like domains. The cytokine
receptor gp130 consists of an Ig-like domain (D1), followed
by a CBM (D2, D3) and three FNIII-like domains (D4, D5,
and D6) (Fig. 1) [8]. The role of the membrane-distal
domains (D1–D3) in ligand binding has been well estab-
lished by functional and structural studies. In response
Correspondence to G. Mu
¨
ller-Newen, Institut fu

membrane-proximal domains of gp130 for receptor activa-
tion in response to ligands that lead to gp130 homodime-
rization. We proposed a particular role for D5 of gp130 in
respect to proper receptor spacing and orientation [18]. In
this study, using gp130 deletion mutants, point mutants and
chimeric receptors, the role of the individual membrane-
proximal domains of gp130 in heterodimerization with the
LIFR is evaluated. A cysteine to alanine mutation in D5 of
gp130 in combination with the LIFR leads to a weak
constitutive activity and an elevated response to stimulation
with LIF. A model for the gp130/LIFR interaction is
proposed, in which D5 of gp130 contacts domain 7 of the
LIFR.
MATERIALS AND METHODS
Enzymes, proteins, antibodies, chemicals,
and cell culture media
Enzymes were purchased from Roche (Mannheim, Ger-
many) and protein A–Sepharose was obtained from
Amersham (Freiburg, Germany). Fugene was obtained
from Roche (Mannheim, Germany). DMEM and antibi-
otics were obtained from Life Technologies (Eggenstein,
Germany); fetal bovine serum was provided by Seromed
(Berlin, Germany). [a-
32
P]deoxyATP was purchased from
Hartmann Analytic (Braunschweig, Germany). Human
rIL-6 was expressed in Escherichia coli, refolded, and
purified as described by Arcone et al.[19].Thespecific
activity was 10
8

streptomycin, 60 mgÆL
)1
penicillin and 5% conditioned
medium from X63Ag-653 BPV-mIL-3 myeloma cells as a
source of IL-3. Simian monkey kidney cells (COS7) were
cultured in DMEM supplemented with 10% fetal bovine
serum, 100 mgÆL
)1
streptomycin, and 60 mgÆL
)1
penicillin.
Cells were grown at 37 °C in a water-saturated atmo-
sphere at 5% CO
2
. BaF3 transfectants were cultured in the
presence of 0.5 lgÆmL
)1
hygromycin if transfected with the
LIFR expression vector pSBC1/2-LIFR/Hygro and
1mgÆmL
)1
G418 if transfected with a pSVL-gp130-expres-
sion vector together with pSV2-Neo.
All cells were regularly checked for the absence of
mycoplasma infection using PCR detection of mycoplasma
DNA.
Plasmid construction
Construction of gp130 wild-type and domain mutant
expression vectors D4, D5, D6 and D5 has been described
elsewhere [18]. The domain exchange mutants gp130 D4

DNA was amplified using the primer pSVL(sense) and an
antisense primer containing the mutation. A second PCR-
fragment was generated using the primers pSVL(antisense)
and a sense primer with the corresponding mutation. These
fragments were isolated, mixed and served as templates for a
fusion PCR using the primers pSVL(sense) and pSVL(anti-
sense). The reaction products were digested with the
restriction enzymes Xho IandBstEII and cloned into the
expression vector gp130-pSVLDEco. The primers used for
the PCR reactions were: pSVL(sense) 5¢-GTGTTACTT
CTGCTCT-3¢; pSVL(antisense) 5¢-TCTAGTTGTGGTT
TGT-3¢; C458A(sense) 5¢-ATACTTGAGTGGGCTGTG
TTATCAG-3¢; C458A(antisense) 5¢-ATCTGATAACAC
AGCCCACTCAAGTAT-3¢; C466A(sense) 5¢-GATAAA
GCACCCGCTATCACAGACTGG-3¢; C466A(antisense)
5¢-CCAGTCTGTGATAGCGGGTGCTTTATCTG-3¢;
C491A(sense) 5¢-GCAGAGAGCAAAGCCTATTTGAT
AACAG-3¢ and C491(antisense) 5¢-TGTTATCAAATAG
GCTTTGCTCTCTG-3¢.
PCRs were performed applying standard procedures. All
plasmids were sequenced using an ABI Prism Automated
sequencer (Applied Biosystems).
The full-length human LIFR cDNA was cloned into
pSBC-1 to yield the mammalian expression vector pSBC-
LIFR as previously described [15]. For the transfection of
BaF3-cells, the bicistronic expression vector pSBC1/2-
LIFR/Hygro was used [15,24].
Transfection of cells
Plasmid DNA was transfected into BaF3-cells by electro-
poration. Thirty micrograms of the bicistronic LIFR

)1
gp130antibodiesB-P4orB-P8or10lgÆmL
)1
LIFR antibody 10B2. Cells were washed with cold NaCl/P
i
/
azide and incubated with R-phycoerythrin-conjugated anti-
(mouse IgG) Fab-fragment at a 1 : 50 dilution. Again, cells
were washed with cold NaCl/P
i
/azide and then resuspended
in 400 lLNaCl/P
i
/azide followed by flow cytometry
analysis using a FACScalibur (Beckton Dickinson).
Electrophoretic mobility shift assay (EMSA)
Cells were incubated at 37 °C for 15 min in the presence of
IL-6/sIL-6R, LIF, OSM or left unstimulated. BaF3-cells
were stimulated with 25 ngÆmL
)1
IL-6 and 1 lgÆmL
)1
sIL-
6R or 50 ngÆmL
)1
LIF or 50 ngÆmL
)1
OSM. COS7 cells
were stimulated with 12.5 ngÆmL
)1

2-mercaptoethanol prior to
stimulation. Immediately after stimulation, cells were washed
twice with ice-cold NaCl/P
i
containing 100 l
M
vanadate.
After addition of 600 lL lysis buffer (10% glycerol, 0.25%
Brij-96, 50 m
M
Tris/HCl, 50 l
M
Na
3
VO
4
,100l
M
EDTA,
1m
M
phenylmethanesulfonyl fluoride, 1 mgÆL
)1
aprotinin,
1mgÆL
)1
leupeptin, pH 8.0) the cells were collected and lysed
for 30 min in a microcentrifuge tube. The lysate was
centrifuged for 1 min at 3000 r.p.m. in an Eppendorf
centrifuge and the supernatant was transferred into a new

dimeric complexes with the LIFR, mutants of gp130, in
which single FNIII-domains are deleted were generated
lacking either D4 (gp130-D4), D5 (gp130-D5) or D6 (gp130-
D6) [18]. These gp130 mutants were coexpressed with the
LIFR in different cell types. The STAT-activation after
stimulation with the cytokines IL-6, LIF or OSM was used
as a measure of signal transduction through the analysed
complexes.
Cells of the murine pre-B cell line BaF3 do not express
endogenous gp130 or LIFR. After stably transfecting these
cells with the respective cDNAs, cell surface expression of
both receptors was detected. After stable transfection of the
deletion constructs gp130D4, gp130D5orgp130D6 together
with the LIFR expression vector in BaF3-cells, the surface
expressions of both receptors were similar to those detected
for wild-type gp130/LIFR transfected cells (Fig. 2A, upper
panel).
After stimulation of these cells with IL-6/sIL-6R, none of
the analysed mutants showed a STAT activation similar to
wild-type receptors (Fig. 2A, lower panel, right). This
confirms the previously reported inactivity of the deletion
mutants in response to the gp130-homodimerizing cytokine
IL-6 [18]. Interestingly, also the formation of active hetero-
dimers with wild-type LIFR in response to LIF or OSM is
strongly reduced or abolished by deletion of individual
membrane-proximal domains of gp130. Thus, in BaF3-cells,
each of the membrane-proximal domains of gp130 is
necessary for the efficient formation of a signal transducing
heterodimeric complex of gp130 and the LIFR.
To ensure that the measured receptor activation after

detected when one of the gp130c deletion constructs was
coexpressed with the LIFR. In COS7 cells under conditions
of receptor overexpression, as in BaF3-cells, each of the
membrane-proximal domains of gp130 is necessary for the
formation of signal transducing heterodimeric complexes of
gp130 and LIFR.
Two explanations for this finding can be discussed. The
first is based on the identical domain architecture of LIFR
and gp130 in the membrane-proximal six domains. This is
likely to result in the same distance between the cell surface
and the ligand-binding epitopes of both receptors. Deletion
of a single domain in the membrane-proximal part of gp130
leads to a shift of the receptor areas involved in ligand
binding closer to the membrane, resulting in the inability of
the receptor chains to form an active receptor dimer.
Additionally, the membrane-proximal domains can act as
contact sites between the signal transducing receptor chains
or can permit the signal competent conformation of gp130
homo- or heterodimers by adjusting a defined position
towards each other. Thus, deletion of a membrane-proximal
domain of gp130 may be without consequence on ligand
binding but lead to a larger distance or a twist of the
cytoplasmic parts of the receptors responsible for signal
transduction.
Replacement of single membrane-proximal
FNIII-domains of gp130 by corresponding domains of
GCSFR leads to different effects on signal transduction
To investigate, if the function of the membrane-proximal
domains of gp130 is limited to ensure the correct spacing
between the CBM and the membrane, each of the domains

tion. In contrast, these cells showed a pronounced STAT1
activation upon stimulation with LIF.
In the heterodimeric LIFR/gp130 receptor system,
different demands are posed to the individual FNIII
domains for signal transduction. Because domain 4 of
gp130 can be replaced by a similar domain of a different
receptor without abrogation of signal transduction, this
points to a spacer role of the domain. Intriguingly, signal
transduction of the D4-GCSFR chimera occurs only after
stimulation with LIF, while after OSM stimulation no
STAT activation can be found in the transfected cells. In
previous experiments [15] we were able to show that
different epitopes in the gp130 CBM are required for after
LIF- and OSM-induced STAT activation. The difference in
signal transduction of the D4-GCSFR chimera after
stimulation with LIF and OSM could point to further
epitopes positioned C-terminally to the CBM that play
specific roles in activation of the receptor by these two
cytokines.
The ligand independent activation of the D5-GCSFR
chimera occurs in both the absence and the presence of
LIFR with identical intensities (compare with [18]). This
suggests that the constitutive activation of this mutant
receptor is due to the formation of homomeric gp130D5
complexes without involvement of LIFR. The observation
of constitutive gp130 activation after replacement of D5 led
to the proposal of a model for gp130 activation [18]. In this
model, D5 is the site for direct contact of two gp130
molecules.
Analysis of dimerization of gp130 mutants:

(dark gray histograms) followed by phycoerythrin-conjugated secondary antibody. As a negative control, mock-transfected cells were treated in the
same way (black histograms). The receptor surface expressions of the cells used for the EMSA in the lower panel are shown. After transfection of
the cells with pSVL-gp130 or pSBC1/2-LIFR/Hygro, the encoded proteins can be detected on the cell surface in similar amounts. (Lower panel)
Stably transfected cells were stimulated for 15 min with IL-6 (25 ngÆmL
)1
in the presence of 1 lgÆmL
)1
sIL-6R), LIF (50 ngÆmL
)1
)orOSM
(50 ngÆmL
)1
) or left unstimulated (–) as indicated. Nuclear extracts were prepared and activated STAT3 and STAT1 homodimers as well as
STAT1/3 heterodimers were detected by EMSA after binding to a labelled oligonucleotide probe (m67SIE). A representative of three independent
experiments is shown. (B) STAT activation in COS7 cells transiently transfected with gp130c, LIFR, LIFR/gp130c, LIFR/gp130D4c,LIFR/
gp130D5c or LIFR/gp130D6c in response to various cytokines. (Left) Forty-eight hours after transfection cells were analysed for receptor surface
expression by flow cytometry. Cells were incubated with gp130 antibody B-P8 (gp130) or with LIFR antibody 10B2 (LIFR) followed by
phycoerythrin-conjugated secondary antibody (black histograms). As a negative control, mock-transfected cells were treated in the same way (gray
histograms). The receptor surface expressions of the cells used for the EMSA in the right panel are shown. Surface expression of gp130c after
transfection of pSVL-gp130c does not influence the LIFR surface expression. Transfection of the LIFR expression vector results in increased LIFR
surface expression without affecting gp130 expression (upper row, left and central histograms). Consequently, transfection of both, gp130c and
LIFR led to strongly increased surface expression of both receptors (upper row, right histograms). The surface expression of the gp130 deletion
constructs was similar with the one of gp130c and did not interfere with LIFR-expression (lower histograms). (Right) Forty-eight hours after
transfection cells were stimulated for 15 min with IL-6 (12.5 ngÆmL
)1
in the presence of 500 ngÆmL
)1
sIL-6R), LIF (20 ngÆmL
)1
)orOSM

in COS7 cells (Fig. 4B). The only monoclonal antibody with
sufficient sensitivity for the detection of gp130 in a
coprecipitation experiment maps to domain 4 of the
extracellular part of the receptor [28]. Therefore, analysis
of the ability of the mutants gp130D4 and gp130D4 to form
complexes with the LIFR by coprecipitation was not
possible with the experimental procedure used in this study.
As the formation of a high affinity complex of LIF, LIFR
and gp130 is a prerequisite for signal transduction upon LIF
stimulation, a coprecipitation analysis of LIFR and
gp130D4 is not meaningful as this chimeric receptor is able
to transduce a signal in response to LIF.
Stimulation-independent formation of complexes with
the LIFR could be not detected for any of the gp130
mutants analysed. All gp130c domain mutants showed a
decreased coprecipitation with the LIFR compared to wild-
type gp130c. Deletion of domain 6 in gp130 led to a
complete loss of coprecipitation of this mutant with the
LIFR. Compared with the deletion mutants gp130D5and
gp130D6, the respective domain replacement mutants
gp130D5 and gp130D6 showed an increased coprecipitation
of LIFR. In all cells transfected with gp130 mutants,
endogenous wild-type gp130 was coprecipitated with LIFR
after LIF stimulation. This served as a control for proper
coprecipitation conditions.
In the case of LIF, ligand binding is a two step process.
First, the cytokine is bound by the specific LIFR with low
affinity (K
d
¼ 1–3 · 10

gp130 and the membrane. Domain 6 therefore is believed to
play a role in the formation of specific contacts between
gp130 and the LIFR.
Fig. 4. Coprecipitation of LIFR with wild-type and mutant gp130. (A)
Coprecipitation of gp130 with the LIFR is ligand dependent. COS7
cells were transfected with LIFR and wild-type gp130 or LIFR and
gp130c or were left untransfected. Forty-eight hours after transfection
cells were stimulated for 15 min with LIF (50 ngÆmL
)1
)orleft
unstimulated (–). After lysis of the cells with Brij buffer the LIFR was
precipitated by addition of 1 lgÆmL
)1
of the specific antiserum sc-659
that is directed against the 19 C-terminal amino acids of the receptor
and thus does not interfere with ligand binding and extracellular
receptor dimerization. The immunoprecipitated proteins were separ-
ated by gel electrophoresis on a 7% SDS/PAGE gel followed by
transfer to a poly(vinylidene difluoride) membrane. Detection of gp130
was performed with the monoclonal antibody B-P4. After removing of
the antibodies from the blot, LIFR was detected using the specific
LIFR-antiserum. (B) gp130 mutants lacking domains 5 or 6 do not
heterodimerize with LIFR in response to LIF. Forty-eight hours after
transfection of COS7 cells with LIFR and the gp130 chimeras copre-
cipitation and detection of LIFR and gp130 was performed as des-
cribed in (A).
2722 A. Timmermann et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Based on our model for gp130 activation [18], we propose
here a model for a ternary gp130/LIFR/LIF complex that is
consistent with the data presented in this work (Fig. 5). The

model of D5, these have been suggested to be involved in the
dimerization and activation of gp130 by formation of
disulfide bonds [30]. The analysis of cysteine residues in
gp130 led to the finding that the two N-terminal cysteines of
D5 (C458 and C466) form an intramolecular disulfide bond,
while the third (C-terminal) cysteine (C491) in this domain
contains a free thiol group. The latter was proposed to be
protected against solvent contact by a loop of eight amino
acids of D5, which is positioned by the disulfide bond
between cysteines C458 and C466 [30].
To analyse the role of the three cysteines in D5 of gp130
in heterodimerization with the LIFR, each of the cysteines
was mutated to alanine. Considering the disulfide bond
between C458 and C466, mutation of one of these amino
acids leads to a free thiol group in the domain (e.g. C466-SH
in the construct gp130C458A). These mutants were intro-
duced into the gp130c construct and together with the LIFR
transiently transfected into COS7 cells. The surface expres-
sion of the mutant receptors were similar to that of wild-
type gp130 (data not shown). Cotransfection of the gp130
mutants C466Ac and C491Ac together with the LIFR did
not lead to a constitutive signal transduction in COS7 cells
(Fig. 6A, upper panel). Also, stimulation of these cells with
OSM did not result in STAT1 activation. In both cases,
stimulation with LIF led to signal transduction, while a
significant STAT1 activation after stimulation with IL-6
was detectable only for the C491A mutant. However,
cotransfection of LIFR and the C458Ac mutant in COS7
cells lad to a weak STAT1 activation independent of
cytokine stimulation. This activation was dramatically

LIF are labelled with II and III.
Ó FEBS 2002 Heterodimerization of gp130 with LIFR (Eur. J. Biochem. 269) 2723
Exchange of this amino acid to alanine leads to the
abrogation of IL-6 signal transduction in COS7 cells
transiently transfected with this mutant. In contrast, upon
coexpression of gp130C458A together with LIFR IL-6
signal transduction is not impaired. Additionally, the
gp130C458A mutant is able to transduce a signal in
response to OSM. Therefore, there does not seem to be an
absolute requirement for this cysteine in gp130 signal
transduction.
To investigate whether the constitutive activity of the
C458A mutant relies on the formation of a disulfide bond
not present in the wild-type receptor, the signal transduction
of this mutant was measured under reducing conditions
(Fig. 6B). These experiments were performed in analogy to
activation and dimerization studies of different receptors
[31,32]. Cells cotransfected with LIFR and gp130C458Ac or
with wild-type gp130c were preincubated in the presence of
2-mercaptoethanol prior to stimulation with LIF. After
incubation of the cells with 2-mercaptoethanol, the surface
expression of both transfected receptors were similar to that
of cells incubated under nonreducing conditions (data not
shown). While the reducing conditions did not lead to a
change in signal transduction in mock (lanes 1–4) and
LIFR/gp130 transfected cells (lanes 5–8), the constitutive
activity and increased sensitivity to LIF of the C458Ac
mutant was abrogated. Instead, the mutant receptor
behaved like wild-type gp130c (lanes 9–12). These findings
point to the formation of a new disulfide bond after

left unstimulated as indicated. Nuclear extracts were prepared and activated STAT1 homodimers were detected by EMSA as decribed in legend to
Fig. 1B. (C) The gp130C458A mutant ligand-independently coprecipitates with the LIFR. Forty-eight hours after transfection coprecipitation of
LIFR and gp130 was performed as described in legend to Fig. 3A. While in LIFR/gp130 transfected cells the coprecipitation of the receptors
depends on stimulation with LIF, it is independent of stimulation in LIFR/gp130C458A transfected cells.
2724 A. Timmermann et al. (Eur. J. Biochem. 269) Ó FEBS 2002
gp130C458Ac is therefore formed independently of cytoki-
ne stimulation. This constitutive complex formation points
to a covalent bonding between the two receptor chains on
the cell surface, as indicated in the activation model based
on these results (Fig. 7). The complex does not have the
conformation necessary for the effective induction of
signalling pathways, although a weak constitutive receptor
activation can be observed, probably due to the increased
proximity of the cytoplasmic parts of the receptors. This
finding highlights the importance of proper receptor orien-
tation in addition to dimerization for receptor activation.
Upon ligand binding, the complex adopts a conformation
able to effectively induce signalling. The complex might
function as a trap for the cytokine, as the dissociation of the
ligand from the receptors is diminished. This would lead to a
prolonged signal transduction via the receptor complex,
resulting in the observed prominent STAT activation.
The elevated signal transduction of the gp130C458A-
mutant together with the LIFR depends on ligand binding
to the preformed receptor complex, which in case of LIF is a
two step process of defined order. This binding order can
explain the observed cytokine specificity of the gp130C458A
mutant, as it can be assumed that also in the preformed
receptor complex LIF first interacts with the LIFR via a site
III, Ig-like domain contact and is than transferred into the

more, we propose that in all cytokine receptors that share
structural homology with gp130 and LIFR like OSMR,
GCSFR and IL-12R, ligand binding leads to a receptor
dimer, in which the C-terminal domains of the CBM are
separated from each other. The three membrane-proximal
FNIII domains function in bringing the transmembrane
and cytoplasmic regions in close proximity in order to
enable signal transduction to occur. More structural data
are required to substantiate our proposed model of gp130/
LIFR activation.
ACKNOWLEDGEMENTS
We thank Dr John Wijdenes (DIACLONE, Besanc¸ on, France) for
providing the gp130 mAbs B-P4 and B-P8 and Dr Vincent Pitard
(CNRS-UMR 5540, Universite
´
de Bordeaux 2, Bordeaux, France)
for providing the LIFR mAb 10B2 used in this study. This work was
supported by grants from the Deutsche Forschungsgemeinschaft
(SFB 542) and the Fonds der Chemischen Industrie (Frankfurt,
Germany).
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